Cravatt BF


Full name : Cravatt Benjamin F

First name : Benjamin F

Mail : Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92307

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Country : USA

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References (212)

Title : Sexually dimorphic effects of monoacylglycerol lipase inhibitor MJN110 on stress-related behaviors and drinking in Marchigian Sardinian alcohol-preferring rats - Vozella_2023_Br.J.Pharmacol__
Author(s) : Vozella V , Cruz B , Feldman HC , Bullard R , Bianchi PC , Natividad LA , Cravatt BF , Zorrilla EP , Ciccocioppo R , Roberto M
Ref : British Journal of Pharmacology , : , 2023
Abstract : BACKGROUND AND PURPOSE: Stress-related disorders are often intertwined with alcohol use disorder (AUD). The endocannabinoid (eCB) system, which comprises the lipid mediators anandamide (AEA) and 2-arachidonoylglycerol (2-AG), plays an important homeostatic role in the regulation of stress circuits and has emerged as a therapeutic target to treat stress disorders and AUD. Extensive research has elucidated the role of AEA, but less is known about 2-AG-mediated signaling. EXPERIMENTAL APPROACH: We pharmacologically enhanced the eCB signaling by inhibiting the 2-AG metabolizing enzyme, monoacylglycerol lipase (MAGL), in male and female Marchigian-Sardinian alcohol preferring (msP) rats, a model of innate alcohol preference and stress hypersensitivity, and control Wistar rats. We tested the acute effect of the selective MAGL inhibitor MJN110 in alleviating symptoms of alcohol drinking, anxiety, irritability, and fear in both male and female rats. KEY RESULTS: A single systemic administration of MJN110 increased 2-AG levels in the central amygdala, prelimbic, and infralimbic cortex, but did not acutely alter alcohol drinking. MAGL inhibition reduced aggressive behaviors in female msPs and increased defensive behaviors in male msPs, during the irritability test. Moreover, in the novelty-induced hypophagia test, MJN110 selectively enhanced palatable food consumption in females, mitigating stress-induced food suppression. Lastly, msP rats showed increased conditioned fear behavior compared to Wistar rats, and MJN110 reduced context-associated conditioned fear responses, but not cue-probed fear expression, in male msPs. CONCLUSION AND IMPLICATIONS: Acute inhibition of MAGL attenuated some stress-related responses in msP rats but not voluntary alcohol drinking. Our results provide new insights into the sex dimorphism documented in stress-induced responses and suggest that sex-specific endocannabinoid-based approaches should be considered in the clinical development of therapeutics.
ESTHER : Vozella_2023_Br.J.Pharmacol__
PubMedSearch : Vozella_2023_Br.J.Pharmacol__
PubMedID: 37488777

Title : Attenuating ABHD17 enhances S- palmitoylation, membrane localization and signal transduction of NOD2 and Crohn's disease-associated variants - Dixon_2023_bioRxiv__
Author(s) : Dixon CL , Martin NR , Niphakis MJ , Cravatt BF , Fairn GD
Ref : Biorxiv , : , 2023
Abstract : NOD2 is an intracellular innate immune receptor that senses bacterial peptidoglycans. Although soluble in the cytosol, a portion of the protein is associated with the plasma membrane and endosomal compartments for microbial surveillance. Palmitoylation of NOD2 by zDHHC5 promotes its membrane recruitment to drive proinflammatory and antimicrobial responses to pathogenic invasion. A depalmitoylation step by an unknown protein, thioesterase, releases NOD2 from membranes into the cytosol, where the protein can then enter a new cycle of palmitoylation-depalmitoylation. Here, we identify alpha/beta -hydrolase domain-containing protein 17 isoforms (ABHD17A, 17B, 17C) as the thioesterases responsible for depalmitoylation of NOD2. Inhibiting ABHD17 increased the plasmalemmal localization of both wild-type NOD2 and a subset of hypo-palmitoylated Crohn's disease-associated variants, resulting in increased NF-kappaB activation and production of pro-inflammatory cytokines in epithelial cells. These results suggest that targeted inhibition of ABHD17 may rescue some Crohn's disease-associated NOD2 variants.
ESTHER : Dixon_2023_bioRxiv__
PubMedSearch : Dixon_2023_bioRxiv__
PubMedID: 38187608

Title : Chemoproteomics-Enabled Identification of 4-Oxo-beta-Lactams as Inhibitors of Dipeptidyl Peptidases 8 and 9 - Carvalho_2022_Angew.Chem.Int.Ed.Engl_61_e202210498
Author(s) : Carvalho LAR , Ross B , Fehr L , Bolgi O , Wohrle S , Lum KM , Podlesainski D , Vieira AC , Kiefersauer R , Felix R , Rodrigues T , Lucas SD , Gross O , Geiss-Friedlander R , Cravatt BF , Huber R , Kaiser M , Moreira R
Ref : Angew Chem Int Ed Engl , : , 2022
Abstract : Dipeptidyl peptidases 8 and 9 (DPP8/9) have gathered interest as drug targets due to their important roles in biological processes like immunity and tumorigenesis. Elucidation of their distinct individual functions remains an ongoing task and could benefit from the availability of novel, chemically diverse and selective chemical tools. Here, we report the activity-based protein profiling (ABPP)-mediated discovery of 4-oxo-beta-lactams as potent, non-substrate-like nanomolar DPP8/9 inhibitors. X-ray crystallographic structures revealed different ligand binding modes for DPP8 and DPP9, including an unprecedented targeting of an extended S2' (eS2') subsite in DPP8. Biological assays confirmed inhibition at both target and cellular levels. Altogether, our integrated chemical proteomics and structure-guided small molecule design approach led to novel DPP8/9 inhibitors with alternative molecular inhibition mechanisms, delivering the highest selectivity index reported to date.
ESTHER : Carvalho_2022_Angew.Chem.Int.Ed.Engl_61_e202210498
PubMedSearch : Carvalho_2022_Angew.Chem.Int.Ed.Engl_61_e202210498
PubMedID: 36089535
Gene_locus related to this paper: human-DPP8 , human-DPP9

Title : In situ identification of cellular drug targets in mammalian tissue - Pang_2022_Cell_185_1793
Author(s) : Pang Z , Schafroth MA , Ogasawara D , Wang Y , Nudell V , Lal NK , Yang D , Wang K , Herbst DM , Ha J , Guijas C , Blankman JL , Cravatt BF , Ye L
Ref : Cell , 185 :1793 , 2022
Abstract : The lack of tools to observe drug-target interactions at cellular resolution in intact tissue has been a major barrier to understanding in vivo drug actions. Here, we develop clearing-assisted tissue click chemistry (CATCH) to optically image covalent drug targets in intact mammalian tissues. CATCH permits specific and robust in situ fluorescence imaging of target-bound drug molecules at subcellular resolution and enables the identification of target cell types. Using well-established inhibitors of endocannabinoid hydrolases and monoamine oxidases, direct or competitive CATCH not only reveals distinct anatomical distributions and predominant cell targets of different drug compounds in the mouse brain but also uncovers unexpected differences in drug engagement across and within brain regions, reflecting rare cell types, as well as dose-dependent target shifts across tissue, cellular, and subcellular compartments that are not accessible by conventional methods. CATCH represents a valuable platform for visualizing in vivo interactions of small molecules in tissue.
ESTHER : Pang_2022_Cell_185_1793
PubMedSearch : Pang_2022_Cell_185_1793
PubMedID: 35483372

Title : Inhibitory Neurotransmission Is Sex-Dependently Affected by Tat Expression in Transgenic Mice and Suppressed by the Fatty Acid Amide Hydrolase Enzyme Inhibitor PF3845 via Cannabinoid Type-1 Receptor Mechanisms - Xu_2022_Cells_11_
Author(s) : Xu C , Yadav-Samudrala BJ , Nath B , Mistry T , Jiang W , Niphakis MJ , Cravatt BF , Mukhopadhyay S , Lichtman AH , Ignatowska-Jankowska BM , Fitting S
Ref : Cells , 11 : , 2022
Abstract : (1) Background. The endocannabinoid (eCB) system, which regulates physiological and cognitive processes, presents a promising therapeutic target for treating HIV-associated neurocognitive disorders (HAND). Here we examine whether upregulating eCB tone has potential protective effects against HIV-1 Tat (a key HIV transactivator of transcription) protein-induced alterations in synaptic activity. (2) Methods. Whole-cell patch-clamp recordings were performed to assess inhibitory GABAergic neurotransmission in prefrontal cortex slices of Tat transgenic male and female mice, in the presence and absence of the fatty acid amide hydrolase (FAAH) enzyme inhibitor PF3845. Western blot and mass spectrometry analyses assessed alterations of cannabinoid receptor and enzyme protein expression as well as endogenous ligands, respectively, to determine the impact of Tat exposure on the eCB system. (3) Results. GABAergic activity was significantly altered upon Tat exposure based on sex, whereas the effectiveness of PF3845 to suppress GABAergic activity in Tat transgenic mice was not altered by Tat or sex and involved CB(1)R-related mechanisms that depended on calcium signaling. Additionally, our data indicated sex-dependent changes for AEA and related non-eCB lipids based on Tat induction. (4) Conclusion. Results highlight sex- and/or Tat-dependent alterations of GABAergic activity and eCB signaling in the prefrontal cortex of Tat transgenic mice and further increase our understanding about the role of FAAH inhibition in neuroHIV.
ESTHER : Xu_2022_Cells_11_
PubMedSearch : Xu_2022_Cells_11_
PubMedID: 35269478

Title : Identification of cell wall synthesis inhibitors active against Mycobacterium tuberculosis by competitive activity-based protein profiling - Li_2022_Cell.Chem.Biol_29_883
Author(s) : Li M , Patel HV , Cognetta AB, 3rd , Smith TC, 2nd , Mallick I , Cavalier JF , Previti ML , Canaan S , Aldridge BB , Cravatt BF , Seeliger JC
Ref : Cell Chemical Biology , 29 :883 , 2022
Abstract : The identification and validation of a small molecule's targets is a major bottleneck in the discovery process for tuberculosis antibiotics. Activity-based protein profiling (ABPP) is an efficient tool for determining a small molecule's targets within complex proteomes. However, how target inhibition relates to biological activity is often left unexplored. Here, we study the effects of 1,2,3-triazole ureas on Mycobacterium tuberculosis (Mtb). After screening -200 compounds, we focus on 4 compounds that form a structure-activity series. The compound with negligible activity reveals targets, the inhibition of which is functionally less relevant for Mtb growth and viability, an aspect not addressed in other ABPP studies. Biochemistry, computational docking, and morphological analysis confirms that active compounds preferentially inhibit serine hydrolases with cell wall and lipid metabolism functions and that disruption of the cell wall underlies biological activity. Our findings show that ABPP identifies the targets most likely relevant to a compound's antibacterial activity.
ESTHER : Li_2022_Cell.Chem.Biol_29_883
PubMedSearch : Li_2022_Cell.Chem.Biol_29_883
PubMedID: 34599873

Title : Profiling of MicroRNA Targets Using Activity-Based Protein Profiling: Linking Enzyme Activity to MicroRNA-185 Function - Filip_2021_Cell.Chem.Biol_28_202
Author(s) : Filip R , Desrochers GF , Lefebvre DM , Reed A , Singaravelu R , Cravatt BF , Pezacki JP
Ref : Cell Chemical Biology , 28 :202 , 2021
Abstract : MicroRNAs (miRNAs) act as cellular signal transducers through repression of protein translation. Elucidating targets using bioinformatics and traditional quantitation methods is often insufficient to uncover global miRNA function. Herein, alteration of protein function caused by miRNA-185 (miR-185), an immunometabolic miRNA, was determined using activity-based protein profiling, transcriptomics, and lipidomics. Fluorophosphonate-based activity-based protein profiling of miR-185-induced changes to human liver cells revealed that exclusively metabolic serine hydrolase enzymes were regulated in activity, some with roles in lipid and endocannabinoid metabolism. Lipidomic analysis linked enzymatic changes to levels of cellular lipid species, such as components of very-low-density lipoprotein particles. Additionally, inhibition of one miR-185 target, monoglyceride lipase, led to decreased hepatitis C virus levels in an infectious model. Overall, the approaches used here were able to identify key functional changes in serine hydrolases caused by miR-185 that are targetable pharmacologically, such that a small molecule inhibitor can recapitulate the miRNA phenotype.
ESTHER : Filip_2021_Cell.Chem.Biol_28_202
PubMedSearch : Filip_2021_Cell.Chem.Biol_28_202
PubMedID: 33450181

Title : Selective inhibition of monoacylglycerol lipase is associated with passive coping behavior and attenuation of stress-induced dopamine release in the medial prefrontal cortex - Pavon_2021_Neurobiol.Stress_14_100293
Author(s) : Pavon FJ , Polis IY , Stouffer DG , Cravatt BF , Roberto M , Martin-Fardon R , Rodriguez de Fonseca F , Parsons LH , Serrano A
Ref : Neurobiol Stress , 14 :100293 , 2021
Abstract : The endocannabinoid system is involved in the regulation of the stress response, but the relative contribution of N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG) and their mechanisms have to be elucidated. In this study, we compared the effects of the pharmacological inhibition of the two major endocannabinoid-degrading enzymes [fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) for AEA and 2-AG, respectively] on stress-coping [forced swim test (FST) and tail suspension test (TST)] and anxiety-like [elevated-plus maze (EPM) and light-dark test (LDT)] behaviors in wild-type and FAAH knockout mice. In vivo microdialysis estimated the effects of FAAH and MAGL inhibition on dopamine (DA) and serotonin (5-HT) levels in the medial prefrontal cortex (mPFC) during an FST. Mice were treated with PF-3845 (FAAH inhibitor), JZL184 (MAGL inhibitor), JZL195 (dual FAAH/MAGL inhibitor) or vehicle. Our data showed that PF-3845 increased latency to immobility and decreased total immobility time in FST, but no effects were observed in TST compared with vehicle-treated wild-type mice. By contrast, JZL184 decreased latency and increased immobility in TST and FST. JZL195 in wild-type mice and JZL184 in FAAH knockout mice reproduced the same passive coping behaviors as JZL184 in wild-type mice in TST and FST. In the microdialysis experiment, FST was associated with increased DA and 5-HT levels in the mPFC. However, JZL184-treated wild-type mice displayed a significant attenuation of forced swim stress-induced DA release compared with vehicle-treated wild-type mice and PF-3845-treated wild-type mice. Finally, FAAH and/or MAGL inhibitors induced robust and consistent anxiolytic-like effects in EPM and LDT. These results suggested differences between FAAH and MAGL inhibition in stress-coping behaviors. Notably, MAGL inhibition induced a consistent avoidant coping behavior and attenuated the stress-induced mPFC DA response in FST. However, more investigation is needed to elucidate the functional association between DA and 2-AG signaling pathways, and the molecular mechanism in the regulation of passive coping strategies during inescapable stress.
ESTHER : Pavon_2021_Neurobiol.Stress_14_100293
PubMedSearch : Pavon_2021_Neurobiol.Stress_14_100293
PubMedID: 33490317
Gene_locus related to this paper: human-MGLL

Title : An abundant biliary metabolite derived from dietary omega-3 polyunsaturated fatty acids regulates triglycerides - Grevengoed_2021_J.Clin.Invest_131_
Author(s) : Grevengoed TJ , Trammell SA , Svenningsen JS , Makarov MV , Nielsen TS , Jacobsen JCB , Treebak JT , Calder PC , Migaud ME , Cravatt BF , Gillum MP
Ref : J Clinical Investigation , 131 : , 2021
Abstract : Omega-3 fatty acids from fish oil reduce triglyceride levels in mammals, yet the mechanisms underlying this effect have not been fully clarified, despite the clinical use of omega-3 ethyl esters to treat severe hypertriglyceridemia and reduce cardiovascular disease risk in humans. Here, we identified in bile a class of hypotriglyceridemic omega-3 fatty acid-derived N-acyl taurines (NATs) that, after dietary omega-3 fatty acid supplementation, increased to concentrations similar to those of steroidal bile acids. The biliary docosahexaenoic acid-containing (DHA-containing) NAT C22:6 NAT was increased in human and mouse plasma after dietary omega-3 fatty acid supplementation and potently inhibited intestinal triacylglycerol hydrolysis and lipid absorption. Supporting this observation, genetic elevation of endogenous NAT levels in mice impaired lipid absorption, whereas selective augmentation of C22:6 NAT levels protected against hypertriglyceridemia and fatty liver. When administered pharmacologically, C22:6 NAT accumulated in bile and reduced high-fat diet-induced, but not sucrose-induced, hepatic lipid accumulation in mice, suggesting that C22:6 NAT is a negative feedback mediator that limits excess intestinal lipid absorption. Thus, biliary omega-3 NATs may contribute to the hypotriglyceridemic mechanism of action of fish oil and could influence the design of more potent omega-3 fatty acid-based therapeutics.
ESTHER : Grevengoed_2021_J.Clin.Invest_131_
PubMedSearch : Grevengoed_2021_J.Clin.Invest_131_
PubMedID: 33507883

Title : Chemical proteomic analysis of palmostatin beta-lactone analogs that affect N-Ras palmitoylation - Suciu_2021_Bioorg.Med.Chem.Lett_53_128414
Author(s) : Suciu RM , Luvaga IK , Hazeen A , Weerasooriya C , Richardson SK , Firestone AJ , Shannon K , Howell AR , Cravatt BF
Ref : Bioorganic & Medicinal Chemistry Lett , 53 :128414 , 2021
Abstract : S-Palmitoylation is a reversible post-translational lipid modification that regulates protein trafficking and signaling. The enzymatic depalmitoylation of proteins is inhibited by the beta-lactones Palmostatin M and B, which have been found to target several serine hydrolases. In efforts to better understand the mechanism of action of Palmostatin M, we describe herein the synthesis, chemical proteomic analysis, and functional characterization of analogs of this compound. We identify Palmostatin M analogs that maintain inhibitory activity in N-Ras depalmitoylation assays while displaying complementary reactivity across the serine hydrolase class as measured by activity-based protein profiling. Active Palmostatin M analogs inhibit the recently characterized ABHD17 subfamily of depalmitoylating enzymes, while sparing other candidate depalmitoylases such as LYPLA1 and LYPLA2. These findings improve our understanding of the structure-activity relationship of Palmostatin M and refine the set of serine hydrolase targets relevant to the compound's effects on N-Ras palmitoylation dynamics.
ESTHER : Suciu_2021_Bioorg.Med.Chem.Lett_53_128414
PubMedSearch : Suciu_2021_Bioorg.Med.Chem.Lett_53_128414
PubMedID: 34666187

Title : Positron Emission Tomography Imaging of the Endocannabinoid System: Opportunities and Challenges in Radiotracer Development - Hou_2021_J.Med.Chem_64_123
Author(s) : Hou L , Rong J , Haider A , Ogasawara D , Varlow C , Schafroth MA , Mu L , Gan J , Xu H , Fowler CJ , Zhang MR , Vasdev N , Ametamey S , Cravatt BF , Wang L , Liang SH
Ref : Journal of Medicinal Chemistry , 64 :123 , 2021
Abstract : The endocannabinoid system (ECS) is involved in a wide range of biological functions and comprises cannabinoid receptors and enzymes responsible for endocannabinoid synthesis and degradation. Over the past 2 decades, significant advances toward developing drugs and positron emission tomography (PET) tracers targeting different components of the ECS have been made. Herein, we summarized the recent development of PET tracers for imaging cannabinoid receptors 1 (CB1R) and 2 (CB2R) as well as the key enzymes monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), particularly focusing on PET neuroimaging applications. State-of-the-art PET tracers for the ECS will be reviewed including their chemical design, pharmacological properties, radiolabeling, as well as preclinical and human PET imaging. In addition, this review addresses the current challenges for ECS PET biomarker development and highlights the important role of PET ligands to study disease pathophysiology as well as to facilitate drug discovery.
ESTHER : Hou_2021_J.Med.Chem_64_123
PubMedSearch : Hou_2021_J.Med.Chem_64_123
PubMedID: 33379862

Title : Prolyl endopeptidase-like is a (thio)esterase involved in mitochondrial respiratory chain function - Rosier_2021_iScience_24_103460
Author(s) : Rosier K , McDevitt MT , Smet J , Floyd BJ , Verschoore M , Marcaida MJ , Bingman CA , Lemmens I , Dal Peraro M , Tavernier J , Cravatt BF , Gounko NV , Vints K , Monnens Y , Bhalla K , Aerts L , Rashan EH , Vanlander AV , Van Coster R , Regal L , Pagliarini DJ , Creemers JWM
Ref : iScience , 24 :103460 , 2021
Abstract : Deficiency of the serine hydrolase prolyl endopeptidase-like (PREPL) causes a recessive metabolic disorder characterized by neonatal hypotonia, feeding difficulties, and growth hormone deficiency. The pathophysiology of PREPL deficiency and the physiological substrates of PREPL remain largely unknown. In this study, we connect PREPL with mitochondrial gene expression and oxidative phosphorylation by analyzing its protein interactors. We demonstrate that the long PREPL(L) isoform localizes to mitochondria, whereas PREPL(S) remains cytosolic. Prepl KO mice showed reduced mitochondrial complex activities and disrupted mitochondrial gene expression. Furthermore, mitochondrial ultrastructure was abnormal in a PREPL-deficient patient and Prepl KO mice. In addition, we reveal that PREPL has (thio)esterase activity and inhibition of PREPL by Palmostatin M suggests a depalmitoylating function. We subsequently determined the crystal structure of PREPL, thereby providing insight into the mechanism of action. Taken together, PREPL is a (thio)esterase rather than a peptidase and PREPL(L) is involved in mitochondrial homeostasis.
ESTHER : Rosier_2021_iScience_24_103460
PubMedSearch : Rosier_2021_iScience_24_103460
PubMedID: 34888501
Gene_locus related to this paper: human-PREPL

Title : Diacylglycerol Lipase-beta Knockout Mice Display a Sex-Dependent Attenuation of Traumatic Brain Injury-Induced Mortality with No Impact on Memory or Other Functional Consequences - O'Brien_2021_Cannabis.Cannabinoid.Res__
Author(s) : O'Brien LD , Smith TL , Donvito G , Cravatt BF , Newton J , Spiegel S , Reeves TM , Phillips LL , Lichtman AH
Ref : Cannabis Cannabinoid Res , : , 2021
Abstract : Background: The endogenous cannabinoid system modulates inflammatory signaling in a variety of pathological states, including traumatic brain injury (TBI). The selective expression of diacylglycerol lipase-beta (DAGL-beta), the 2-arachidonylglycerol biosynthetic enzyme, on resident immune cells of the brain (microglia) and the role of this pathway in neuroinflammation, suggest that this enzyme may contribute to TBI-induced neuroinflammation. Accordingly, we tested whether DAGL-beta(-/-) mice would show a protective phenotype from the deleterious consequences of TBI on cognitive and neurological motor functions. Materials and Methods: DAGL-beta(-/-) and -beta(+/+) mice were subjected to the lateral fluid percussion model of TBI and assessed for learning and memory in the Morris water maze (MWM) Fixed Platform (reference memory) and Reversal (cognitive flexibility) tasks, as well as in a cued MWM task to infer potential sensorimotor/motivational deficits. In addition, subjects were assessed for motor behavior (Rotarod and the Neurological Severity Score assays) and in the light/dark box and the elevated plus maze to infer whether these manipulations affected anxiety-like behavior. Finally, we also examined whether brain injury disrupts the ceramide/sphingolipid lipid signaling system and if DAGL-beta deletion offers protection. Results: TBI disrupted all measures of neurological motor function and reduced body weight, but did not affect body temperature or performance in common assays used to infer anxiety. TBI also impaired performance in MWM Fixed Platform and Reversal tasks, but did not affect cued MWM performance. Although no differences were found between DAGL-beta(-/-) and -beta(+/+) mice in any of these measures, male DAGL-beta(-/-) mice displayed an unexpected survival-protective phenotype, which persisted at increased injury severities. In contrast, TBI did not elicit mortality in female mice regardless of genotype. TBI also produced significant changes in sphingolipid profiles (a family of lipids, members of which have been linked to both apoptotic and antiapoptotic pathways), in which DAGL-beta deletion modestly altered levels of select species. Conclusions: These findings indicate that although DAGL-beta does not play a necessary role in TBI-induced cognitive and neurological function, it appears to contribute to the increased vulnerability of male mice to TBI-induced mortality, whereas female mice show high survival rates irrespective of DAGL-beta expression.
ESTHER : O'Brien_2021_Cannabis.Cannabinoid.Res__
PubMedSearch : O'Brien_2021_Cannabis.Cannabinoid.Res__
PubMedID: 34142866
Gene_locus related to this paper: human-DAGLB , mouse-DGLB

Title : Novel Reversible-Binding PET Ligands for Imaging Monoacylglycerol Lipase Based on the Piperazinyl Azetidine Scaffold - Rong_2021_J.Med.Chem__
Author(s) : Rong J , Mori W , Xia X , Schafroth MA , Zhao C , Van RS , Yamasaki T , Chen J , Xiao Z , Haider A , Ogasawara D , Hiraishi A , Shao T , Zhang Y , Chen Z , Pang F , Hu K , Xie L , Fujinaga M , Kumata K , Gou Y , Fang Y , Gu S , Wei H , Bao L , Xu H , Collier TL , Shao Y , Carson RE , Cravatt BF , Wang L , Zhang MR , Liang SH
Ref : Journal of Medicinal Chemistry , : , 2021
Abstract : Monoacylglycerol lipase (MAGL) is a 33 kDa serine protease primarily responsible for hydrolyzing 2-arachidonoylglycerol into the proinflammatory eicosanoid precursor arachidonic acid in the central nervous system. Inhibition of MAGL constitutes an attractive therapeutic concept for treating psychiatric disorders and neurodegenerative diseases. Herein, we present the design and synthesis of multiple reversible MAGL inhibitor candidates based on a piperazinyl azetidine scaffold. Compounds 10 and 15 were identified as the best-performing reversible MAGL inhibitors by pharmacological evaluations, thus channeling their radiolabeling with fluorine-18 in high radiochemical yields and favorable molar activity. Furthermore, evaluation of [(18)F]10 and [(18)F]15 ([(18)F]MAGL-2102) by autoradiography and positron emission tomography (PET) imaging in rodents and nonhuman primates demonstrated favorable brain uptakes, heterogeneous radioactivity distribution, good specific binding, and adequate brain kinetics, and [(18)F]15 demonstrated a better performance. In conclusion, [(18)F]15 was found to be a suitable PET radioligand for the visualization of MAGL, harboring potential for the successful translation into humans.
ESTHER : Rong_2021_J.Med.Chem__
PubMedSearch : Rong_2021_J.Med.Chem__
PubMedID: 34569803
Gene_locus related to this paper: human-MGLL

Title : Monoacylglycerol Lipase Inhibitor MJN110 Reduces Neuronal Hyperexcitability, Restores Dendritic Arborization Complexity, and Regulates Reward-Related Behavior in Presence of HIV-1 Tat - League_2021_Front.Neurol_12_651272
Author(s) : League AF , Gorman BL , Hermes DJ , Johnson CT , Jacobs IR , Yadav-Samudrala BJ , Poklis JL , Niphakis MJ , Cravatt BF , Lichtman AH , Ignatowska-Jankowska BM , Fitting S
Ref : Front Neurol , 12 :651272 , 2021
Abstract : While current therapeutic strategies for people living with human immunodeficiency virus type 1 (HIV-1) suppress virus replication peripherally, viral proteins such as transactivator of transcription (Tat) enter the central nervous system early upon infection and contribute to chronic inflammatory conditions even alongside antiretroviral treatment. As demand grows for supplemental strategies to combat virus-associated pathology presenting frequently as HIV-associated neurocognitive disorders (HAND), the present study aimed to characterize the potential utility of inhibiting monoacylglycerol lipase (MAGL) activity to increase inhibitory activity at cannabinoid receptor-type 1 receptors through upregulation of 2-arachidonoylglycerol (2-AG) and downregulation of its degradation into proinflammatory metabolite arachidonic acid (AA). The MAGL inhibitor MJN110 significantly reduced intracellular calcium and increased dendritic branching complexity in Tat-treated primary frontal cortex neuron cultures. Chronic MJN110 administration in vivo increased 2-AG levels in the prefrontal cortex (PFC) and striatum across Tat(+) and Tat(-) groups and restored PFC N-arachidonoylethanolamine (AEA) levels in Tat(+) subjects. While Tat expression significantly increased rate of reward-related behavioral task acquisition in a novel discriminative stimulus learning and cognitive flexibility assay, MJN110 altered reversal acquisition specifically in Tat(+) mice to rates indistinguishable from Tat(-) controls. Collectively, our results suggest a neuroprotective role of MAGL inhibition in reducing neuronal hyperexcitability, restoring dendritic arborization complexity, and mitigating neurocognitive alterations driven by viral proteins associated with latent HIV-1 infection.
ESTHER : League_2021_Front.Neurol_12_651272
PubMedSearch : League_2021_Front.Neurol_12_651272
PubMedID: 34484091
Gene_locus related to this paper: human-MGLL

Title : Sex-dependent effects of endocannabinoid modulation of conditioned fear extinction in rats - Morena_2021_Br.J.Pharmacol_178_983
Author(s) : Morena M , Nastase AS , Santori A , Cravatt BF , Shansky RM , Hill MN
Ref : British Journal of Pharmacology , 178 :983 , 2021
Abstract : BACKGROUND AND PURPOSE: Women are twice as likely as men to develop post-traumatic stress disorder (PTSD) making the search for biological mechanisms underlying these gender disparities especially crucial. One of the hallmark symptoms of PTSD is an alteration in the ability to extinguish fear responses to trauma-associated cues. In male rodents, the endocannabinoid system can modulate fear extinction and has been suggested as a therapeutic target for PTSD. However, whether and how the endocannabinoid system may modulate fear expression and extinction in females remains unknown. EXPERIMENTAL APPROACH: To answer this question, we pharmacologically manipulated endocannabinoid signalling in male and female rats prior to extinction of auditory conditioned fear and measured both passive (freezing) and active (darting) conditioned responses. KEY RESULTS: Surprisingly, we found that acute systemic inhibition of the endocannabinoid anandamide (AEA) or 2-arachidonoyl glycerol (2-AG) hydrolysis did not significantly alter fear expression or extinction in males. However, the same manipulations in females produced diverging effects. Increased AEA signalling at vanilloid TRPV1 receptors impaired fear memory extinction. In contrast, inhibition of 2-AG hydrolysis promoted active over passive fear responses acutely via activation of cannabinoid(1) (CB(1) ) receptors. Measurement of AEA and 2-AG levels after extinction training revealed sex- and brain region-specific changes. CONCLUSION AND IMPLICATIONS: We provide the first evidence that AEA and 2-AG signalling affect fear expression and extinction in females in opposite directions. These findings are relevant to future research on sex differences in mechanisms of fear extinction and may help develop sex-specific therapeutics to treat trauma-related disorders.
ESTHER : Morena_2021_Br.J.Pharmacol_178_983
PubMedSearch : Morena_2021_Br.J.Pharmacol_178_983
PubMedID: 33314038

Title : Development of a highly-specific (18)F-labeled irreversible positron emission tomography tracer for monoacylglycerol lipase mapping - Chen_2021_Acta.Pharm.Sin.B_11_1686
Author(s) : Chen Z , Mori W , Rong J , Schafroth MA , Shao T , Van RS , Ogasawara D , Yamasaki T , Hiraishi A , Hatori A , Chen J , Zhang Y , Hu K , Fujinaga M , Sun J , Yu Q , Collier TL , Shao Y , Cravatt BF , Josephson L , Zhang MR , Liang SH
Ref : Acta Pharm Sin B , 11 :1686 , 2021
Abstract : As a serine hydrolase, monoacylglycerol lipase (MAGL) is principally responsible for the metabolism of 2-arachidonoylglycerol (2-AG) in the central nervous system (CNS), leading to the formation of arachidonic acid (AA). Dysfunction of MAGL has been associated with multiple CNS disorders and symptoms, including neuroinflammation, cognitive impairment, epileptogenesis, nociception and neurodegenerative diseases. Inhibition of MAGL provides a promising therapeutic direction for the treatment of these conditions, and a MAGL positron emission tomography (PET) probe would greatly facilitate preclinical and clinical development of MAGL inhibitors. Herein, we design and synthesize a small library of fluoropyridyl-containing MAGL inhibitor candidates. Pharmacological evaluation of these candidates by activity-based protein profiling identified 14 as a lead compound, which was then radiolabeled with fluorine-18 via a facile S(N)Ar reaction to form 2-[(18)F]fluoropyridine scaffold. Good blood-brain barrier permeability and high in vivo specific binding was demonstrated for radioligand [(18)F]14 (also named as [(18)F]MAGL-1902). This work may serve as a roadmap for clinical translation and further design of potent (18)F-labeled MAGL PET tracers.
ESTHER : Chen_2021_Acta.Pharm.Sin.B_11_1686
PubMedSearch : Chen_2021_Acta.Pharm.Sin.B_11_1686
PubMedID: 34221877
Gene_locus related to this paper: human-MGLL

Title : ABHD17 regulation of plasma membrane palmitoylation and N-Ras-dependent cancer growth - Remsberg_2021_Nat.Chem.Biol__
Author(s) : Remsberg JR , Suciu RM , Zambetti NA , Hanigan TW , Firestone AJ , Inguva A , Long A , Ngo N , Lum KM , Henry CL , Richardson SK , Predovic M , Huang B , Dix MM , Howell AR , Niphakis MJ , Shannon K , Cravatt BF
Ref : Nat Chemical Biology , : , 2021
Abstract : Multiple Ras proteins, including N-Ras, depend on a palmitoylation/depalmitoylation cycle to regulate their subcellular trafficking and oncogenicity. General lipase inhibitors such as Palmostatin M (Palm M) block N-Ras depalmitoylation, but lack specificity and target several enzymes displaying depalmitoylase activity. Here, we describe ABD957, a potent and selective covalent inhibitor of the ABHD17 family of depalmitoylases, and show that this compound impairs N-Ras depalmitoylation in human acute myeloid leukemia (AML) cells. ABD957 produced partial effects on N-Ras palmitoylation compared with Palm M, but was much more selective across the proteome, reflecting a plasma membrane-delineated action on dynamically palmitoylated proteins. Finally, ABD957 impaired N-Ras signaling and the growth of NRAS-mutant AML cells in a manner that synergizes with MAP kinase kinase (MEK) inhibition. Our findings uncover a surprisingly restricted role for ABHD17 enzymes as regulators of the N-Ras palmitoylation cycle and suggest that ABHD17 inhibitors may have value as targeted therapies for NRAS-mutant cancers.
ESTHER : Remsberg_2021_Nat.Chem.Biol__
PubMedSearch : Remsberg_2021_Nat.Chem.Biol__
PubMedID: 33927411
Gene_locus related to this paper: human-ABHD17A , human-ABHD17B , human-ABHD17C

Title : 3-Oxo-beta-sultam as a Sulfonylating Chemotype for Inhibition of Serine Hydrolases and Activity-Based Protein Profiling - Carvalho_2020_ACS.Chem.Biol_15_878
Author(s) : Carvalho LAR , Almeida VT , Brito JA , Lum KM , Oliveira TF , Guedes RC , Goncalves LM , Lucas SD , Cravatt BF , Archer M , Moreira R
Ref : ACS Chemical Biology , 15 :878 , 2020
Abstract : 3-Oxo-beta-sultams are four-membered ring ambident electrophiles that can react with nucleophiles either at the carbonyl carbon or at the sulfonyl sulfur atoms, and that have been reported to inhibit serine hydrolases via acylation of the active-site serine residue. We have developed a panel of 3-oxo-beta-sultam inhibitors and show, through crystallographic data, that they are regioselective sulfonylating electrophiles, covalently binding to the catalytic serine of human and porcine elastases through the sulfur atom. Application of 3-oxo-beta-sultam-derived activity-based probes in a human proteome revealed their potential to label disease-related serine hydrolases and proteasome subunits. Activity-based protein profiling applications of 3-oxo-beta-sultams should open up new opportunities to investigate these classes of enzymes in complex proteomes and expand the toolbox of available sulfur-based covalent protein modifiers in chemical biology.
ESTHER : Carvalho_2020_ACS.Chem.Biol_15_878
PubMedSearch : Carvalho_2020_ACS.Chem.Biol_15_878
PubMedID: 32176480

Title : AIG1 and ADTRP are endogenous hydrolases of fatty acid esters of hydroxy fatty acids (FAHFAs) in mice - Erikci_2020_J.Biol.Chem_295_5891
Author(s) : Erikci Ertunc M , Kok BP , Parsons WH , Wang JG , Tan D , Donaldson CJ , Pinto AFM , Vaughan JM , Ngo N , Lum KM , Henry CL , Coppola AR , Niphakis MJ , Cravatt BF , Saez E , Saghatelian A
Ref : Journal of Biological Chemistry , 295 :5891 , 2020
Abstract : Fatty acid esters of hydroxy fatty acids (FAHFAs) are a newly discovered class of signaling lipids with anti-inflammatory and anti-diabetic properties. However, the endogenous regulation of FAHFAs remains a pressing but unanswered question. Here, using MS-based FAHFA hydrolysis assays, LC-MS-based lipidomics analyses, and activity-based protein profiling, we found that androgen-induced gene 1 (AIG1) and androgen-dependent TFPI-regulating protein (ADTRP), two threonine hydrolases, control FAHFA levels in vivo in both genetic and pharmacologic mouse models. Tissues from mice lacking ADTRP (Adtrp-KO), or both AIG1 and ADTRP (DKO) had higher concentrations of FAHFAs particularly isomers with the ester bond at the 9(th) carbon due to decreased FAHFA hydrolysis activity. The levels of other lipid classes were unaltered indicating that AIG1 and ADTRP specifically hydrolyze FAHFAs. Complementing these genetic studies, we also identified a dual AIG1/ADTRP inhibitor, ABD-110207, which is active in vivo Acute treatment of WT mice with ABD-110207 resulted in elevated FAHFA levels, further supporting the notion that AIG1 and ADTRP activity control endogenous FAHFA levels. However, loss of AIG1/ADTRP did not mimic the changes associated with pharmacologically administered FAHFAs on extent of upregulation of FAHFA levels, glucose tolerance, or insulin sensitivity in mice, indicating that therapeutic strategies should weigh more on FAHFA administration. Together, these findings identify AIG1 and ADTRP as the first endogenous FAHFA hydrolases identified and provide critical genetic and chemical tools for further characterization of these enzymes and endogenous FAHFAs to unravel their physiological functions and roles in health and disease.
ESTHER : Erikci_2020_J.Biol.Chem_295_5891
PubMedSearch : Erikci_2020_J.Biol.Chem_295_5891
PubMedID: 32152231

Title : Blockade of the Lysophosphatidylserine Lipase ABHD12 Potentiates Ferroptosis in Cancer Cells - Kathman_2020_ACS.Chem.Biol_15_871
Author(s) : Kathman SG , Boshart J , Jing H , Cravatt BF
Ref : ACS Chemical Biology , 15 :871 , 2020
Abstract : Ferroptosis is a type of cell death caused by the pathogenic accumulation of lipid hydroperoxides. Pharmacological mechanisms to induce ferroptosis may provide a way to kill cancer cells that are resistant to other forms of cell death like apoptosis. Nonetheless, the proteins that regulate ferroptotic sensitivity in cancer cells remain incompletely understood. Here, we screened a panel of inhibitors of serine hydrolases-an enzyme class important for regulating lipid metabolism-for potentiation of ferroptosis in HT1080 fibrosarcoma cells. We found that DO264, a selective inhibitor of the lyso- and ox-phosphatidylserine (PS) lipase ABHD12, enhances ferroptotic death caused by RSL3, an inhibitor of the lipid peroxidase GPX4. RSL3-induced ferroptosis was also potentiated by genetic disruption of ABHD12. Metabolomic experiments revealed that, in addition to elevated lyso-PS, ABHD12-inactivated cells show higher quantities of arachidonate (C20:4)-containing PS and 2-arachidonoyl glycerol, pointing to potential oxidation-sensitive lipid mediators of ferroptosis regulated by ABHD12.
ESTHER : Kathman_2020_ACS.Chem.Biol_15_871
PubMedSearch : Kathman_2020_ACS.Chem.Biol_15_871
PubMedID: 32195565
Gene_locus related to this paper: human-ABHD12

Title : Discovery of small-molecule enzyme activators by activity-based protein profiling - Kok_2020_Nat.Chem.Biol_16_997
Author(s) : Kok BP , Ghimire S , Kim W , Chatterjee S , Johns T , Kitamura S , Eberhardt J , Ogasawara D , Xu J , Sukiasyan A , Kim SM , Godio C , Bittencourt JM , Cameron M , Galmozzi A , Forli S , Wolan DW , Cravatt BF , Boger DL , Saez E
Ref : Nat Chemical Biology , 16 :997 , 2020
Abstract : Activity-based protein profiling (ABPP) has been used extensively to discover and optimize selective inhibitors of enzymes. Here, we show that ABPP can also be implemented to identify the converse-small-molecule enzyme activators. Using a kinetically controlled, fluorescence polarization-ABPP assay, we identify compounds that stimulate the activity of LYPLAL1-a poorly characterized serine hydrolase with complex genetic links to human metabolic traits. We apply ABPP-guided medicinal chemistry to advance a lead into a selective LYPLAL1 activator suitable for use in vivo. Structural simulations coupled to mutational, biochemical and biophysical analyses indicate that this compound increases LYPLAL1's catalytic activity likely by enhancing the efficiency of the catalytic triad charge-relay system. Treatment with this LYPLAL1 activator confers beneficial effects in a mouse model of diet-induced obesity. These findings reveal a new mode of pharmacological regulation for this large enzyme family and suggest that ABPP may aid discovery of activators for additional enzyme classes.
ESTHER : Kok_2020_Nat.Chem.Biol_16_997
PubMedSearch : Kok_2020_Nat.Chem.Biol_16_997
PubMedID: 32514184
Gene_locus related to this paper: human-LYPLAL1

Title : ABHD12 and LPCAT3 Interplay Regulates a Lyso-phosphatidylserine-C20:4 Phosphatidylserine Lipid Network Implicated in Neurological Disease - Ichu_2020_Biochemistry_59_1793
Author(s) : Ichu TA , Reed A , Ogasawara D , Ulanovskaya O , Roberts A , Aguirre CA , Bar-Peled L , Gao J , Germain J , Barbas S , Masuda K , Conti B , Tontonoz P , Cravatt BF
Ref : Biochemistry , 59 :1793 , 2020
Abstract : PHARC (polyneuropathy, hearing loss, cerebellar ataxia, retinitis pigmentosa, and cataract) is a human neurological disorder caused by deleterious mutations in the ABHD12 gene, which encodes an integral membrane lyso-phosphatidylserine (lyso-PS) lipase. Pharmacological or genetic disruption of ABHD12 leads to higher levels of lyso-PS lipids in human cells and the central nervous system (CNS) of mice. ABHD12 loss also causes rapid rewiring of PS content, resulting in selective increases in the level of arachidonoyl (C20:4) PS and decreases in the levels of other PS species. The biochemical basis for ABHD12-dependent PS remodeling and its pathophysiological significance remain unknown. Here, we show that genetic deletion of the lysophospholipid acyltransferase LPCAT3 blocks accumulation of brain C20:4 PS in mice lacking ABHD12 and concurrently produces hyper-increases in the level of lyso-PS in these animals. These lipid changes correlate with exacerbated auditory dysfunction and brain microgliosis in mice lacking both ABHD12 and LPCAT3. Taken together, our findings reveal that ABHD12 and LPCAT3 coordinately regulate lyso-PS and C20:4 PS content in the CNS and point to lyso-PS lipids as the likely bioactive metabolites contributing to PHARC-related neuropathologies.
ESTHER : Ichu_2020_Biochemistry_59_1793
PubMedSearch : Ichu_2020_Biochemistry_59_1793
PubMedID: 32364701
Gene_locus related to this paper: human-ABHD12

Title : ABHD4-dependent developmental anoikis safeguards the embryonic brain - Laszlo_2020_Nat.Commun_11_4363
Author(s) : Laszlo ZI , Lele Z , Zoldi M , Miczan V , Mogor F , Simon GM , Mackie K , Kacskovics I , Cravatt BF , Katona I
Ref : Nat Commun , 11 :4363 , 2020
Abstract : A specialized neurogenic niche along the ventricles accumulates millions of progenitor cells in the developing brain. After mitosis, fate-committed daughter cells delaminate from this germinative zone. Considering the high number of cell divisions and delaminations taking place during embryonic development, brain malformations caused by ectopic proliferation of misplaced progenitor cells are relatively rare. Here, we report that a process we term developmental anoikis distinguishes the pathological detachment of progenitor cells from the normal delamination of daughter neuroblasts in the developing mouse neocortex. We identify the endocannabinoid-metabolizing enzyme abhydrolase domain containing 4 (ABHD4) as an essential mediator for the elimination of pathologically detached cells. Consequently, rapid ABHD4 downregulation is necessary for delaminated daughter neuroblasts to escape from anoikis. Moreover, ABHD4 is required for fetal alcohol-induced apoptosis, but not for the well-established form of developmentally controlled programmed cell death. These results suggest that ABHD4-mediated developmental anoikis specifically protects the embryonic brain from the consequences of sporadic delamination errors and teratogenic insults.
ESTHER : Laszlo_2020_Nat.Commun_11_4363
PubMedSearch : Laszlo_2020_Nat.Commun_11_4363
PubMedID: 32868797
Gene_locus related to this paper: human-ABHD4 , mouse-abhd4

Title : The novel MAGL inhibitor MJN110 enhances responding to reward-predictive incentive cues by activation of CB1 receptors - Feja_2020_Neuropharmacol_162_107814
Author(s) : Feja M , Leigh MPK , Baindur AN , McGraw JJ , Wakabayashi KT , Cravatt BF , Bass CE
Ref : Neuropharmacology , 162 :107814 , 2020
Abstract : CB1 receptor antagonists disrupt operant responding for food and drug reinforcers, and cue-induced reinstatement of cocaine and heroin seeking. Conversely, enhancing endocannabinoid signaling, particularly 2-arachidonyl glycerol (2-AG), by inhibition of monoacyl glycerol lipase (MAGL), may facilitate some aspects of reward seeking. To determine how endocannabinoid signaling affects responding to reward-predictive cues, we employed an operant task that allows us to parse the incentive motivational properties of cues. Rats were required to nosepoke during an intermittent audiovisual incentive cue (IC) to obtain a 10% sucrose reward. The CB1 receptor antagonist, rimonabant, dose-dependently decreased the response ratio (rewarded ICs/total presented) and active nosepokes per IC, while it increased the latency to respond to the cue and obtain the reward, indicating an overall decrease in both the choice and vigor of responding. Yet rats persisted in entering the reward cup. Using a modified version of the task, the novel MAGL inhibitor MJN110 increased the response ratio, decreased the latencies to respond to the IC and enhanced active nosepokes per IC, indicating a facilitation of cue-induced reward seeking. These effects were blocked by a subthreshold dose of rimonabant. Finally, MJN110 did not alter consumption of freely available sucrose within volumes obtained in the operant task. Together these data demonstrate blocking endocannabinoid tone at the CB1 receptor attenuates the ability of cues to induce reward seeking, while some aspects of motivation for the reward are retained. Conversely, enhancing 2-AG signaling at CB1 receptors facilitates IC responding and increases the motivational properties of the IC.
ESTHER : Feja_2020_Neuropharmacol_162_107814
PubMedSearch : Feja_2020_Neuropharmacol_162_107814
PubMedID: 31628934
Gene_locus related to this paper: human-MGLL

Title : The Endocannabinoid System Alleviates Pain in a Murine Model of Cancer-Induced Bone Pain - Thompson_2020_J.Pharmacol.Exp.Ther_373_230
Author(s) : Thompson AL , Grenald SA , Ciccone HA , BassiriRad N , Niphakis MJ , Cravatt BF , Largent-Milnes TM , Vanderah TW
Ref : Journal of Pharmacology & Experimental Therapeutics , 373 :230 , 2020
Abstract : Metastatic breast cancer is prevalent worldwide, and one of the most common sites of metastasis is long bones. Of patients with disease, the major symptom is pain, yet current medications fail to adequately result in analgesic efficacy and present major undesirable adverse effects. In our study, we investigate the potential of a novel monoacylglycerol lipase (MAGL) inhibitor, MJN110, in a murine model of cancer-induced bone pain. Literature has previously demonstrated that MAGL inhibitors function to increase the endogenous concentrations of 2-arachydonylglycerol, which then activates CB1 and CB2 receptors to inhibit inflammation and pain. We demonstrate that administration of MJN110 significantly and dose dependently alleviates spontaneous pain behavior during acute administration compared with vehicle control. In addition, MJN110 maintains its efficacy in a chronic-dosing paradigm over the course of 7 days without signs of receptor sensitization. In vitro analysis of MJN110 demonstrated a dose-dependent and significant decrease in cell viability and proliferation of 66.1 breast adenocarcinoma cells to a greater extent than KML29, an alternate MAGL inhibitor, or the CB2 agonist JWH015. Chronic administration of the compound did not appear to affect tumor burden, as evidenced by radiograph or histologic analysis. Together, these data support the application for MJN110 as a novel therapeutic for cancer-induced bone pain. SIGNIFICANCE STATEMENT: Current standard of care for metastatic breast cancer pain is opioid-based therapies with adjunctive chemotherapy, which have highly addictive and other deleterious side effects. The need for effective, non-opioid-based therapies is essential, and harnessing the endogenous cannabinoid system is proving to be a new target to treat various types of pain conditions. We present a novel drug targeting the endogenous cannabinoid system that is effective at reducing pain in a mouse model of metastatic breast cancer to bone.
ESTHER : Thompson_2020_J.Pharmacol.Exp.Ther_373_230
PubMedSearch : Thompson_2020_J.Pharmacol.Exp.Ther_373_230
PubMedID: 32054717
Gene_locus related to this paper: human-MGLL

Title : Inhibition of monoacylglycerol lipase reduces nicotine reward in the conditioned place preference test in male mice - Muldoon_2020_Neuropharmacol__108170
Author(s) : Muldoon PP , Akinola LS , Schlosburg JE , Lichtman AH , Sim-Selley LJ , Mahadevan A , Cravatt BF , Damaj MI
Ref : Neuropharmacology , :108170 , 2020
Abstract : Nicotine, the primary psychoactive component in tobacco, plays a major role in the initiation and maintenance of tobacco dependence and addiction, a leading cause of preventable death worldwide. An essential need thus exists for more effective pharmacotherapies for nicotine-use cessation. Previous reports suggest that pharmacological and genetic blockade of CB1 receptors attenuate nicotine reinforcement and reward; while exogenous agonists enhanced these abuse-related behaviors. In this study, we utilized complementary genetic and pharmacologic approaches to test the hypothesis that increasing the levels of the endocannabinoid 2-arachindonoylglycerol (2-AG), will enhance nicotine reward by stimulating neuronal CB1 receptors. Contrary to our hypothesis, we found that inhibition of monoacylglycerol lipase (MAGL), the primary catabolic enzyme of 2-AG, attenuates nicotine conditioned place preference (CPP) in mice, through a non-CB1 receptor-mediated mechanism. MAGL inhibition did not alter palatable food reward or Lithium Chloride (LiCl) aversion. In support of our findings, repeated MAGL inhibition did not induce a reduction in CB1 brain receptor levels or hinder function. To explore the potential mechanism of action, we investigated if MAGL inhibition affected other fatty acid levels in our CPP paradigm. Indeed, MAGL inhibition caused a concomitant decrease in arachidonic acid (AA) levels in various brain regions of interest, suggesting an AA cascade-dependent mechanism. This idea is supported by dose-dependent attenuation of nicotine preference by the selective COX-2 inhibitors valdecoxib and LM-4131. Collectively, these findings, along with our reported studies on nicotine withdrawal, suggest that inhibition of MAGL represents a promising new target for the development of pharmacotherapies to treat nicotine dependence.
ESTHER : Muldoon_2020_Neuropharmacol__108170
PubMedSearch : Muldoon_2020_Neuropharmacol__108170
PubMedID: 32479813
Gene_locus related to this paper: human-MGLL , mouse-MGLL

Title : Genetic disruption of N-RasG12D palmitoylation perturbs hematopoiesis and prevents myeloid transformation in mice - Zambetti_2020_Blood_135_1772
Author(s) : Zambetti NA , Firestone AJ , Remsberg JR , Huang BJ , Wong JC , Long AM , Predovic M , Suciu RM , Inguva A , Kogan SC , Haigis KM , Cravatt BF , Shannon K
Ref : Blood , 135 :1772 , 2020
Abstract : Oncogenic RAS mutations pose substantial challenges for rational drug discovery. Sequence variations within the hypervariable region of Ras isoforms underlie differential posttranslational modification and subcellular trafficking, potentially resulting in selective vulnerabilities. Specifically, inhibiting the palmitoylation/depalmitoylation cycle is an appealing strategy for treating NRAS mutant cancers, particularly as normal tissues would retain K-Ras4b function for physiologic signaling. The role of endogenous N-RasG12D palmitoylation in signal transduction, hematopoietic differentiation, and myeloid transformation is unknown, and addressing these key questions will inform efforts to develop mechanism-based therapies. To evaluate the palmitoylation/depalmitoylation cycle as a candidate drug target in an in vivo disease-relevant model system, we introduced a C181S mutation into a conditional NrasG12D "knock-in" allele. The C181S second-site amino acid substitution abrogated myeloid transformation by NrasG12D, which was associated with mislocalization of the nonpalmitoylated N-Ras mutant protein, reduced Raf/MEK/ERK signaling, and alterations in hematopoietic stem and progenitor populations. Furthermore, hematologic malignancies arising in NrasG12D/G12D,C181S compound heterozygous mice invariably acquired revertant mutations that restored cysteine 181. Together, these studies validate the palmitoylation cycle as a promising therapeutic target in NRAS mutant cancers.
ESTHER : Zambetti_2020_Blood_135_1772
PubMedSearch : Zambetti_2020_Blood_135_1772
PubMedID: 32219446

Title : Single-Cell Profiling and SCOPE-Seq Reveal Lineage Dynamics of Adult Ventricular-Subventricular Zone Neurogenesis and NOTUM as a Key Regulator - Mizrak_2020_Cell.Rep_31_107805
Author(s) : Mizrak D , Bayin NS , Yuan J , Liu Z , Suciu RM , Niphakis MJ , Ngo N , Lum KM , Cravatt BF , Joyner AL , Sims PA
Ref : Cell Rep , 31 :107805 , 2020
Abstract : In the adult ventricular-subventricular zone (V-SVZ), neural stem cells (NSCs) generate new olfactory bulb (OB) neurons and glia throughout life. To map adult neuronal lineage progression, we profiled >56,000 V-SVZ and OB cells by single-cell RNA sequencing (scRNA-seq). Our analyses reveal the molecular diversity of OB neurons, including fate-mapped neurons, lineage progression dynamics, and an NSC intermediate enriched for Notum, which encodes a secreted WNT antagonist. SCOPE-seq technology, which links live-cell imaging with scRNA-seq, uncovers cell-size transitions during NSC differentiation and preferential NOTUM binding to proliferating neuronal precursors. Consistently, application of NOTUM protein in slice cultures and pharmacological inhibition of NOTUM in slice cultures and in vivo demonstrated that NOTUM negatively regulates V-SVZ proliferation. Timely, context-dependent neurogenesis demands adaptive signaling among neighboring progenitors. Our findings highlight a critical regulatory state during NSC activation marked by NOTUM, which attenuates WNT-stimulated proliferation in NSC progeny.
ESTHER : Mizrak_2020_Cell.Rep_31_107805
PubMedSearch : Mizrak_2020_Cell.Rep_31_107805
PubMedID: 32579931
Gene_locus related to this paper: human-NOTUM

Title : N-Acyl pyrazoles: Effective and tunable inhibitors of serine hydrolases - Otrubova_2019_Bioorg.Med.Chem_27_1693
Author(s) : Otrubova K , Chatterjee S , Ghimire S , Cravatt BF , Boger DL
Ref : Bioorganic & Medicinal Chemistry , 27 :1693 , 2019
Abstract : A series of N-acyl pyrazoles was examined as candidate serine hydrolase inhibitors in which the active site acylating reactivity and the leaving group ability of the pyrazole could be tuned not only through the nature of the acyl group (reactivity: amide > carbamate > urea), but also through pyrazole C4 substitution with electron-withdrawing or electron-donating substituents. Their impact on enzyme inhibitory activity displayed pronounced effects with the activity improving substantially as one alters both the nature of the reacting carbonyl group (urea > carbamate > amide) and the pyrazole C4 substituent (CN > H > Me). It was further demonstrated that the acyl chain of the N-acyl pyrazole ureas can be used to tailor the potency and selectivity of the inhibitor class to a targeted serine hydrolase. Thus, elaboration of the acyl chain of pyrazole-based ureas provided remarkably potent, irreversible inhibitors of fatty acid amide hydrolase (FAAH, apparent K(i) = 100-200 pM), dual inhibitors of FAAH and monoacylglycerol hydrolase (MGLL), or selective inhibitors of MGLL (IC(50) = 10-20 nM) while simultaneously minimizing off-target activity (e.g., ABHD6 and KIAA1363).
ESTHER : Otrubova_2019_Bioorg.Med.Chem_27_1693
PubMedSearch : Otrubova_2019_Bioorg.Med.Chem_27_1693
PubMedID: 30879861

Title : Diacylglycerol Lipase-Alpha Regulates Hippocampal-Dependent Learning and Memory Processes in Mice - Schurman_2019_J.Neurosci_39_5949
Author(s) : Schurman LD , Carper MC , Moncayo LV , Ogasawara D , Richardson K , Yu L , Liu X , Poklis JL , Liu QS , Cravatt BF , Lichtman AH
Ref : Journal of Neuroscience , 39 :5949 , 2019
Abstract : Diacylglycerol lipase-alpha (DAGL-alpha), the principal biosynthetic enzyme of the endogenous cannabinoid 2-arachidonylglycerol (2-AG) on neurons, plays a key role in CB(1) receptor-mediated synaptic plasticity and hippocampal neurogenesis, but its contribution to global hippocampal-mediated processes remains unknown. Thus, the present study examines the role that DAGL-alpha plays on LTP in hippocampus, as well as in hippocampal-dependent spatial learning and memory tasks, and on the production of endocannabinoid and related lipids through the use of complementary pharmacologic and genetic approaches to disrupt this enzyme in male mice. Here we show that DAGL-alpha gene deletion or pharmacological inhibition disrupts LTP in CA1 of the hippocampus but elicits varying magnitudes of behavioral learning and memory deficits in mice. In particular, DAGL-alpha(-/-) mice display profound impairments in the Object Location assay and Morris Water Maze (MWM) acquisition engaging in nonspatial search strategies. In contrast, WT mice administered the DAGL-alpha inhibitor DO34 show delays in MWM acquisition and reversal learning, but no deficits in expression, extinction, forgetting, or perseveration processes in this task, as well as no impairment in Object Location. The deficits in synaptic plasticity and MWM performance occur in concert with decreased 2-AG and its major lipid metabolite (arachidonic acid), but increases of a 2-AG diacylglycerol precursor in hippocampus, PFC, striatum, and cerebellum. These novel behavioral and electrophysiological results implicate a direct and perhaps selective role of DAGL-alpha in the integration of new spatial information.SIGNIFICANCE STATEMENT Here we show that genetic deletion or pharmacologic inhibition of diacylglycerol lipase-alpha (DAGL-alpha) impairs hippocampal CA1 LTP, differentially disrupts spatial learning and memory performance in Morris water maze (MWM) and Object Location tasks, and alters brain levels of endocannabinoids and related lipids. Whereas DAGL-alpha(-/-) mice exhibit profound phenotypic spatial memory deficits, a DAGL inhibitor selectively impairs the integration of new information in MWM acquisition and reversal tasks, but not memory processes of expression, extinction, forgetting, or perseveration, and does not affect performance in the Objection Location task. The findings that constitutive or short-term DAGL-alpha disruption impairs learning and memory at electrophysiological and selective in vivo levels implicate this enzyme as playing a key role in the integration of new spatial information.
ESTHER : Schurman_2019_J.Neurosci_39_5949
PubMedSearch : Schurman_2019_J.Neurosci_39_5949
PubMedID: 31127001
Gene_locus related to this paper: mouse-q6wqj1

Title : N-acyl taurines are endogenous lipid messengers that improve glucose homeostasis - Grevengoed_2019_Proc.Natl.Acad.Sci.U.S.A_116_24770
Author(s) : Grevengoed TJ , Trammell SAJ , McKinney MK , Petersen N , Cardone RL , Svenningsen JS , Ogasawara D , Nexoe-Larsen CC , Knop FK , Schwartz TW , Kibbey RG , Cravatt BF , Gillum MP
Ref : Proc Natl Acad Sci U S A , 116 :24770 , 2019
Abstract : Fatty acid amide hydrolase (FAAH) degrades 2 major classes of bioactive fatty acid amides, the N-acylethanolamines (NAEs) and N-acyl taurines (NATs), in central and peripheral tissues. A functional polymorphism in the human FAAH gene is linked to obesity and mice lacking FAAH show altered metabolic states, but whether these phenotypes are caused by elevations in NAEs or NATs is unknown. To overcome the problem of concurrent elevation of NAEs and NATs caused by genetic or pharmacological disruption of FAAH in vivo, we developed an engineered mouse model harboring a single-amino acid substitution in FAAH (S268D) that selectively disrupts NAT, but not NAE, hydrolytic activity. The FAAH-S268D mice accordingly show substantial elevations in NATs without alterations in NAE content, a unique metabolic profile that correlates with heightened insulin sensitivity and GLP-1 secretion. We also show that N-oleoyl taurine (C18:1 NAT), the most abundant NAT in human plasma, decreases food intake, improves glucose tolerance, and stimulates GPR119-dependent GLP-1 and glucagon secretion in mice. Together, these data suggest that NATs act as a class of lipid messengers that improve postprandial glucose regulation and may have potential as investigational metabolites to modify metabolic disease.
ESTHER : Grevengoed_2019_Proc.Natl.Acad.Sci.U.S.A_116_24770
PubMedSearch : Grevengoed_2019_Proc.Natl.Acad.Sci.U.S.A_116_24770
PubMedID: 31740614

Title : Discovery and Optimization of Selective and in Vivo Active Inhibitors of the Lysophosphatidylserine Lipase alpha\/beta-Hydrolase Domain-Containing 12 (ABHD12) - Ogasawara_2019_J.Med.Chem_62_1643
Author(s) : Ogasawara D , Ichu TA , Jing H , Hulce JJ , Reed A , Ulanovskaya OA , Cravatt BF
Ref : Journal of Medicinal Chemistry , 62 :1643 , 2019
Abstract : ABHD12 is a membrane-bound hydrolytic enzyme that acts on the lysophosphatidylserine (lyso-PS) and lysophosphatidylinositol (lyso-PI) classes of immunomodulatory lipids. Human and mouse genetic studies point to a key role for the ABHD12-(lyso)-PS/PI pathway in regulating (neuro)immunological functions in both the central nervous system and periphery. Selective inhibitors of ABHD12 would offer valuable pharmacological probes to complement genetic models of ABHD12-regulated (lyso)-PS/PI metabolism and signaling. Here, we provide a detailed description of the discovery and activity-based protein profiling (ABPP) guided optimization of reversible thiourea inhibitors of ABHD12 that culminated in the identification of DO264 as a potent, selective, and in vivo active ABHD12 inhibitor. We also show that DO264, but not a structurally related inactive control probe (S)-DO271, augments inflammatory cytokine production from human THP-1 macrophage cells. The in vitro and in vivo properties of DO264 designate this compound as a suitable chemical probe for studying the biological functions of ABHD12-(lyso)-PS/PI pathways.
ESTHER : Ogasawara_2019_J.Med.Chem_62_1643
PubMedSearch : Ogasawara_2019_J.Med.Chem_62_1643
PubMedID: 30720278
Gene_locus related to this paper: human-ABHD12

Title : Inhibition of monoacylglycerol lipase, an anti-inflammatory and antifibrogenic strategy in the liver - Habib_2019_Gut_68_522
Author(s) : Habib A , Chokr D , Wan J , Hegde P , Mabire M , Siebert M , Ribeiro-Parenti L , Le Gall M , Letteron P , Pilard N , Mansouri A , Brouillet A , Tardelli M , Weiss E , Le Faouder P , Guillou H , Cravatt BF , Moreau R , Trauner M , Lotersztajn S
Ref : Gut , 68 :522 , 2019
Abstract : OBJECTIVE: Sustained inflammation originating from macrophages is a driving force of fibrosis progression and resolution. Monoacylglycerol lipase (MAGL) is the rate-limiting enzyme in the degradation of monoacylglycerols. It is a proinflammatory enzyme that metabolises 2-arachidonoylglycerol, an endocannabinoid receptor ligand, into arachidonic acid. Here, we investigated the impact of MAGL on inflammation and fibrosis during chronic liver injury. DESIGN: C57BL/6J mice and mice with global invalidation of MAGL (MAGL (-/-) ), or myeloid-specific deletion of either MAGL (MAGL(Mye-/-)), ATG5 (ATG(Mye-/-)) or CB2 (CB2(Mye-/-)), were used. Fibrosis was induced by repeated carbon tetrachloride (CCl(4)) injections or bile duct ligation (BDL). Studies were performed on peritoneal or bone marrow-derived macrophages and Kupffer cells. RESULTS: MAGL (-/-) or MAGL(Mye-/-) mice exposed to CCl(4) or subjected to BDL were more resistant to inflammation and fibrosis than wild-type counterparts. Therapeutic intervention with MJN110, an MAGL inhibitor, reduced hepatic macrophage number and inflammatory gene expression and slowed down fibrosis progression. MAGL inhibitors also accelerated fibrosis regression and increased Ly-6C(low) macrophage number. Antifibrogenic effects exclusively relied on MAGL inhibition in macrophages, since MJN110 treatment of MAGL(Mye-/-) BDL mice did not further decrease liver fibrosis. Cultured macrophages exposed to MJN110 or from MAGL(Mye-/-) mice displayed reduced cytokine secretion. These effects were independent of the cannabinoid receptor 2, as they were preserved in CB2(Mye-/-) mice. They relied on macrophage autophagy, since anti-inflammatory and antifibrogenic effects of MJN110 were lost in ATG5(Mye-/-) BDL mice, and were associated with increased autophagic flux and autophagosome biosynthesis in macrophages when MAGL was pharmacologically or genetically inhibited. CONCLUSION: MAGL is an immunometabolic target in the liver. MAGL inhibitors may show promising antifibrogenic effects during chronic liver injury.
ESTHER : Habib_2019_Gut_68_522
PubMedSearch : Habib_2019_Gut_68_522
PubMedID: 30301768

Title : Design, Synthesis, and Evaluation of (18)F-Labeled Monoacylglycerol Lipase Inhibitors as Novel Positron Emission Tomography Probes - Chen_2019_J.Med.Chem_62_8866
Author(s) : Chen Z , Mori W , Fu H , Schafroth MA , Hatori A , Shao T , Zhang G , Van RS , Zhang Y , Hu K , Fujinaga M , Wang L , Belov V , Ogasawara D , Giffenig P , Deng X , Rong J , Yu Q , Zhang X , Papisov MI , Shao Y , Collier TL , Ma JA , Cravatt BF , Josephson L , Zhang MR , Liang SH
Ref : Journal of Medicinal Chemistry , 62 :8866 , 2019
Abstract : Dysfunction of monoacylglycerol lipase (MAGL) is associated with several psychopathological disorders, including drug addiction and neurodegenerative diseases. Herein we design, synthesize, and evaluate several irreversible fluorine-containing MAGL inhibitors for positron emission tomography (PET) ligand development. Compound 6 (identified from a therapeutic agent) was advanced for (18)F-labeling via a novel spirocyclic iodonium ylide (SCIDY) strategy, which demonstrated high brain permeability and excellent specific binding. This work supports further development of novel (18)F-labeled MAGL PET probes.
ESTHER : Chen_2019_J.Med.Chem_62_8866
PubMedSearch : Chen_2019_J.Med.Chem_62_8866
PubMedID: 31518130

Title : Deficiency of Monoacylglycerol Lipase Enhances IgM Plasma Levels and Limits Atherogenesis in a CB2-Dependent Manner -
Author(s) : Guillamat Prats R , Rami M , Ring L , Rinne P , Lauer E , Lenglet S , Thomas A , Pagano S , Vuilleumier N , Cravatt BF , Weber C , Faussner A , Steffens S
Ref : Thromb Haemost , 119 :348 , 2019
PubMedID: 30609444

Title : Design, Synthesis, and Evaluation of Reversible and Irreversible Monoacylglycerol Lipase Positron Emission Tomography (PET) Tracers Using a Tail Switching Strategy on a Piperazinyl Azetidine Skeleton - Chen_2019_J.Med.Chem_62_3336
Author(s) : Chen Z , Mori W , Deng X , Cheng R , Ogasawara D , Zhang G , Schafroth MA , Dahl K , Fu H , Hatori A , Shao T , Zhang Y , Yamasaki T , Zhang X , Rong J , Yu Q , Hu K , Fujinaga M , Xie L , Kumata K , Gou Y , Chen J , Gu S , Bao L , Wang L , Collier TL , Vasdev N , Shao Y , Ma JA , Cravatt BF , Fowler C , Josephson L , Zhang MR , Liang SH
Ref : Journal of Medicinal Chemistry , 62 :3336 , 2019
Abstract : Monoacylglycerol lipase (MAGL) is a serine hydrolase that degrades 2-arachidonoylglycerol (2-AG) in the endocannabinoid system (eCB). Selective inhibition of MAGL has emerged as a potential therapeutic approach for the treatment of diverse pathological conditions, including chronic pain, inflammation, cancer, and neurodegeneration. Herein, we disclose a novel array of reversible and irreversible MAGL inhibitors by means of "tail switching" on a piperazinyl azetidine scaffold. We developed a lead irreversible-binding MAGL inhibitor 8 and reversible-binding compounds 17 and 37, which are amenable for radiolabeling with (11)C or (18)F. [(11)C]8 ([(11)C]MAGL-2-11) exhibited high brain uptake and excellent binding specificity in the brain toward MAGL. Reversible radioligands [(11)C]17 ([(11)C]PAD) and [(18)F]37 ([(18)F]MAGL-4-11) also demonstrated excellent in vivo binding specificity toward MAGL in peripheral organs. This work may pave the way for the development of MAGL-targeted positron emission tomography tracers with tunability in reversible and irreversible binding mechanisms.
ESTHER : Chen_2019_J.Med.Chem_62_3336
PubMedSearch : Chen_2019_J.Med.Chem_62_3336
PubMedID: 30829483
Gene_locus related to this paper: human-MGLL

Title : Pharmacological convergence reveals a lipid pathway that regulates C. elegans lifespan - Chen_2019_Nat.Chem.Biol_15_453
Author(s) : Chen AL , Lum KM , Lara-Gonzalez P , Ogasawara D , Cognetta AB, 3rd , To A , Parsons WH , Simon GM , Desai A , Petrascheck M , Bar-Peled L , Cravatt BF
Ref : Nat Chemical Biology , 15 :453 , 2019
Abstract : Phenotypic screening has identified small-molecule modulators of aging, but the mechanism of compound action often remains opaque due to the complexities of mapping protein targets in whole organisms. Here, we combine a library of covalent inhibitors with activity-based protein profiling to coordinately discover bioactive compounds and protein targets that extend lifespan in Caenorhabditis elegans. We identify JZL184-an inhibitor of the mammalian endocannabinoid (eCB) hydrolase monoacylglycerol lipase (MAGL or MGLL)-as a potent inducer of longevity, a result that was initially perplexing as C. elegans does not possess an MAGL ortholog. We instead identify FAAH-4 as a principal target of JZL184 and show that this enzyme, despite lacking homology with MAGL, performs the equivalent metabolic function of degrading eCB-related monoacylglycerides in C. elegans. Small-molecule phenotypic screening thus illuminates pure pharmacological connections marking convergent metabolic functions in distantly related organisms, implicating the FAAH-4/monoacylglyceride pathway as a regulator of lifespan in C. elegans.
ESTHER : Chen_2019_Nat.Chem.Biol_15_453
PubMedSearch : Chen_2019_Nat.Chem.Biol_15_453
PubMedID: 30911178

Title : Ethanol-induced alterations in endocannabinoids and relevant neurotransmitters in the nucleus accumbens of fatty acid amide hydrolase knockout mice - Pavon_2019_Addict.Biol_24_1204
Author(s) : Pavon FJ , Serrano A , Stouffer DG , Polis I , Roberto M , Cravatt BF , Martin-Fardon R , Rodriguez de Fonseca F , Parsons LH
Ref : Addict Biol , 24 :1204 , 2019
Abstract : Deletion of fatty acid amide hydrolase (FAAH), enzyme responsible for degrading endocannabinoids, increases alcohol consumption and preference. However, there is a lack of data on neurochemical events in mice exposed to alcohol in the absence of FAAH. Extracellular levels of endocannabinoids and relevant neurotransmitters were measured by in vivo microdialysis in the nucleus accumbens (NAc) of FAAH knockout (KO) and wild-type (WT) mice during an ethanol (EtOH; 2 g/kg, ip) challenge in EtOH-naive and repeated (r) EtOH-treated mice. In both genotypes, EtOH treatment caused no changes in baseline endocannabinoid levels, although FAAH KO mice displayed higher baseline N-arachidonoylethanolamine levels than WT mice. EtOH challenge caused a sustained increase in 2-arachidonoylglycerol (2-AG) levels in EtOH-naive WT mice but not in FAAH KO mice. In contrast, 2-AG levels were decreased following EtOH challenge in (r)EtOH-treated mice in both genotypes. Whereas (r)EtOH-treated mice showed higher baseline dopamine and serotonin levels than EtOH-naive mice in WT mice, these differences were attenuated in FAAH KO mice. Significant differences in baseline gamma-aminobutyric acid (GABA) and glutamate levels by EtOH history were observed in WT mice but not in FAAH KO mice. Moreover, opposed effects on glutamate response were observed after EtOH challenge in EtOH-naive and (r)EtOH-treated FAAH KO mice. Finally, FAAH deletion failed to show EtOH-induced locomotion sensitivity. These data provide evidence of a potential influence of 2-AG in the neurochemical response to EtOH exposure in the NAc.
ESTHER : Pavon_2019_Addict.Biol_24_1204
PubMedSearch : Pavon_2019_Addict.Biol_24_1204
PubMedID: 30421483

Title : Notum produced by Paneth cells attenuates regeneration of aged intestinal epithelium - Pentinmikko_2019_Nature_571_398
Author(s) : Pentinmikko N , Iqbal S , Mana M , Andersson S , Cognetta AB, 3rd , Suciu RM , Roper J , Luopajarvi K , Markelin E , Gopalakrishnan S , Smolander OP , Naranjo S , Saarinen T , Juuti A , Pietilainen K , Auvinen P , Ristimaki A , Gupta N , Tammela T , Jacks T , Sabatini DM , Cravatt BF , Yilmaz OH , Katajisto P
Ref : Nature , 571 :398 , 2019
Abstract : A decline in stem cell function impairs tissue regeneration during ageing, but the role of the stem-cell-supporting niche in ageing is not well understood. The small intestine is maintained by actively cycling intestinal stem cells that are regulated by the Paneth cell niche(1,2). Here we show that the regenerative potential of human and mouse intestinal epithelium diminishes with age owing to defects in both stem cells and their niche. The functional decline was caused by a decrease in stemness-maintaining Wnt signalling due to production of Notum, an extracellular Wnt inhibitor, in aged Paneth cells. Mechanistically, high activity of mammalian target of rapamycin complex 1 (mTORC1) in aged Paneth cells inhibits activity of peroxisome proliferator activated receptor alpha (PPAR-alpha)(3), and lowered PPAR-alpha activity increased Notum expression. Genetic targeting of Notum or Wnt supplementation restored function of aged intestinal organoids. Moreover, pharmacological inhibition of Notum in mice enhanced the regenerative capacity of aged stem cells and promoted recovery from chemotherapy-induced damage. Our results reveal a role of the stem cell niche in ageing and demonstrate that targeting of Notum can promote regeneration of aged tissues.
ESTHER : Pentinmikko_2019_Nature_571_398
PubMedSearch : Pentinmikko_2019_Nature_571_398
PubMedID: 31292548
Gene_locus related to this paper: human-NOTUM , mouse-notum

Title : Endocannabinoid regulation of homeostatic feeding and stress-induced alterations in food intake in male rats - Sticht_2019_Br.J.Pharmacol_176_1524
Author(s) : Sticht MA , Lau DJ , Keenan CM , Cavin JB , Morena M , Vemuri VK , Makriyannis A , Cravatt BF , Sharkey KA , Hill MN
Ref : British Journal of Pharmacology , 176 :1524 , 2019
Abstract : BACKGROUND AND PURPOSE: Stress is known to reduce food intake. Many aspects of the stress response and feeding are regulated by the endocannabinoid system, but the roles of anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) in stress-induced anorexia are unclear. EXPERIMENTAL APPROACH: Effects of acute restraint stress on endocannabinoids were investigated in male Sprague-Dawley rats. Systemic and central pharmacological inhibition of fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL) was used to assess the effects of elevated AEA and 2-AG on homeostatic feeding and on food consumption after stress. Animals were pretreated with the FAAH inhibitor, PF-04457845, or the MAGL inhibitor, MJN110, before 2 h acute restraint stress or 2 h homecage period without food. KEY RESULTS: Restraint stress decreased hypothalamic and circulating AEA, with no effect in the gastrointestinal tract, while 2-AG content in the jejunum (but not duodenum) was reduced. PF-04457845 (30 microg), given i.c.v., attenuated stress-induced anorexia via CB(1) receptors, but reduced homeostatic feeding in unstressed animals through an unknown mechanism. On the other hand, systemic administration of MJN110 (10 ) reduced feeding, regardless of stress or feeding status and inhibited basal intestinal transit in unstressed rats. The ability of MAGL inhibition to reduce feeding in combination with stress was independent of CB(1) receptor signalling in the gut as the peripherally restricted CB(1) receptor antagonist, AM6545 did not block this effect. CONCLUSIONS AND IMPLICATIONS: Our data reveal diverse roles for 2-AG and AEA in homeostatic feeding and changes in energy intake following stress. LINKED ARTICLES: This article is part of a themed section on 8(th) European Workshop on Cannabinoid Research. To view the other articles in this section visit
ESTHER : Sticht_2019_Br.J.Pharmacol_176_1524
PubMedSearch : Sticht_2019_Br.J.Pharmacol_176_1524
PubMedID: 30051485

Title : Deficient endocannabinoid signaling in the central amygdala contributes to alcohol dependence-related anxiety-like behavior and excessive alcohol intake - Serrano_2018_Neuropsychopharmacology_43_1840
Author(s) : Serrano A , Pavon FJ , Buczynski MW , Schlosburg J , Natividad LA , Polis IY , Stouffer DG , Zorrilla EP , Roberto M , Cravatt BF , Martin-Fardon R , Rodriguez de Fonseca F , Parsons LH
Ref : Neuropsychopharmacology , 43 :1840 , 2018
Abstract : Negative emotional states that are associated with excessive alcohol intake, particularly anxiety-like states, have been linked to opponent processes in the central nucleus of the amygdala (CeA), affecting stress-related transmitters and monoamines. This study extends these observations to include endocannabinoid signaling in alcohol-dependent animals. Rats and mice were exposed to chronic intermittent alcohol with vapor inhalation or liquid diet to induce dependence. In vivo microdialysis was used to estimate interstitial concentrations of endocannabinoids [N-arachidonoylethanolamine (anandamide; AEA) and 2-arachidonoylglycerol (2-AG)] and amino acids (glutamate and GABA) in rat CeA. Additionally, we evaluated the inhibition of endocannabinoids clearance enzymes [monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase] on anxiety-like behavior and alcohol consumption in alcohol-dependent rats and mice. Results revealed that alcohol dependence produced decreases in baseline 2-AG dialysate levels and increases in baseline levels of glutamate and GABA. Acute alcohol abstinence induced an enhancement of these dependence-induced effects and the levels of 2-AG and GABA were restored upon alcohol re-exposure. Additional studies showed that the increased CeA 2-AG levels induced by restraint stress and alcohol self-administration were blunted in alcohol-dependent rats. Pharmacological studies in rats and mice showed that anxiety-like behavior and alcohol consumption were increased in alcohol-dependent animals, and these behavioral effects were attenuated mainly by MAGL inhibitors [MJN110 (10 and 20 mg/kg) in rats and JZL184 (1 and 3 mg/kg) in mice]. The present results suggest a key role for endocannabinoid signaling in motivational neuroadaptations during alcohol dependence, in which a deficiency in CeA 2-AG signaling in alcohol-dependent animals is linked to stress and excessive alcohol consumption.
ESTHER : Serrano_2018_Neuropsychopharmacology_43_1840
PubMedSearch : Serrano_2018_Neuropsychopharmacology_43_1840
PubMedID: 29748627

Title : Selective blockade of the lyso-PS lipase ABHD12 stimulates immune responses in vivo - Ogasawara_2018_Nat.Chem.Biol_14_1099
Author(s) : Ogasawara D , Ichu TA , Vartabedian VF , Benthuysen J , Jing H , Reed A , Ulanovskaya OA , Hulce JJ , Roberts A , Brown S , Rosen H , Teijaro JR , Cravatt BF
Ref : Nat Chemical Biology , 14 :1099 , 2018
Abstract : ABHD12 metabolizes bioactive lysophospholipids, including lysophosphatidylserine (lyso-PS). Deleterious mutations in human ABHD12 cause the neurological disease PHARC, and ABHD12(-/-) mice display PHARC-like phenotypes, including hearing loss, along with elevated brain lyso-PS and features of stimulated innate immune cell function. Here, we develop a selective and in vivo-active inhibitor of ABHD12 termed DO264 and show that this compound elevates lyso-PS in mouse brain and primary human macrophages. Unlike ABHD12(-/-) mice, adult mice treated with DO264 exhibited minimal perturbations in auditory function. On the other hand, both DO264-treated and ABHD12(-/-) mice displayed heightened immunological responses to lymphocytic choriomeningitis virus (LCMV) clone 13 infection that manifested as severe lung pathology with elevated proinflammatory chemokines. These results reveal similarities and differences in the phenotypic impact of pharmacological versus genetic blockade of ABHD12 and point to a key role for this enzyme in regulating immunostimulatory lipid pathways in vivo.
ESTHER : Ogasawara_2018_Nat.Chem.Biol_14_1099
PubMedSearch : Ogasawara_2018_Nat.Chem.Biol_14_1099
PubMedID: 30420694
Gene_locus related to this paper: human-ABHD12

Title : Monoacylglycerol Lipase Inhibitors Reverse Paclitaxel-Induced Nociceptive Behavior and Proinflammatory Markers in a Mouse Model of Chemotherapy-Induced Neuropathy - Curry_2018_J.Pharmacol.Exp.Ther_366_169
Author(s) : Curry ZA , Wilkerson JL , Bagdas D , Kyte SL , Patel N , Donvito G , Mustafa MA , Poklis JL , Niphakis MJ , Hsu KL , Cravatt BF , Gewirtz DA , Damaj MI , Lichtman AH
Ref : Journal of Pharmacology & Experimental Therapeutics , 366 :169 , 2018
Abstract : Although paclitaxel effectively treats various cancers, its debilitating peripheral neuropathic pain side effects often persist long after treatment has ended. Therefore, a compelling need exists for the identification of novel pharmacologic strategies to mitigate this condition. As inhibitors of monoacylglycerol lipase (MAGL), the primary hydrolytic enzyme of the endogenous cannabinoid, 2-arachidonyolglycerol, produces antinociceptive effects in numerous rodent models of pain, we investigated whether inhibitors of this enzyme (i.e., JZL184 and MJN110) would reverse paclitaxel-induced mechanical allodynia in mice. These drugs dose dependently reversed allodynia with respective ED(50) values (95% confidence limit) of 8.4 (5.2-13.6) and 1.8 (1.0-3.3) mg/kg. Complementary genetic and pharmacologic approaches revealed that the antiallodynic effects of each drug require both cannabinoid receptors, CB(1) and CB(2) MJN110 reduced paclitaxel-mediated increased expression of monocyte chemoattractant protein-1 (MCP-1, CCL2) and phospho-p38 MAPK in dorsal root ganglia as well as MCP-1 in spinal dorsal horn. Whereas the antinociceptive effects of high dose JZL184 (40 mg/kg) underwent tolerance following 6 days of repeated dosing, repeated administration of a threshold dose (i.e., 4 mg/kg) completely reversed paclitaxel-induced allodynia. In addition, we found that the administration of MJN110 to control mice lacked intrinsic rewarding effects in the conditioned place preference (CPP) paradigm. However, it produced a CPP in paclitaxel-treated animals, suggesting a reduced paclitaxel-induced aversive state. Importantly, JZL184 did not alter the antiproliferative and apoptotic effects of paclitaxel in A549 and H460 non-small cell lung cancer cells. Taken together, these data indicate that MAGL inhibitors reverse paclitaxel-induced neuropathic pain without interfering with chemotherapeutic efficacy.
ESTHER : Curry_2018_J.Pharmacol.Exp.Ther_366_169
PubMedSearch : Curry_2018_J.Pharmacol.Exp.Ther_366_169
PubMedID: 29540562

Title : Re-examining the potential of targeting ABHD6 in multiple sclerosis: Efficacy of systemic and peripherally restricted inhibitors in experimental autoimmune encephalomyelitis - Manterola_2018_Neuropharmacol_141_181
Author(s) : Manterola A , Bernal-Chico A , Cipriani R , Ruiz A , Perez-Samartin A , Moreno-Rodriguez M , Hsu KL , Cravatt BF , Brown JM , Rodriguez-Puertas R , Matute C , Mato S
Ref : Neuropharmacology , 141 :181 , 2018
Abstract : alpha/beta-Hydrolase domain-containing 6 (ABHD6) contributes to the hydrolysis of the major endocannabinoid 2-arachidonoylglycerol (2-AG) in the central nervous system (CNS) and in the periphery. ABHD6 blockade has been proposed as novel strategy to treat multiple sclerosis (MS), based on the observation that the inhibitor WWL70 exerts protective anti-inflammatory effects in experimental autoimmune encephalomyelitis (EAE). According to recent data, WWL70 exhibits off-target anti-inflammatory activity in microglial cells and the potential of ABHD6 as drug target in MS remains controversial. Here we further investigated the role of ABHD6 during autoimmune demyelination by comparing the efficacy of two novel inhibitors with different CNS permeability in vivo. Preventive treatment with the systemically active inhibitor KT182 ameliorated the neurological signs of EAE during the time-course of disease. By contrast, administration of the peripherally restricted compound KT203 was ineffective in attenuating EAE symptomatology. Both inhibitors failed to improve corticospinal tract conduction latency and to attenuate inflammation at EAE recovery phase, despite being equally active at targeting brain ABHD6. Chronic administration of KT182 was associated to a partial loss of brain CB(1) receptor coupling ability, suggesting the engagement of CB(1) receptor-mediated mechanisms during the EAE disease progression. In cultured neurons, KT182 attenuated NMDA-stimulated excitotoxicity and mitochondrial calcium overload. However, these protective effects were not attributable to ABHD6, as they were not mimicked by the alternative inhibitors KT203, KT195 and WWL70. These results indicate that ABHD6 blockade exerts only modest therapeutic effects against autoimmune demyelination and call into question its utility as novel drug target in MS.
ESTHER : Manterola_2018_Neuropharmacol_141_181
PubMedSearch : Manterola_2018_Neuropharmacol_141_181
PubMedID: 30171986
Gene_locus related to this paper: human-ABHD6

Title : In Vitro and in Vivo Evaluation of (11)C-Labeled Azetidinecarboxylates for Imaging Monoacylglycerol Lipase by PET Imaging Studies - Cheng_2018_J.Med.Chem_61_2278
Author(s) : Cheng R , Mori W , Ma L , Alhouayek M , Hatori A , Zhang Y , Ogasawara D , Yuan G , Chen Z , Zhang X , Shi H , Yamasaki T , Xie L , Kumata K , Fujinaga M , Nagai Y , Minamimoto T , Svensson M , Wang L , Du Y , Ondrechen MJ , Vasdev N , Cravatt BF , Fowler C , Zhang MR , Liang SH
Ref : Journal of Medicinal Chemistry , 61 :2278 , 2018
Abstract : Monoacylglycerol lipase (MAGL) is the principle enzyme for metabolizing endogenous cannabinoid ligand 2-arachidonoyglycerol (2-AG). Blockade of MAGL increases 2-AG levels, resulting in subsequent activation of the endocannabinoid system, and has emerged as a novel therapeutic strategy to treat drug addiction, inflammation, and neurodegenerative diseases. Herein we report a new series of MAGL inhibitors, which were radiolabeled by site-specific labeling technologies, including (11)C-carbonylation and spirocyclic iodonium ylide (SCIDY) radiofluorination. The lead compound [(11)C]10 (MAGL-0519) demonstrated high specific binding and selectivity in vitro and in vivo. We also observed unexpected washout kinetics with these irreversible radiotracers, in which in vivo evidence for turnover of the covalent residue was unveiled between MAGL and azetidine carboxylates. This work may lead to new directions for drug discovery and PET tracer development based on azetidine carboxylate inhibitor scaffold.
ESTHER : Cheng_2018_J.Med.Chem_61_2278
PubMedSearch : Cheng_2018_J.Med.Chem_61_2278
PubMedID: 29481079

Title : The Spastic Paraplegia-Associated Phospholipase DDHD1 Is a Primary Brain Phosphatidylinositol Lipase - Inloes_2018_Biochemistry_57_5759
Author(s) : Inloes JM , Jing H , Cravatt BF
Ref : Biochemistry , 57 :5759 , 2018
Abstract : Deleterious mutations in the serine hydrolase DDHD domain containing 1 (DDHD1) cause the SPG28 subtype of the neurological disease hereditary spastic paraplegia (HSP), which is characterized by axonal neuropathy and gait impairments. DDHD1 has been shown to display PLA1-type phospholipase activity with a preference for phosphatidic acid. However, the endogenous lipid pathways regulated by DDHD1 in vivo remain poorly understood. Here we use a combination of untargeted and targeted metabolomics to compare the lipid content of brain tissue from DDHD1(+/+) and DDHD1(-/-) mice, revealing that DDHD1 inactivation causes a substantial decrease in the level of polyunsaturated lysophosphatidylinositol (LPI) lipids and a corresponding increase in the level of phosphatidylinositol (PI) lipids. Levels of other phospholipids were mostly unchanged, with the exception of decreases in the levels of select polyunsaturated lysophosphatidylserine (LPS) and lysophosphatidylcholine lipids and a striking remodeling of PI phosphates (e.g., PIP and PIP2) in DDHD1(-/-) brain tissue. Biochemical assays confirmed that DDHD1 hydrolyzes PI/PS to LPI/LPS with sn-1 selectivity and accounts for a substantial fraction of the PI/PS lipase activity in mouse brain tissue. These data indicate that DDHD1 is a principal regulator of bioactive LPI and other lysophospholipids, as well as PI phosphates, in the mammalian nervous system, pointing to a potential role for these lipid pathways in HSP.
ESTHER : Inloes_2018_Biochemistry_57_5759
PubMedSearch : Inloes_2018_Biochemistry_57_5759
PubMedID: 30221923

Title : Functional Contribution of the Spastic Paraplegia-Related Triglyceride Hydrolase DDHD2 to the Formation and Content of Lipid Droplets - Inloes_2018_Biochemistry_57_827
Author(s) : Inloes JM , Kiosses WB , Wang H , Walther TC , Farese RV, Jr. , Cravatt BF
Ref : Biochemistry , 57 :827 , 2018
Abstract : Deleterious mutations in the serine lipase DDHD2 are a causative basis of complex hereditary spastic paraplegia (HSP, subtype SPG54) in humans. We recently found that DDHD2 is a principal triglyceride hydrolase in the central nervous system (CNS) and that genetic deletion of this enzyme in mice leads to ectopic lipid droplet (LD) accumulation in neurons throughout the brain. Nonetheless, how HSP-related mutations in DDHD2 relate to triglyceride metabolism and LD formation remains poorly understood. Here, we have characterized a set of HSP-related mutations in DDHD2 and found that they disrupt triglyceride hydrolase activity in vitro and impair the capacity of DDHD2 to protect cells from LD accumulation following exposure to free fatty acid, an outcome that was also observed with a DDHD2-selective inhibitor. We furthermore isolated and characterized LDs from brain tissue of DDHD2(-/-) mice, revealing that they contain both established LD-associated proteins identified previously in other organs and CNS-enriched proteins, including several proteins with genetic links to human neurological disease. These data, taken together, indicate that the genetic inactivation of DDHD2, as caused by HSP-associated mutations, substantially perturbs lipid homeostasis and the formation and content of LDs, underscoring the importance of triglyceride metabolism for normal CNS function and the key role that DDHD2 plays in this process.
ESTHER : Inloes_2018_Biochemistry_57_827
PubMedSearch : Inloes_2018_Biochemistry_57_827
PubMedID: 29278326

Title : Discovery of Modulators of Adipocyte Physiology Using Fully Functionalized Fragments - Galmozzi_2018_Methods.Mol.Biol_1787_115
Author(s) : Galmozzi A , Parker CG , Kok BP , Cravatt BF , Saez E
Ref : Methods Mol Biol , 1787 :115 , 2018
Abstract : Defects in adipocyte function associated with obesity drive the development of systemic insulin resistance and type 2 diabetes. Agents that correct obesity-linked adipocyte dysfunction serve as useful insulin sensitizers in humans, as is exemplified by the thiazolidinediones (TZDs). We have developed a new platform that integrates advanced chemoproteomics with phenotypic screening to identify small molecules that promote differentiation and lipid storage in adipocytes, and, in tandem, their molecular target(s). These molecules mimic the activity of TZDs in culture and thus may also serve as insulin sensitizers in vivo. Central to this platform is the use of fully functionalized fragment (FFF) probes that consist of a variable, fragment-like recognition element linked to an alkyne-diazirine group that enables the photoactivated capture of probe-bound proteins directly in living cells and subsequent copper-catalyzed azide-alkyne cycloaddition to reporter tags for enrichment and identification of these probe-bound proteins by mass spectrometry. This platform, which can be adapted to diverse screens and cell types beyond adipocytes, has the potential to uncover new biological pathways amenable to pharmacological modulation that may impact human disease.
ESTHER : Galmozzi_2018_Methods.Mol.Biol_1787_115
PubMedSearch : Galmozzi_2018_Methods.Mol.Biol_1787_115
PubMedID: 29736714

Title : Fatty acid amide hydrolase (FAAH) inactivation confers enhanced sensitivity to nicotine-induced dopamine release in the mouse nucleus accumbens - Pavon_2018_Addict.Biol_23_723
Author(s) : Pavon FJ , Serrano A , Sidhpura N , Polis I , Stouffer D , de Fonseca FR , Cravatt BF , Martin-Fardon R , Parsons LH
Ref : Addict Biol , 23 :723 , 2018
Abstract : Nicotine exerts its rewarding effects by promoting an increase in dopamine (DA) release in the nucleus accumbens (NAc), and this process is influenced by the endocannabinoid system. Fatty acid amide hydrolase (FAAH) is the main enzyme responsible for the degradation of the endocannabinoid anandamide and other non-cannabinoid N-acylethanolamines. Previous research has reported that both genetic deletion and pharmacological inhibition of FAAH enhance nicotine-induced conditioned place preference at low doses. We conducted a microdialysis study to characterize nicotine-induced changes in DA and serotonin (5-HT) levels in the NAc of FAAH knockout (KO) mice using a conditioned place preference-like paradigm with three nicotine doses (0.1, 1 and 10 mg/kg, s.c.). Additionally, the effects of the selective FAAH inhibitor PF-3845 (10 mg/kg, i.p.) were also examined. Our data indicated that compared with wild-type mice, genetic deletion of FAAH selectively enhanced the effect of low-dose nicotine on DA release (p < 0.001) and resulted in a strong post-nicotine elevation in DA levels (p < 0.01). However, there were no differences between the genotypes at higher doses. Furthermore, FAAH KO mice displayed a moderate enhancement of the effect of low-dose nicotine on NAc 5-HT release (p < 0.05), with no differences between the genotypes at higher doses. Compared with vehicle-pretreated mice, mice pretreated with PF-3845 displayed an enhancement of the effect of low-dose nicotine on NAc DA release (p < 0.001), which resulted in a sustained increase in DA levels (p < 0.05). Similar to FAAH KO mice, PF-3845-pretreated mice displayed a moderate enhancement of the effect of low-dose nicotine on NAc 5-HT release (p < 0.01). These observations in mice suggest that enhanced nicotine-induced NAc DA release might contribute to increased sensitivity to the conditioned rewarding effects of low-dose nicotine following FAAH inhibition, which has been previously reported. Future studies combining behavioral and neurochemical approaches are needed to elucidate the precise mechanism of these effects.
ESTHER : Pavon_2018_Addict.Biol_23_723
PubMedSearch : Pavon_2018_Addict.Biol_23_723
PubMedID: 28660730

Title : Stress Promotes Drug Seeking Through Glucocorticoid-Dependent Endocannabinoid Mobilization in the Prelimbic Cortex - McReynolds_2018_Biol.Psychiatry_84_85
Author(s) : McReynolds JR , Doncheck EM , Li Y , Vranjkovic O , Graf EN , Ogasawara D , Cravatt BF , Baker DA , Liu QS , Hillard CJ , Mantsch JR
Ref : Biological Psychiatry , 84 :85 , 2018
Abstract : BACKGROUND: Clinical reports suggest that rather than directly driving cocaine use, stress may create a biological context within which other triggers for drug use become more potent. We hypothesize that stress-induced increases in corticosterone "set the stage" for relapse by promoting endocannabinoid-induced attenuation of inhibitory transmission in the prelimbic cortex (PL). METHODS: We have established a rat model for these stage-setting effects of stress. In this model, neither a stressor (electric footshock) nor stress-level corticosterone treatment alone reinstates cocaine seeking following self-administration and extinction, but each treatment potentiates reinstatement in response to an otherwise subthreshold cocaine priming dose (2.5 mg/kg, intraperitoneal). The contributions of endocannabinoid signaling in the PL to the effects of stress-level corticosterone on PL neurotransmission and cocaine seeking were determined using intra-PL microinfusions. Endocannabinoid-dependent effects of corticosterone on inhibitory synaptic transmission in the rat PL were determined using whole-cell recordings in layer V pyramidal neurons. RESULTS: Corticosterone application attenuated inhibitory synaptic transmission in the PL via cannabinoid receptor type 1 (CB1R)- and 2-arachidonoylglycerol-dependent inhibition of gamma-aminobutyric acid release without altering postsynaptic responses. The ability of systemic stress-level corticosterone treatment to potentiate cocaine-primed reinstatement was recapitulated by intra-PL injection of corticosterone, the CB1R agonist WIN 55,212-2, or the monoacylglycerol lipase inhibitor URB602. Corticosterone effects on reinstatement were attenuated by intra-PL injections of either the CB1R antagonist, AM251, or the diacylglycerol lipase inhibitor, DO34. CONCLUSIONS: These findings suggest that stress-induced increases in corticosterone promote cocaine seeking by mobilizing 2-arachidonoylglycerol in the PL, resulting in CB1R-mediated attenuation of inhibitory transmission in this brain region.
ESTHER : McReynolds_2018_Biol.Psychiatry_84_85
PubMedSearch : McReynolds_2018_Biol.Psychiatry_84_85
PubMedID: 29100630

Title : Selective Irreversible Inhibitors of the Wnt-Deacylating Enzyme NOTUM Developed by Activity-Based Protein Profiling - Suciu_2018_ACS.Med.Chem.Lett_9_563
Author(s) : Suciu RM , Cognetta AB, 3rd , Potter ZE , Cravatt BF
Ref : ACS Med Chem Lett , 9 :563 , 2018
Abstract : Wnt proteins are secreted morphogens that play critical roles in embryonic development and tissue remodeling in adult organisms. Aberrant Wnt signaling contributes to diseases such as cancer. Wnts are modified by an unusual O-fatty acylation event (O-linked palmitoleoylation of a conserved serine) that is required for binding to Frizzled receptors. O-Palmitoleoylation of Wnts is introduced by the porcupine (PORCN) acyltransferase and removed by the serine hydrolase NOTUM. PORCN inhibitors are under development for oncology, while NOTUM inhibitors have potential for treating degenerative diseases. Here, we describe the use of activity-based protein profiling (ABPP) to discover and advance a class of N-hydroxyhydantoin (NHH) carbamates that potently and selectively inhibit NOTUM. An optimized NHH carbamate inhibitor, ABC99, preserves Wnt-mediated cell signaling in the presence of NOTUM and was also converted into an ABPP probe for visualizing NOTUM in native biological systems.
ESTHER : Suciu_2018_ACS.Med.Chem.Lett_9_563
PubMedSearch : Suciu_2018_ACS.Med.Chem.Lett_9_563
PubMedID: 29937983
Gene_locus related to this paper: human-NOTUM

Title : Role of interleukin 1-beta in the inflammatory response in a fatty acid amide hydrolase-knockout mouse model of Alzheimer's disease - Aparicio_2018_Biochem.Pharmacol_157_202
Author(s) : Aparicio N , Grande MT , Ruiz de Martin Esteban S , Lopez A , Ruiz-Perez G , Amores M , Vazquez C , Martinez-Relimpio AM , Pazos MR , Cravatt BF , Tolon RM , Romero J
Ref : Biochemical Pharmacology , 157 :202 , 2018
Abstract : The search for novel therapies for the treatment of Alzheimer's disease is an urgent need, due to the current paucity of available pharmacological tools and the recent failures obtained in clinical trials. Among other strategies, the modulation of amyloid-triggered neuroinflammation by the endocannabinoid system seems of relevance. Previous data indicate that the enhancement of the endocannabinoid tone through the inhibition of the enzymes responsible for the degradation of their main endogenous ligands may render beneficial effects. Based on previously reported data, in which we described a paradoxical effect of the genetic deletion of the fatty acid amide hydrolase, we here aimed to expand our knowledge on the role of the endocannabinoid system in the context of Alzheimer's disease. To that end, we inhibited the production of interleukin-1beta, one of the main inflammatory cytokines involved in the neuroinflammation triggered by amyloid peptides, in a transgenic mouse model of this disease by using minocycline, a drug known to impair the synthesis of this cytokine. Our data suggest that interleukin-1beta may be instrumental in order to achieve the beneficial effects derived of fatty acid amide hydrolase genetic inactivation. This could be appreciated at the molecular (cytokine expression, amyloid production, plaque deposition) as well as behavioral levels (memory impairment). We here describe a previously unknown link between the endocannabinoid system and interleukin-1beta in the context of Alzheimer's disease that open new possibilities for the development of novel therapeutics.
ESTHER : Aparicio_2018_Biochem.Pharmacol_157_202
PubMedSearch : Aparicio_2018_Biochem.Pharmacol_157_202
PubMedID: 30195729

Title : Mice lacking lipid droplet-associated hydrolase, a gene linked to human prostate cancer, have normal cholesterol ester metabolism - Kory_2017_J.Lipid.Res_58_226
Author(s) : Kory N , Grond S , Kamat SS , Li Z , Krahmer N , Chitraju C , Zhou P , Frohlich F , Semova I , Ejsing C , Zechner R , Cravatt BF , Farese RV, Jr. , Walther TC
Ref : J Lipid Res , 58 :226 , 2017
Abstract : Variations in the gene LDAH (C2ORF43), which encodes lipid droplet-associated hydrolase (LDAH), are among few loci associated with human prostate cancer. Homologs of LDAH have been identified as proteins of lipid droplets (LDs). LDs are cellular organelles that store neutral lipids, such as triacylglycerols and sterol esters, as precursors for membrane components and as reservoirs of metabolic energy. LDAH is reported to hydrolyze cholesterol esters and to be important in macrophage cholesterol ester metabolism. Here, we confirm that LDAH is localized to LDs in several model systems. We generated a murine model in which Ldah is disrupted but found no evidence for a major function of LDAH in cholesterol ester or triacylglycerol metabolism in vivo, nor a role in energy or glucose metabolism. Our data suggest that LDAH is not a major cholesterol ester hydrolase, and an alternative metabolic function may be responsible for its possible effect on development of prostate cancer.
ESTHER : Kory_2017_J.Lipid.Res_58_226
PubMedSearch : Kory_2017_J.Lipid.Res_58_226
PubMedID: 27836991
Gene_locus related to this paper: human-LDAH , mouse-Ldah

Title : Omega-3 fatty acid epoxides are autocrine mediators that control the magnitude of IgE-mediated mast cell activation - Shimanaka_2017_Nat.Med_23_1287
Author(s) : Shimanaka Y , Kono N , Taketomi Y , Arita M , Okayama Y , Tanaka Y , Nishito Y , Mochizuki T , Kusuhara H , Adibekian A , Cravatt BF , Murakami M , Arai H
Ref : Nat Med , 23 :1287 , 2017
Abstract : Critical to the function of mast cells in immune responses including allergy is their production of lipid mediators, among which only omega-6 (omega-6) arachidonate-derived eicosanoids have been well characterized. Here, by employing comprehensive lipidomics, we identify omega-3 (omega-3) fatty acid epoxides as new mast cell-derived lipid mediators and show that they are produced by PAF-AH2, an oxidized-phospholipid-selective phospholipase A2. Genetic or pharmacological deletion of PAF-AH2 reduced the steady-state production of omega-3 epoxides, leading to attenuated mast cell activation and anaphylaxis following FcvarepsilonRI cross-linking. Mechanistically, the omega-3 epoxides promote IgE-mediated activation of mast cells by downregulating Srcin1, a Src-inhibitory protein that counteracts FcvarepsilonRI signaling, through a pathway involving PPARg. Thus, the PAF-AH2-omega-3 epoxide-Srcin1 axis presents new potential drug targets for allergic diseases.
ESTHER : Shimanaka_2017_Nat.Med_23_1287
PubMedSearch : Shimanaka_2017_Nat.Med_23_1287
PubMedID: 29035365
Gene_locus related to this paper: human-PAFAH2

Title : Inhibition of the endocannabinoid-regulating enzyme monoacylglycerol lipase elicits a CB1 receptor-mediated discriminative stimulus in mice - Owens_2017_Neuropharmacol_125_80
Author(s) : Owens RA , Mustafa MA , Ignatowska-Jankowska BM , Damaj MI , Beardsley PM , Wiley JL , Niphakis MJ , Cravatt BF , Lichtman AH
Ref : Neuropharmacology , 125 :80 , 2017
Abstract : Substantial challenges exist for investigating the cannabinoid receptor type 1 (CB1)-mediated discriminative stimulus effects of the endocannabinoids, 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (anandamide; AEA), compared with exogenous CB1 receptor agonists, such as Delta(9)-tetrahydrocannabinol (THC) and the synthetic cannabinoid CP55,940. Specifically, each endocannabinoid is rapidly degraded by the respective hydrolytic enzymes, monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH). Whereas MAGL inhibitors partially substitute for THC and fully substitute for CP55,940, FAAH inhibitors do not substitute for either drug. Interestingly, combined FAAH-MAGL inhibition results in full THC substitution, and the dual FAAH-MAGL inhibitor SA-57 serves as its own discriminative training stimulus. Because MAGL inhibitors fully substitute for SA-57, we tested whether the selective MAGL inhibitor MJN110 would serve as a training stimulus. Twelve of 13 C57BL/6J mice learned to discriminate MJN110 from vehicle, and the CB1 receptor antagonist rimonabant dose-dependently blocked its discriminative stimulus. CP55,940, SA-57, and another MAGL inhibitor JZL184, fully substituted for MJN110. In contrast, the FAAH inhibitor PF-3845 failed to substitute for the MJN110 discriminative stimulus, but produced a 1.6 (1.1-2.2; 95% confidence interval) leftward shift in the MJN110 dose-response curve. Inhibitors of other relevant enzymes (i.e., ABHD6, COX-2) and nicotine did not engender substitution. Diazepam partially substituted for MJN110, but rimonabant failed to block this partial effect. These findings suggest that MAGL normally throttles 2-AG stimulation of CB1 receptors to a magnitude insufficient to produce cannabimimetic subjective effects. Accordingly, inhibitors of this enzyme may release this endogenous brake producing effects akin to those produced by exogenously administered cannabinoids.
ESTHER : Owens_2017_Neuropharmacol_125_80
PubMedSearch : Owens_2017_Neuropharmacol_125_80
PubMedID: 28673548

Title : Investigation of Diacylglycerol Lipase Alpha Inhibition in the Mouse Lipopolysaccharide Inflammatory Pain Model - Wilkerson_2017_J.Pharmacol.Exp.Ther_363_394
Author(s) : Wilkerson JL , Donvito G , Grim TW , Abdullah RA , Ogasawara D , Cravatt BF , Lichtman AH
Ref : Journal of Pharmacology & Experimental Therapeutics , 363 :394 , 2017
Abstract : Diacylglycerol lipase (DAGL) alpha and beta, the major biosynthetic enzymes of the endogenous cannabinoid (endocannabinoid) 2-arachidonylglycerol (2-AG), are highly expressed in the nervous system and immune system, respectively. Genetic deletion or pharmacological inhibition of DAGL-beta protects against lipopolysaccharide (LPS)-induced inflammatory responses in mouse peritoneal macrophages and reverses LPS-induced allodynia in mice. To gain insight into the contribution of DAGL-alpha in LPS-induced allodynia, we tested global knockout mice as well as DO34, a dual DAGL-alpha/beta inhibitor. Intraperitoneal administration of DO34 (30 mg/kg) significantly decreased whole-brain levels of 2-AG ( approximately 83%), anandamide ( approximately 42%), and arachidonic acid ( approximately 58%). DO34 dose-dependently reversed mechanical and cold allodynia, and these antinociceptive effects did not undergo tolerance after 6 days of repeated administration. In contrast, DO34 lacked acute thermal antinociceptive, motor, and hypothermal pharmacological effects in naive mice. As previously reported, DAGL-beta (-/-) mice displayed a protective phenotype from LPS-induced allodynia. However, DAGL-alpha (-/-) mice showed full allodynic responses, similar to their wild-type littermates. Interestingly, DO34 (30 mg/kg) fully reversed LPS-induced allodynia in DAGL-alpha (+/+) and (-/-) mice, but did not affect the antinociceptive phenotype of DAGL-beta (-/-) mice in this model, indicating a DAGL-alpha-independent site of action. These findings suggest that DAGL-alpha and DAGL-beta play distinct roles in LPS-induced nociception. Whereas DAGL-alpha appears to be dispensable for the development and expression of LPS-induced nociception, DAGL-beta inhibition represents a promising strategy to treat inflammatory pain.
ESTHER : Wilkerson_2017_J.Pharmacol.Exp.Ther_363_394
PubMedSearch : Wilkerson_2017_J.Pharmacol.Exp.Ther_363_394
PubMedID: 28970359
Gene_locus related to this paper: mouse-q6wqj1

Title : The endocannabinoid hydrolysis inhibitor SA-57: Intrinsic antinociceptive effects, augmented morphine-induced antinociception, and attenuated heroin seeking behavior in mice - Wilkerson_2017_Neuropharmacol_114_156
Author(s) : Wilkerson JL , Ghosh S , Mustafa M , Abdullah RA , Niphakis MJ , Cabrera R , Maldonado RL , Cravatt BF , Lichtman AH
Ref : Neuropharmacology , 114 :156 , 2017
Abstract : Although opioids are highly efficacious analgesics, their abuse potential and other untoward side effects diminish their therapeutic utility. The addition of non-opioid analgesics offers a promising strategy to reduce required antinociceptive opioid doses that concomitantly reduce opioid-related side effects. Inhibitors of the primary endocannabinoid catabolic enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) show opioid-sparing effects in preclinical models of pain. As simultaneous inhibition of these enzymes elicits enhanced antinociceptive effects compared with single enzyme inhibition, the present study tested whether the dual FAAH-MAGL inhibitor SA-57 [4-[2-(4-chlorophenyl)ethyl]-1-piperidinecarboxylic acid 2-(methylamino)-2-oxoethyl ester] produces morphine-sparing antinociceptive effects, without major side effects associated with either drug class. SA-57 dose-dependently reversed mechanical allodynia in the constriction injury (CCI) of the sciatic nerve model of neuropathic pain and carrageenan inflammatory pain model. As previously reported, SA-57 was considerably more potent in elevating anandamide (AEA) than 2-arachidonyl glycerol (2-AG) in brain. Its anti-allodynic effects required cannabinoid (CB)1 and CB2 receptors; however, only CB2 receptors were necessary for the anti-edematous effects in the carrageenan assay. Although high doses of SA-57 alone were required to produce antinociception, low doses of this compound, which elevated AEA and did not affect 2-AG brain levels, augmented the antinociceptive effects of morphine, but lacked cannabimimetic side effects. Because of the high abuse liability of opioids and implication of the endocannabinoid system in the reinforcing effects of opioids, the final experiment tested whether SA-57 would alter heroin seeking behavior. Strikingly, SA-57 reduced heroin-reinforced nose poke behavior and the progressive ratio break point for heroin. In conclusion, the results of the present study suggest that inhibition of endocannabinoid degradative enzymes represents a promising therapeutic approach to decrease effective doses of opioids needed for clinical pain control, and may also possess therapeutic potential to reduce opioid abuse.
ESTHER : Wilkerson_2017_Neuropharmacol_114_156
PubMedSearch : Wilkerson_2017_Neuropharmacol_114_156
PubMedID: 27890602

Title : Regulation of calcium release from the endoplasmic reticulum by the serine hydrolase ABHD2 - Yun_2017_Biochem.Biophys.Res.Commun_490_1226
Author(s) : Yun B , Lee H , Powell R , Reisdorph N , Ewing H , Gelb MH , Hsu KL , Cravatt BF , Leslie CC
Ref : Biochemical & Biophysical Research Communications , 490 :1226 , 2017
Abstract : The serine hydrolase inhibitors pyrrophenone and KT195 inhibit cell death induced by A23187 and H2O2 by blocking the release of calcium from the endoplasmic reticulum and mitochondrial calcium uptake. The effect of pyrrophenone and KT195 on these processes is not due to inhibition of their known targets, cytosolic phospholipase A2 and alpha/beta-hydrolase domain-containing (ABHD) 6, respectively, but represent off-target effects. To identify targets of KT195, fibroblasts were treated with KT195-alkyne to covalently label protein targets followed by click chemistry with biotin azide, enrichment on streptavidin beads and tryptic peptide analysis by mass spectrometry. Although several serine hydrolases were identified, alpha/beta-hydrolase domain-containing 2 (ABHD2) was the only target in which both KT195 and pyrrophenone competed for binding to KT195-alkyne. ABHD2 is a serine hydrolase with a predicted transmembrane domain consistent with its pull-down from the membrane proteome. Subcellular fractionation showed localization of ABHD2 to the endoplasmic reticulum but not to mitochondria or mitochondrial-associated membranes. Knockdown of ABHD2 with shRNA attenuated calcium release from the endoplasmic reticulum, mitochondrial calcium uptake and cell death in fibroblasts stimulated with A23187. The results describe a novel mechanism for regulating calcium transfer from the endoplasmic reticulum to mitochondria that involves the serine hydrolase ABHD2.
ESTHER : Yun_2017_Biochem.Biophys.Res.Commun_490_1226
PubMedSearch : Yun_2017_Biochem.Biophys.Res.Commun_490_1226
PubMedID: 28684316
Gene_locus related to this paper: human-ABHD2

Title : Design of Benzoxathiazin-3-one 1,1-Dioxides as a New Class of Irreversible Serine Hydrolase Inhibitors: Discovery of a Uniquely Selective PNPLA4 Inhibitor - Kornahrens_2017_J.Am.Chem.Soc_139_7052
Author(s) : Kornahrens AF , Cognetta AB, 3rd , Brody DM , Matthews ML , Cravatt BF , Boger DL
Ref : Journal of the American Chemical Society , 139 :7052 , 2017
Abstract : The design and examination of 4,1,2-benzoxathiazin-3-one 1,1-dioxides as candidate serine hydrolase inhibitors are disclosed, and represent the synthesis and study of a previously unexplored heterocycle. This new class of activated cyclic carbamates provided selective irreversible inhibition of a small subset of serine hydrolases without release of a leaving group, does not covalently modify active site catalytic cysteine and lysine residues of other enzyme classes, and was found to be amenable to predictable structural modifications that modulate intrinsic reactivity or active site recognition. Even more remarkable and within the small pilot series of candidate inhibitors examined in an initial study, an exquisitely selective inhibitor for a poorly characterized serine hydrolase (PNPLA4, patatin-like phospholipase domain-containing protein 4) involved in adipocyte triglyceride homeostasis was discovered.
ESTHER : Kornahrens_2017_J.Am.Chem.Soc_139_7052
PubMedSearch : Kornahrens_2017_J.Am.Chem.Soc_139_7052
PubMedID: 28498651

Title : Multicomponent mapping of boron chemotypes furnishes selective enzyme inhibitors - Tan_2017_Nat.Commun_8_1760
Author(s) : Tan J , Cognetta AB, 3rd , Diaz DB , Lum KM , Adachi S , Kundu S , Cravatt BF , Yudin AK
Ref : Nat Commun , 8 :1760 , 2017
Abstract : Heteroatom-rich organoboron compounds have attracted attention as modulators of enzyme function. Driven by the unmet need to develop chemoselective access to boron chemotypes, we report herein the synthesis of alpha- and beta-aminocyano(MIDA)boronates from borylated carbonyl compounds. Activity-based protein profiling of the resulting beta-aminoboronic acids furnishes selective and cell-active inhibitors of the (ox)lipid-metabolizing enzyme alpha/beta-hydrolase domain 3 (ABHD3). The most potent compound displays nanomolar in vitro and in situ IC50 values and fully inhibits ABHD3 activity in human cells with no detectable cross-reactivity against other serine hydrolases. These findings demonstrate that synthetic methods that enhance the heteroatom diversity of boron-containing molecules within a limited set of scaffolds accelerate the discovery of chemical probes of human enzymes.
ESTHER : Tan_2017_Nat.Commun_8_1760
PubMedSearch : Tan_2017_Nat.Commun_8_1760
PubMedID: 29170371
Gene_locus related to this paper: human-ABHD3 , mouse-abhd3

Title : Triazole Ureas Act as Diacylglycerol Lipase Inhibitors and Prevent Fasting-Induced Refeeding - Deng_2017_J.Med.Chem_60_428
Author(s) : Deng H , Kooijman S , van den Nieuwendijk AM , Ogasawara D , van der Wel T , van Dalen F , Baggelaar MP , Janssen FJ , van den Berg RJ , den Dulk H , Cravatt BF , Overkleeft HS , Rensen PC , van der Stelt M
Ref : Journal of Medicinal Chemistry , 60 :428 , 2017
Abstract : Triazole ureas constitute a versatile class of irreversible inhibitors that target serine hydrolases in both cells and animal models. We have previously reported that triazole ureas can act as selective and CNS-active inhibitors for diacylglycerol lipases (DAGLs), enzymes responsible for the biosynthesis of 2-arachidonoylglycerol (2-AG) that activates cannabinoid CB1 receptor. Here, we report the enantio- and diastereoselective synthesis and structure-activity relationship studies. We found that 2,4-substituted triazole ureas with a biphenylmethanol group provided the most optimal scaffold. Introduction of a chiral ether substituent on the 5-position of the piperidine ring provided ultrapotent inhibitor 38 (DH376) with picomolar activity. Compound 38 temporarily reduces fasting-induced refeeding of mice, thereby emulating the effect of cannabinoid CB1-receptor inverse agonists. This was mirrored by 39 (DO34) but also by the negative control compound 40 (DO53) (which does not inhibit DAGL), which indicates the triazole ureas may affect the energy balance in mice through multiple molecular targets.
ESTHER : Deng_2017_J.Med.Chem_60_428
PubMedSearch : Deng_2017_J.Med.Chem_60_428
PubMedID: 27992221

Title : Activity-based protein profiling reveals off-target proteins of the FAAH inhibitor BIA 10-2474 - van Esbroeck_2017_Science_356_1084
Author(s) : van Esbroeck ACM , Janssen APA , Cognetta AB, 3rd , Ogasawara D , Shpak G , van der Kroeg M , Kantae V , Baggelaar MP , de Vrij FMS , Deng H , Allara M , Fezza F , Lin Z , van der Wel T , Soethoudt M , Mock ED , den Dulk H , Baak IL , Florea BI , Hendriks G , De Petrocellis L , Overkleeft HS , Hankemeier T , De Zeeuw CI , Di Marzo V , Maccarrone M , Cravatt BF , Kushner SA , van der Stelt M
Ref : Science , 356 :1084 , 2017
Abstract : A recent phase 1 trial of the fatty acid amide hydrolase (FAAH) inhibitor BIA 10-2474 led to the death of one volunteer and produced mild-to-severe neurological symptoms in four others. Although the cause of the clinical neurotoxicity is unknown, it has been postulated, given the clinical safety profile of other tested FAAH inhibitors, that off-target activities of BIA 10-2474 may have played a role. Here we use activity-based proteomic methods to determine the protein interaction landscape of BIA 10-2474 in human cells and tissues. This analysis revealed that the drug inhibits several lipases that are not targeted by PF04457845, a highly selective and clinically tested FAAH inhibitor. BIA 10-2474, but not PF04457845, produced substantial alterations in lipid networks in human cortical neurons, suggesting that promiscuous lipase inhibitors have the potential to cause metabolic dysregulation in the nervous system.
ESTHER : van Esbroeck_2017_Science_356_1084
PubMedSearch : van Esbroeck_2017_Science_356_1084
PubMedID: 28596366

Title : A Screen for Protein-Protein Interactions in Live Mycobacteria Reveals a Functional Link between the Virulence-Associated Lipid Transporter LprG and the Mycolyltransferase Antigen 85A - Touchette_2017_ACS.Infect.Dis_3_336
Author(s) : Touchette MH , Van Vlack ER , Bai L , Kim J , Cognetta AB, 3rd , Previti ML , Backus KM , Martin DW , Cravatt BF , Seeliger JC
Ref : ACS Infect Dis , 3 :336 , 2017
Abstract : Outer membrane lipids in pathogenic mycobacteria are important for virulence and survival. Although the biosynthesis of these lipids has been extensively studied, mechanisms responsible for their assembly in the outer membrane are not understood. In the study of Gram-negative outer membrane assembly, protein-protein interactions define transport mechanisms, but analogous interactions have not been explored in mycobacteria. Here we identified interactions with the lipid transport protein LprG. Using site-specific photo-cross-linking in live mycobacteria, we mapped three major interaction interfaces within LprG. We went on to identify proteins that cross-link at the entrance to the lipid binding pocket, an area likely relevant to LprG transport function. We verified LprG site-specific interactions with two hits, the conserved lipoproteins LppK and LppI. We further showed that LprG interacts physically and functionally with the mycolyltransferase Ag85A, as loss of either protein leads to similar defects in cell growth and mycolylation. Overall, our results support a model in which protein interactions coordinate multiple pathways in outer membrane biogenesis and connect lipid biosynthesis to transport.
ESTHER : Touchette_2017_ACS.Infect.Dis_3_336
PubMedSearch : Touchette_2017_ACS.Infect.Dis_3_336
PubMedID: 28276676
Gene_locus related to this paper: myctu-a85a

Title : Neuronal and Astrocytic Monoacylglycerol Lipase Limit the Spread of Endocannabinoid Signaling in the Cerebellum - Chen_2016_eNeuro_3_
Author(s) : Chen Y , Liu X , Vickstrom CR , Liu MJ , Zhao L , Viader A , Cravatt BF , Liu QS
Ref : eNeuro , 3 : , 2016
Abstract : Endocannabinoids are diffusible lipophilic molecules that may spread to neighboring synapses. Monoacylglycerol lipase (MAGL) is the principal enzyme that degrades the endocannabinoid 2-arachidonoylglycerol (2-AG). Using knock-out mice in which MAGL is deleted globally or selectively in neurons and astrocytes, we investigated the extent to which neuronal and astrocytic MAGL limit the spread of 2-AG-mediated retrograde synaptic depression in cerebellar slices. A brief tetanic stimulation of parallel fibers in the molecular layer induced synaptically evoked suppression of excitation (SSE) in Purkinje cells, and both neuronal and astrocytic MAGL contribute to the termination of this form of endocannabinoid-mediated synaptic depression. The spread of SSE among Purkinje cells occurred only after global knock-out of MAGL or pharmacological blockade of either MAGL or glutamate uptake, but no spread was detected following neuron- or astrocyte-specific deletion of MAGL. The spread of endocannabinoid signaling was also influenced by the spatial pattern of synaptic stimulation, because it did not occur at spatially dispersed parallel fiber synapses induced by stimulating the granular layer. The tetanic stimulation of parallel fibers did not induce endocannabinoid-mediated synaptic suppression in Golgi cells even after disruption of MAGL and glutamate uptake, suggesting that heightened release of 2-AG by Purkinje cells does not spread the retrograde signal to parallel fibers that innervate Golgi cells. These results suggest that both neuronal and astrocytic MAGL limit the spatial diffusion of 2-AG and confer synapse-specificity of endocannabinoid signaling.
ESTHER : Chen_2016_eNeuro_3_
PubMedSearch : Chen_2016_eNeuro_3_
PubMedID: 27182552

Title : Branched Fatty Acid Esters of Hydroxy Fatty Acids Are Preferred Substrates of the MODY8 Protein Carboxyl Ester Lipase - Kolar_2016_Biochemistry_55_4636
Author(s) : Kolar MJ , Kamat SS , Parsons WH , Homan EA , Maher T , Peroni OD , Syed I , Fjeld K , Molven A , Kahn BB , Cravatt BF , Saghatelian A
Ref : Biochemistry , 55 :4636 , 2016
Abstract : A recently discovered class of endogenous mammalian lipids, branched fatty acid esters of hydroxy fatty acids (FAHFAs), possesses anti-diabetic and anti-inflammatory activities. Here, we identified and validated carboxyl ester lipase (CEL), a pancreatic enzyme hydrolyzing cholesteryl esters and other dietary lipids, as a FAHFA hydrolase. Variants of CEL have been linked to maturity-onset diabetes of the young, type 8 (MODY8), and to chronic pancreatitis. We tested the FAHFA hydrolysis activity of the CEL MODY8 variant and found a modest increase in activity as compared with that of the normal enzyme. Together, the data suggest that CEL might break down dietary FAHFAs.
ESTHER : Kolar_2016_Biochemistry_55_4636
PubMedSearch : Kolar_2016_Biochemistry_55_4636
PubMedID: 27509211
Gene_locus related to this paper: human-CEL

Title : Effects of fatty acid amide hydrolase (FAAH) inhibitors on working memory in rats - Panlilio_2016_Psychopharmacology.(Berl)_233_1879
Author(s) : Panlilio LV , Thorndike EB , Nikas SP , Alapafuja SO , Bandiera T , Cravatt BF , Makriyannis A , Piomelli D , Goldberg SR , Justinova Z
Ref : Psychopharmacology (Berl) , 233 :1879 , 2016
Abstract : RATIONALE: Manipulations of the endocannabinoid system could potentially produce therapeutic effects with minimal risk of adverse cannabis-like side effects. Inhibitors of fatty acid amide hydrolase (FAAH) increase endogenous levels of the cannabinoid-receptor agonist, anandamide, and show promise for treating a wide range of disorders. However, their effects on learning and memory have not been fully characterized. OBJECTIVES: We determined the effects of five structurally different FAAH inhibitors in an animal model of working memory known to be sensitive to impairment by delta-9 tetrahydrocannabinol (THC). METHODS: A delayed nonmatching-to-position procedure was used in rats. Illuminated nosepoke holes were used to provide sample cues (left versus right) and record responses (correct versus incorrect) after delays ranging from 0 to 28 s. Various test drugs were given acutely up to two times per week before daily sessions. RESULTS: One FAAH inhibitor, AM3506 (3 mg/kg), decreased accuracy in the memory task. Four other FAAH inhibitors (URB597, URB694, PF-04457845, and ARN14633) and a monoacylglycerol lipase inhibitor (JZL184, which blocks the degradation of the endocannabinoid 2-arachidonoylglycerol) had no effect. Testing of AM3506 in combination with antagonists for receptors known to be affected by anandamide and other fatty acid amides indicated that the impairment induced by AM3506 was mediated by cannabinoid CB1 receptors, and not by alpha-type peroxisome proliferator-activated receptors (PPAR-alpha) or vanilloid transient receptor potential cation channels (TRPV1). CONCLUSIONS: FAAH inhibitors differ with respect to their potential for memory impairment, abuse liability, and probably other cannabis-like effects, and they should be evaluated individually for specific therapeutic and adverse effects.
ESTHER : Panlilio_2016_Psychopharmacology.(Berl)_233_1879
PubMedSearch : Panlilio_2016_Psychopharmacology.(Berl)_233_1879
PubMedID: 26558620

Title : A chemical proteomic atlas of brain serine hydrolases identifies cell type-specific pathways regulating neuroinflammation - Viader_2016_Elife_5_
Author(s) : Viader A , Ogasawara D , Joslyn CM , Sanchez-Alavez M , Mori S , Nguyen W , Conti B , Cravatt BF
Ref : Elife , 5 : , 2016
Abstract : Metabolic specialization among major brain cell types is central to nervous system function and determined in large part by the cellular distribution of enzymes. Serine hydrolases are a diverse enzyme class that plays fundamental roles in CNS metabolism and signaling. Here, we perform an activity-based proteomic analysis of primary mouse neurons, astrocytes, and microglia to furnish a global portrait of the cellular anatomy of serine hydrolases in the brain. We uncover compelling evidence for the cellular compartmentalization of key chemical transmission pathways, including the functional segregation of endocannabinoid (eCB) biosynthetic enzymes diacylglycerol lipase-alpha (DAGLalpha) and -beta (DAGLbeta) to neurons and microglia, respectively. Disruption of DAGLbeta perturbed eCB-eicosanoid crosstalk specifically in microglia and suppressed neuroinflammatory events in vivo independently of broader effects on eCB content. Mapping the cellular distribution of metabolic enzymes thus identifies pathways for regulating specialized inflammatory responses in the brain while avoiding global alterations in CNS function.
ESTHER : Viader_2016_Elife_5_
PubMedSearch : Viader_2016_Elife_5_
PubMedID: 26779719

Title : Discriminative Stimulus Properties of the Endocannabinoid Catabolic Enzyme Inhibitor SA-57 in Mice - Owens_2016_J.Pharmacol.Exp.Ther_358_306
Author(s) : Owens RA , Ignatowska-Jankowska B , Mustafa M , Beardsley PM , Wiley JL , Jali A , Selley DE , Niphakis MJ , Cravatt BF , Lichtman AH
Ref : Journal of Pharmacology & Experimental Therapeutics , 358 :306 , 2016
Abstract : Whereas the inhibition of fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL), the respective major hydrolytic enzymes of N-arachidonoyl ethanolamine (AEA) and 2-arachidonoylglycerol (2-AG), elicits no or partial substitution for Delta(9)-tetrahydrocannabinol (THC) in drug-discrimination procedures, combined inhibition of both enzymes fully substitutes for THC, as well as produces a constellation of cannabimimetic effects. The present study tested whether C57BL/6J mice would learn to discriminate the dual FAAH-MAGL inhibitor SA-57 (4-[2-(4-chlorophenyl)ethyl]-1-piperidinecarboxylic acid 2-(methylamino)-2-oxoethyl ester) from vehicle in the drug-discrimination paradigm. In initial experiments, 10 mg/kg SA-57 fully substituted for CP55,940 ((-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cycl ohexanol), a high-efficacy CB1 receptor agonist in C57BL/6J mice and for AEA in FAAH (-/-) mice. Most (i.e., 23 of 24) subjects achieved criteria for discriminating SA-57 (10 mg/kg) from vehicle within 40 sessions, with full generalization occurring 1 to 2 hours postinjection. CP55,940, the dual FAAH-MAGL inhibitor JZL195 (4-nitrophenyl 4-(3-phenoxybenzyl)piperazine-1-carboxylate), and the MAGL inhibitors MJN110 (2,5-dioxopyrrolidin-1-yl 4-(bis(4-chlorophenyl)methyl)piperazine-1-carboxylate) and JZL184 (4-[Bis(1,3-benzodioxol-5-yl)hydroxymethyl]-1-piperidinecarboxylic acid 4-nitrophenyl ester) fully substituted for SA-57. Although the FAAH inhibitors PF-3845 ((N-3-pyridinyl-4-[[3-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenyl]methyl]-1-pipe ridinecarboxamide) and URB597 (cyclohexylcarbamic acid 3'-(aminocarbonyl)-[1,1'-biphenyl]-3-yl ester) did not substitute for SA-57, PF-3845 produced a 2-fold leftward shift in the MJN110 substitution dose-response curve. In addition, the CB1 receptor antagonist rimonabant blocked the generalization of SA-57, as well as substitution of CP55,940, JZL195, MJN110, and JZL184. These findings suggest that MAGL inhibition plays a major role in the CB1 receptor-mediated SA-57 training dose, which is further augmented by FAAH inhibition.
ESTHER : Owens_2016_J.Pharmacol.Exp.Ther_358_306
PubMedSearch : Owens_2016_J.Pharmacol.Exp.Ther_358_306
PubMedID: 27307500

Title : Elevation of 2-AG by monoacylglycerol lipase inhibition in the visceral insular cortex interferes with anticipatory nausea in a rat model - Limebeer_2016_Behav.Neurosci_130_261
Author(s) : Limebeer CL , Rock EM , Puvanenthirarajah N , Niphakis MJ , Cravatt BF , Parker LA
Ref : Behavioral Neuroscience , 130 :261 , 2016
Abstract : Anticipatory nausea (AN) is a conditioned nausea reaction experienced by chemotherapy patients upon returning to the clinic. Currently, there are no specific treatments for this phenomenon, with the classic antiemetic treatments (e.g., ondansetron) providing no relief. The rat model of AN, contextually elicited conditioned gaping reactions in rats, provides a tool for assessing potential treatments for this difficult to treat disorder. Systemically administered drugs which elevate the endocannabinoids, anandamide (AEA) and 2-arachidonoyl glycerol (2-AG), by interfering with their respective degrading enzymes, fatty acid amide hydrolase (FAAH) and monoacyl glycerol lipase (MAGL) interfere with AN in the rat model. We have shown that MAGL inhibition within the visceral insular cortex (VIC) interferes with acute nausea in the gaping model (Sticht et al., 2015). Here we report that bilateral infusion of the MAGL inhibitor, MJN110 (but neither the FAAH inhibitor, PF3845, nor ondansetron) into the VIC suppressed contextually elicited conditioned gaping, and this effect was reversed by coadministration of the CB1 antagonist, AM251. These findings suggest that 2-AG within the VIC plays a critical role in the regulation of both acute nausea and AN. Because there are currently no specific therapeutics for chemotherapy patients that develop anticipatory nausea, MAGL inhibition by MJN110 may be a candidate treatment. (PsycINFO Database Record
ESTHER : Limebeer_2016_Behav.Neurosci_130_261
PubMedSearch : Limebeer_2016_Behav.Neurosci_130_261
PubMedID: 26974857

Title : AIG1 and ADTRP are atypical integral membrane hydrolases that degrade bioactive FAHFAs - Parsons_2016_Nat.Chem.Biol_12_367
Author(s) : Parsons WH , Kolar MJ , Kamat SS , Cognetta AB, 3rd , Hulce JJ , Saez E , Kahn BB , Saghatelian A , Cravatt BF
Ref : Nat Chemical Biology , 12 :367 , 2016
Abstract : Enzyme classes may contain outlier members that share mechanistic, but not sequence or structural, relatedness with more common representatives. The functional annotation of such exceptional proteins can be challenging. Here, we use activity-based profiling to discover that the poorly characterized multipass transmembrane proteins AIG1 and ADTRP are atypical hydrolytic enzymes that depend on conserved threonine and histidine residues for catalysis. Both AIG1 and ADTRP hydrolyze bioactive fatty acid esters of hydroxy fatty acids (FAHFAs) but not other major classes of lipids. We identify multiple cell-active, covalent inhibitors of AIG1 and show that these agents block FAHFA hydrolysis in mammalian cells. These results indicate that AIG1 and ADTRP are founding members of an evolutionarily conserved class of transmembrane threonine hydrolases involved in bioactive lipid metabolism. More generally, our findings demonstrate how chemical proteomics can excavate potential cases of convergent or parallel protein evolution that defy conventional sequence- and structure-based predictions.
ESTHER : Parsons_2016_Nat.Chem.Biol_12_367
PubMedSearch : Parsons_2016_Nat.Chem.Biol_12_367
PubMedID: 27018888

Title : The Selective Monoacylglycerol Lipase Inhibitor MJN110 Produces Opioid-Sparing Effects in a Mouse Neuropathic Pain Model - Wilkerson_2016_J.Pharmacol.Exp.Ther_357_145
Author(s) : Wilkerson JL , Niphakis MJ , Grim TW , Mustafa MA , Abdullah RA , Poklis JL , Dewey WL , Akbarali H , Banks ML , Wise LE , Cravatt BF , Lichtman AH
Ref : Journal of Pharmacology & Experimental Therapeutics , 357 :145 , 2016
Abstract : Serious clinical liabilities associated with the prescription of opiates for pain control include constipation, respiratory depression, pruritus, tolerance, abuse, and addiction. A recognized strategy to circumvent these side effects is to combine opioids with other antinociceptive agents. The combination of opiates with the primary active constituent of cannabis (Delta(9)-tetrahydrocannabinol) produces enhanced antinociceptive actions, suggesting that cannabinoid receptor agonists can be opioid sparing. Here, we tested whether elevating the endogenous cannabinoid 2-arachidonoylglycerol through the inhibition of its primary hydrolytic enzyme monoacylglycerol lipase (MAGL), will produce opioid-sparing effects in the mouse chronic constriction injury (CCI) of the sciatic nerve model of neuropathic pain. The dose-response relationships of i.p. administration of morphine and the selective MAGL inhibitor 2,5-dioxopyrrolidin-1-yl 4-(bis(4-chlorophenyl)methyl)piperazine-1-carboxylate (MJN110) were tested alone and in combination at equieffective doses for reversal of CCI-induced mechanical allodynia and thermal hyperalgesia. The respective ED50 doses (95% confidence interval) of morphine and MJN110 were 2.4 (1.9-3.0) mg/kg and 0.43 (0.23-0.79) mg/kg. Isobolographic analysis of these drugs in combination revealed synergistic antiallodynic effects. Acute antinociceptive effects of the combination of morphine and MJN110 required mu-opioid, CB1, and CB2 receptors. This combination did not reduce gastric motility or produce subjective cannabimimetic effects in the drug discrimination assay. Importantly, combinations of MJN110 and morphine given repeatedly (i.e., twice a day for 6 days) continued to produce antiallodynic effects with no evidence of tolerance. Taken together, these findings suggest that MAGL inhibition produces opiate-sparing events with diminished tolerance, constipation, and cannabimimetic side effects.
ESTHER : Wilkerson_2016_J.Pharmacol.Exp.Ther_357_145
PubMedSearch : Wilkerson_2016_J.Pharmacol.Exp.Ther_357_145
PubMedID: 26791602

Title : Robust anti-nociceptive effects of monoacylglycerol lipase inhibition in a model of osteoarthritis pain - Burston_2016_Br.J.Pharmacol_173_3134
Author(s) : Burston JJ , Mapp PI , Sarmad S , Barrett DA , Niphakis MJ , Cravatt BF , Walsh DA , Chapman V
Ref : British Journal of Pharmacology , 173 :3134 , 2016
Abstract : BACKGROUND AND PURPOSE: Chronic pain is often a symptom of knee osteoarthritis (OA) for which current analgesics are either inadequate or are associated with serious side effects. The endocannabinoid system may offer alternative targets for pain relief. We evaluated the effects of a potent and selective monoacylglycerol (MAG) lipase inhibitor (MJN110) on OA pain behaviour, spinal mechanisms of action and joint histopathology in the rat. EXPERIMENTAL APPROACH: Intra-articular injection of monosodium iodoacetate (MIA) models OA pain and mimics clinical joint pathology. Effects of MJN110 on MIA-induced weight-bearing asymmetry and lowered paw withdrawal thresholds (PWTs), changes in spinal gene expression and brain levels of relevant lipids were determined. KEY
RESULTS: Acute MJN110 (5 ) significantly reversed MIA-induced weight-bearing asymmetry (MIA/vehicle: 68 +/- 6 g; MIA/MJN110: 35 +/- 4 g) and lowered ipsilateral PWTs (MIA/vehicle: 7 +/- 0.8 g; MIA/MJN110: 11 +/- 0.6 g), via both CB1 and CB2 receptors. Repeated treatment with MJN110 (5 ) resulted in anti-nociceptive tolerance. A lower dose of MJN110 (1 ) acutely inhibited pain behaviour, which was maintained for 1 week of repeated administration but had no effect on joint histology. MJN110 significantly inhibited expression of membrane-associated PGE synthase-1 in the ipsilateral dorsal horn of the spinal cord of MIA rats, compared with vehicle-treated MIA rats. Both doses of MJN110 significantly elevated brain levels of the endocannabinoid 2-arachidonoylglycerol. CONCLUSIONS AND IMPLICATIONS: Our data support further assessment of the therapeutic potential of MAG lipase inhibitors for the treatment of OA pain.
ESTHER : Burston_2016_Br.J.Pharmacol_173_3134
PubMedSearch : Burston_2016_Br.J.Pharmacol_173_3134
PubMedID: 27501482

Title : An in vivo multiplexed small-molecule screening platform - Gruner_2016_Nat.Methods_13_883
Author(s) : Gruner BM , Schulze CJ , Yang D , Ogasawara D , Dix MM , Rogers ZN , Chuang CH , McFarland CD , Chiou SH , Brown JM , Cravatt BF , Bogyo M , Winslow MM
Ref : Nat Methods , 13 :883 , 2016
Abstract : Phenotype-based small-molecule screening is a powerful method to identify molecules that regulate cellular functions. However, such screens are generally performed in vitro under conditions that do not necessarily model complex physiological conditions or disease states. Here, we use molecular cell barcoding to enable direct in vivo phenotypic screening of small-molecule libraries. The multiplexed nature of this approach allows rapid in vivo analysis of hundreds to thousands of compounds. Using this platform, we screened >700 covalent inhibitors directed toward hydrolases for their effect on pancreatic cancer metastatic seeding. We identified multiple hits and confirmed the relevant target of one compound as the lipase ABHD6. Pharmacological and genetic studies confirmed the role of this enzyme as a regulator of metastatic fitness. Our results highlight the applicability of this multiplexed screening platform for investigating complex processes in vivo.
ESTHER : Gruner_2016_Nat.Methods_13_883
PubMedSearch : Gruner_2016_Nat.Methods_13_883
PubMedID: 27617390

Title : Rapid and profound rewiring of brain lipid signaling networks by acute diacylglycerol lipase inhibition - Ogasawara_2016_Proc.Natl.Acad.Sci.U.S.A_113_26
Author(s) : Ogasawara D , Deng H , Viader A , Baggelaar MP , Breman A , den Dulk H , van den Nieuwendijk AM , Soethoudt M , van der Wel T , Zhou J , Overkleeft HS , Sanchez-Alavez M , Mori S , Nguyen W , Conti B , Liu X , Chen Y , Liu QS , Cravatt BF , van der Stelt M
Ref : Proc Natl Acad Sci U S A , 113 :26 , 2016
Abstract : Diacylglycerol lipases (DAGLalpha and DAGLbeta) convert diacylglycerol to the endocannabinoid 2-arachidonoylglycerol. Our understanding of DAGL function has been hindered by a lack of chemical probes that can perturb these enzymes in vivo. Here, we report a set of centrally active DAGL inhibitors and a structurally related control probe and their use, in combination with chemical proteomics and lipidomics, to determine the impact of acute DAGL blockade on brain lipid networks in mice. Within 2 h, DAGL inhibition produced a striking reorganization of bioactive lipids, including elevations in DAGs and reductions in endocannabinoids and eicosanoids. We also found that DAGLalpha is a short half-life protein, and the inactivation of DAGLs disrupts cannabinoid receptor-dependent synaptic plasticity and impairs neuroinflammatory responses, including lipopolysaccharide-induced anapyrexia. These findings illuminate the highly interconnected and dynamic nature of lipid signaling pathways in the brain and the central role that DAGL enzymes play in regulating this network.
ESTHER : Ogasawara_2016_Proc.Natl.Acad.Sci.U.S.A_113_26
PubMedSearch : Ogasawara_2016_Proc.Natl.Acad.Sci.U.S.A_113_26
PubMedID: 26668358
Gene_locus related to this paper: human-DAGLA , human-DAGLB

Title : Chemical Proteomic Profiling of Human Methyltransferases - Horning_2016_J.Am.Chem.Soc_138_13335
Author(s) : Horning BD , Suciu RM , Ghadiri DA , Ulanovskaya OA , Matthews ML , Lum KM , Backus KM , Brown SJ , Rosen H , Cravatt BF
Ref : Journal of the American Chemical Society , 138 :13335 , 2016
Abstract : Methylation is a fundamental mechanism used in Nature to modify the structure and function of biomolecules, including proteins, DNA, RNA, and metabolites. Methyl groups are predominantly installed into biomolecules by a large and diverse class of S-adenosyl methionine (SAM)-dependent methyltransferases (MTs), of which there are approximately 200 known or putative members in the human proteome. Deregulated MT activity contributes to numerous diseases, including cancer, and several MT inhibitors are in clinical development. Nonetheless, a large fraction of the human MT family remains poorly characterized, underscoring the need for new technologies to characterize MTs and their inhibitors in native biological systems. Here, we describe a suite of S-adenosyl homocysteine (SAH) photoreactive probes and their application in chemical proteomic experiments to profile and enrich a large number of MTs (>50) from human cancer cell lysates with remarkable specificity over other classes of proteins. We further demonstrate that the SAH probes can enrich MT-associated proteins and be used to screen for and assess the selectivity of MT inhibitors, leading to the discovery of a covalent inhibitor of nicotinamide N-methyltransferase (NNMT), an enzyme implicated in cancer and metabolic disorders. The chemical proteomics probes and methods for their utilization reported herein should prove of value for the functional characterization of MTs, MT complexes, and MT inhibitors in mammalian biology and disease.
ESTHER : Horning_2016_J.Am.Chem.Soc_138_13335
PubMedSearch : Horning_2016_J.Am.Chem.Soc_138_13335
PubMedID: 27689866

Title : Just add water: cannabinoid discrimination in a water T-maze with FAAH(-\/-) and FAAH(+\/+) mice - Wiley_2016_Behav.Pharmacol_27_479
Author(s) : Wiley JL , Lefever TW , Pulley NS , Marusich JA , Cravatt BF , Lichtman AH
Ref : Behav Pharmacol , 27 :479 , 2016
Abstract : Incomplete overlap in the discriminative stimulus effects of Delta-tetrahydrocannabinol (THC) and the endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol has been reported in food-reinforced tasks. The aim of this study was to examine cannabinoid discriminative stimulus effects in a nonappetitive procedure. Adult male mice lacking the gene for AEA's major metabolic enzyme, fatty acid amide hydrolase (FAAH), and FAAH mice were trained to discriminate THC or AEA in a water T-maze, in which the response was swimming to an escape platform on the injection-appropriate side. JZL184, a monoacylglycerol lipase inhibitor, was also tested. FAAH mice showed faster acquisition than FAAH mice. THC and AEA fully substituted, with only minor cross-procedure potency variations. Incomplete substitution of JZL184 was observed in THC-trained FAAH mice in the water-maze task, as contrasted with full substitution in a food-reinforced nose-poke procedure. Stress-induced changes in AEA and/or 2-arachidonoylglycerol concentrations in the brain may have mediated this attenuation. JZL184 also partially substituted in AEA-trained FAAH mice in the water maze, suggesting incomplete overlap in the stimulus effects of AEA and JZL184. Through the use of a novel water-maze procedure, the present study supports the work of previous behavioral pharmacologists in showing the robustness of the discrimination paradigm.
ESTHER : Wiley_2016_Behav.Pharmacol_27_479
PubMedSearch : Wiley_2016_Behav.Pharmacol_27_479
PubMedID: 27385208

Title : Diacylglycerol lipase beta inhibition reverses nociceptive behaviour in mouse models of inflammatory and neuropathic pain - Wilkerson_2016_Br.J.Pharmacol_173_1678
Author(s) : Wilkerson JL , Ghosh S , Bagdas D , Mason BL , Crowe MS , Hsu KL , Wise LE , Kinsey SG , Damaj MI , Cravatt BF , Lichtman AH
Ref : British Journal of Pharmacology , 173 :1678 , 2016
Abstract : BACKGROUND AND PURPOSE: Inhibition of diacylglycerol lipase (DGL)beta prevents LPS-induced pro-inflammatory responses in mouse peritoneal macrophages. Thus, the present study tested whether DGLbeta inhibition reverses allodynic responses of mice in the LPS model of inflammatory pain, as well as in neuropathic pain models. EXPERIMENTAL APPROACH: Initial experiments examined the cellular expression of DGLbeta and inflammatory mediators within the LPS-injected paw pad. DAGL-beta (-/-) mice or wild-type mice treated with the DGLbeta inhibitor KT109 were assessed in the LPS model of inflammatory pain. Additional studies examined the locus of action for KT109-induced antinociception, its efficacy in chronic constrictive injury (CCI) of sciatic nerve and chemotherapy-induced neuropathic pain (CINP) models. KEY
RESULTS: Intraplantar LPS evoked mechanical allodynia that was associated with increased expression of DGLbeta, which was co-localized with increased TNF-alpha and prostaglandins in paws. DAGL-beta (-/-) mice or KT109-treated wild-type mice displayed reductions in LPS-induced allodynia. Repeated KT109 administration prevented the expression of LPS-induced allodynia, without evidence of tolerance. Intraplantar injection of KT109 into the LPS-treated paw, but not the contralateral paw, reversed the allodynic responses. However, i.c.v. or i.t. administration of KT109 did not alter LPS-induced allodynia. Finally, KT109 also reversed allodynia in the CCI and CINP models and lacked discernible side effects (e.g. gross motor deficits, anxiogenic behaviour or gastric ulcers). CONCLUSIONS AND IMPLICATIONS: These findings suggest that local inhibition of DGLbeta at the site of inflammation represents a novel avenue to treat pathological pain, with no apparent untoward side effects.
ESTHER : Wilkerson_2016_Br.J.Pharmacol_173_1678
PubMedSearch : Wilkerson_2016_Br.J.Pharmacol_173_1678
PubMedID: 26915789

Title : Double Dissociation of Monoacylglycerol Lipase Inhibition and CB1 Antagonism in the Central Amygdala, Basolateral Amygdala, and the Interoceptive Insular Cortex on the Affective Properties of Acute Naloxone-Precipitated Morphine Withdrawal in Rats - Wills_2016_Neuropsychopharmacology_41_1865
Author(s) : Wills KL , Petrie GN , Millett G , Limebeer CL , Rock EM , Niphakis MJ , Cravatt BF , Parker LA
Ref : Neuropsychopharmacology , 41 :1865 , 2016
Abstract : Both CB1 receptor antagonism and agonism, in particular by 2-arachidonyl glycerol (2-AG), have been shown to reduce somatic symptoms of morphine withdrawal (MWD). Here we evaluated the effects of both systemic pretreatment with the monoacylglycerol lipase (MAGL) inhibitor MJN110 (which selectively elevates 2-AG) and central administration of both MJN110 and the CB1 antagonist (AM251) on the affective properties of MWD. Acute MWD induced place aversion occurs when naloxone is administered 24 h following a single exposure to a high dose of morphine. Systemic pretreatment with the MAGL inhibitor, MJN110, prevented the aversive effects of acute MWD by a CB1 receptor-dependent mechanism. Furthermore, in a double dissociation, AM251 infusions into the central amygdala, but MJN110 infusions into the basolateral amygdala, interfered with the naloxone-precipitated MWD induced place aversion. As well, MJN110, but not AM251, infusions into the interoceptive insular cortex (a region known to be activated in acute MWD) also prevented the establishment of the place aversion by a CB1 mechanism of action. These findings reveal the respective sites of action of systemically administered MJN110 and AM251 in regulating the aversive effects of MWD.
ESTHER : Wills_2016_Neuropsychopharmacology_41_1865
PubMedSearch : Wills_2016_Neuropsychopharmacology_41_1865
PubMedID: 26647976

Title : Endocannabinoid regulation of nausea is mediated by 2-arachidonoylglycerol (2-AG) in the rat visceral insular cortex - Sticht_2016_Neuropharmacol_102_92
Author(s) : Sticht MA , Limebeer CL , Rafla BR , Abdullah RA , Poklis JL , Ho W , Niphakis MJ , Cravatt BF , Sharkey KA , Lichtman AH , Parker LA
Ref : Neuropharmacology , 102 :92 , 2016
Abstract : Cannabinoid (CB) agonists suppress nausea in humans and animal models; yet, their underlying neural substrates remain largely unknown. Evidence suggests that the visceral insular cortex (VIC) plays a critical role in nausea. Given the expression of CB1 receptors and the presence of endocannabinoids in this brain region, we hypothesized that the VIC endocannabinoid system regulates nausea. In the present study, we assessed whether inhibiting the primary endocannabinoid hydrolytic enzymes in the VIC reduces acute lithium chloride (LiCl)-induced conditioned gaping, a rat model of nausea. We also quantified endocannabinoid levels during an episode of nausea, and assessed VIC neuronal activation using the marker, c-Fos. Local inhibition of monoacylglycerol lipase (MAGL), the main hydrolytic enzyme of 2-arachidonylglycerol (2-AG), reduced acute nausea through a CB1 receptor mechanism, whereas inhibition of fatty acid amide hydrolase (FAAH), the primary catabolic enzyme of anandamide (AEA), was without effect. Levels of 2-AG were also selectively elevated in the VIC during an episode of nausea. Inhibition of MAGL robustly increased 2-AG in the VIC, while FAAH inhibition had no effect on AEA. Finally, we demonstrated that inhibition of MAGL reduced VIC Fos immunoreactivity in response to LiCl treatment. Taken together, these findings provide compelling evidence that acute nausea selectively increases 2-AG in the VIC, and suggests that 2-AG signaling within the VIC regulates nausea by reducing neuronal activity in this forebrain region.
ESTHER : Sticht_2016_Neuropharmacol_102_92
PubMedSearch : Sticht_2016_Neuropharmacol_102_92
PubMedID: 26541329

Title : Coordinated regulation of endocannabinoid-mediated retrograde synaptic suppression in the cerebellum by neuronal and astrocytic monoacylglycerol lipase - Liu_2016_Sci.Rep_6_35829
Author(s) : Liu X , Chen Y , Vickstrom CR , Li Y , Viader A , Cravatt BF , Liu QS
Ref : Sci Rep , 6 :35829 , 2016
Abstract : The endocannabinoid 2-arachidonoylglycerol (2-AG) mediates retrograde synaptic depression including depolarization-induced suppression of excitation (DSE) and inhibition (DSI). 2-AG is degraded primarily by monoacylglycerol lipase (MAGL), which is expressed in neurons and astrocytes. Using knockout mice in which MAGL is deleted globally or selectively in neurons or astrocytes, we investigated the relative contribution of neuronal and astrocytic MAGL to the termination of DSE and DSI in Purkinje cells (PCs) in cerebellar slices. We report that neuronal MAGL plays a predominant role in terminating DSE at climbing fiber (CF) to PC synapses, while both neuronal and astrocytic MAGL significantly contributes to the termination of DSE at parallel fiber (PF) to PC synapses and DSI at putative Stellate cell to PC synapses. Thus, DSE and DSI at different synapses is not uniformly affected by global and cell type-specific knockout of MAGL. Additionally, MAGL global knockout, but not cell type-specific knockout, caused tonic activation and partial desensitization of the CB1 receptor at PF-PC synapses. This tonic CB1 activation is mediated by 2-AG since it was blocked by the diacylglycerol lipase inhibitor DO34. Together, these results suggest that both neuronal and astrocytic MAGL contribute to 2-AG clearance and prevent CB1 receptor over-stimulation in the cerebellum.
ESTHER : Liu_2016_Sci.Rep_6_35829
PubMedSearch : Liu_2016_Sci.Rep_6_35829
PubMedID: 27775008

Title : Diacylglycerol lipase disinhibits VTA dopamine neurons during chronic nicotine exposure - Buczynski_2016_Proc.Natl.Acad.Sci.U.S.A_113_1086
Author(s) : Buczynski MW , Herman MA , Hsu KL , Natividad LA , Irimia C , Polis IY , Pugh H , Chang JW , Niphakis MJ , Cravatt BF , Roberto M , Parsons LH
Ref : Proc Natl Acad Sci U S A , 113 :1086 , 2016
Abstract : Chronic nicotine exposure (CNE) alters synaptic transmission in the ventral tegmental area (VTA) in a manner that enhances dopaminergic signaling and promotes nicotine use. The present experiments identify a correlation between enhanced production of the endogenous cannabinoid 2-arachidonoylglycerol (2-AG) and diminished release of the inhibitory neurotransmitter GABA in the VTA following CNE. To study the functional role of on-demand 2-AG signaling in GABAergic synapses, we used 1,2,3-triazole urea compounds to selectively inhibit 2-AG biosynthesis by diacylglycerol lipase (DAGL). The potency and selectivity of these inhibitors were established in rats in vitro (rat brain proteome), ex vivo (brain slices), and in vivo (intracerebroventricular administration) using activity-based protein profiling and targeted metabolomics analyses. Inhibition of DAGL (2-AG biosynthesis) rescues nicotine-induced VTA GABA signaling following CNE. Conversely, enhancement of 2-AG signaling in naive rats by inhibiting 2-AG degradation recapitulates the loss of nicotine-induced GABA signaling evident following CNE. DAGL inhibition reduces nicotine self-administration without disrupting operant responding for a nondrug reinforcer or motor activity. Collectively, these findings provide a detailed characterization of selective inhibitors of rat brain DAGL and demonstrate that excessive 2-AG signaling contributes to a loss of inhibitory GABAergic constraint of VTA excitability following CNE.
ESTHER : Buczynski_2016_Proc.Natl.Acad.Sci.U.S.A_113_1086
PubMedSearch : Buczynski_2016_Proc.Natl.Acad.Sci.U.S.A_113_1086
PubMedID: 26755579

Title : Differential effects of endocannabinoid catabolic inhibitors on morphine withdrawal in mice - Gamage_2015_Drug.Alcohol.Depend_146_7
Author(s) : Gamage TF , Ignatowska-Jankowska BM , Muldoon PP , Cravatt BF , Damaj MI , Lichtman AH
Ref : Drug Alcohol Depend , 146 :7 , 2015
Abstract : BACKGROUND: Inhibition of endocannabinoid catabolic enzymes fatty acid amide hydrolase (FAAH) and/or monoacylglycerol lipase (MAGL) reduces somatic morphine withdrawal signs, but its effects on aversive aspects of withdrawal are unknown. The present study investigated whether Delta(9)-tetrahydrocannabinol (THC), the MAGL inhibitor JZL184, the FAAH inhibitor PF-3845, or the dual FAAH/MAGL inhibitor SA-57 would reduce acquisition of morphine withdrawal-induced conditioned place avoidance (CPA) and jumping.
METHODS: Mice were implanted with placebo or 75 mg morphine pellets, 48 h later injected with naloxone or saline and placed in the conditioning apparatus, and assessed for CPA at 72 h. Subjects were also observed for jumping behavior following naloxone challenge.
RESULTS: Naloxone (0.056 mg/kg) produced robust CPA in morphine-pelleted, but not placebo-pelleted, mice. Morphine pretreatment prevented the occurrence of withdrawal CPA and withdrawal jumping, while clonidine (an alpha2 adrenergic receptor agonist) only blocked withdrawal CPA. THC, JZL184, and SA-57 significantly reduced the percentage of mice that jumped during the conditioning session, but did not affect acquisition of withdrawal CPA. PF-3845 did not reduce morphine withdrawal CPA or jumping. Finally, neither THC nor the endocannabinoid catabolic enzyme inhibitors in non-dependent mice elicited a conditioned place preference or aversion.
CONCLUSIONS: These findings suggest that inhibiting endocannabinoid catabolic enzymes reduces somatic morphine withdrawal signs, but not aversive aspects as inferred in the CPA paradigm. The observation that non-dependent mice administered inhibitors of endocannabinoid degradation did not display place preferences is consistent with the idea that that endocannabinoid catabolic enzymes might be targeted therapeutically, with reduced risk of abuse.
ESTHER : Gamage_2015_Drug.Alcohol.Depend_146_7
PubMedSearch : Gamage_2015_Drug.Alcohol.Depend_146_7
PubMedID: 25479915

Title : Delta9-tetrahydrocannabinol and endocannabinoid degradative enzyme inhibitors attenuate intracranial self-stimulation in mice - Wiebelhaus_2015_J.Pharmacol.Exp.Ther_352_195
Author(s) : Wiebelhaus JM , Grim TW , Owens RA , Lazenka MF , Sim-Selley LJ , Abdullah RA , Niphakis MJ , Vann RE , Cravatt BF , Wiley JL , Negus SS , Lichtman AH
Ref : Journal of Pharmacology & Experimental Therapeutics , 352 :195 , 2015
Abstract : A growing body of evidence implicates endogenous cannabinoids as modulators of the mesolimbic dopamine system and motivated behavior. Paradoxically, the reinforcing effects of Delta(9)-tetrahydrocannabinol (THC), the primary psychoactive constituent of cannabis, have been difficult to detect in preclinical rodent models. In this study, we investigated the impact of THC and inhibitors of the endocannabinoid hydrolytic enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) on operant responding for electrical stimulation of the medial forebrain bundle [intracranial self-stimulation (ICSS)], which is known to activate the mesolimbic dopamine system. These drugs were also tested in assays of operant responding for food reinforcement and spontaneous locomotor activity. THC and the MAGL inhibitor JZL184 (4-[bis(1,3-benzodioxol-5-yl)hydroxymethyl]-1-piperidinecarboxylic acid 4-nitrophenyl ester) attenuated operant responding for ICSS and food, and also reduced spontaneous locomotor activity. In contrast, the FAAH inhibitor PF-3845 (N-3-pyridinyl-4-[[3-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenyl]methyl]-1-piper idinecarboxamide) was largely without effect in these assays. Consistent with previous studies showing that combined inhibition of FAAH and MAGL produces a substantially greater cannabimimetic profile than single enzyme inhibition, the dual FAAH-MAGL inhibitor SA-57 (4-[2-(4-chlorophenyl)ethyl]-1-piperidinecarboxylic acid 2-(methylamino)-2-oxoethyl ester) produced a similar magnitude of ICSS depression as that produced by THC. ICSS attenuation by JZL184 was associated with increased brain levels of 2-arachidonoylglycerol (2-AG), whereas peak effects of SA-57 were associated with increased levels of both N-arachidonoylethanolamine (anandamide) and 2-AG. The cannabinoid receptor type 1 receptor antagonist rimonabant, but not the cannabinoid receptor type 2 receptor antagonist SR144528, blocked the attenuating effects of THC, JZL184, and SA-57 on ICSS. Thus, THC, MAGL inhibition, and dual FAAH-MAGL inhibition not only reduce ICSS, but also decrease other reinforced and nonreinforced behaviors.
ESTHER : Wiebelhaus_2015_J.Pharmacol.Exp.Ther_352_195
PubMedSearch : Wiebelhaus_2015_J.Pharmacol.Exp.Ther_352_195
PubMedID: 25398241

Title : Simultaneous inhibition of fatty acid amide hydrolase and monoacylglycerol lipase shares discriminative stimulus effects with Delta9-tetrahydrocannabinol in mice - Hruba_2015_J.Pharmacol.Exp.Ther_353_261
Author(s) : Hruba L , Seillier A , Zaki A , Cravatt BF , Lichtman AH , Giuffrida A , McMahon LR
Ref : Journal of Pharmacology & Experimental Therapeutics , 353 :261 , 2015
Abstract : Monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) inhibitors exert preclinical effects indicative of therapeutic potential (i.e., analgesia). However, the extent to which MAGL and FAAH inhibitors produce unwanted effects remains unclear. Here, FAAH and MAGL inhibition was examined separately and together in a Delta(9)-tetrahydrocannabinol (Delta(9)-THC; 5.6 mg/kg i.p.) discrimination assay predictive of subjective effects associated with cannabis use, and the relative contribution of N-arachidonoyl ethanolamine (AEA) and 2-arachidonoylglycerol (2-AG) in the prefrontal cortex, hippocampus, and caudate putamen to those effects was examined. Delta(9)-THC dose-dependently increased Delta(9)-THC appropriate responses (ED50 value = 2.8 mg/kg), whereas the FAAH inhibitors PF-3845 [N-3-pyridinyl-4-[[3-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenyl]methyl]-1-piper idinecarboxamide] and URB597 [(3'-(aminocarbonyl)[1,1'-biphenyl]-3-yl)-cyclohexylcarbamate] or a MAGL inhibitor JZL184 [4-nitrophenyl-4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxyl ate] alone did not substitute for the Delta(9)-THC discriminative stimulus. The nonselective FAAH/MAGL inhibitors SA-57 [4-[2-(4-chlorophenyl)ethyl]-1-piperidinecarboxylic acid 2-(methylamino)-2-oxoethyl ester] and JZL195 [4-nitrophenyl 4-(3-phenoxybenzyl)piperazine-1-carboxylate] fully substituted for Delta(9)-THC with ED50 values equal to 2.4 and 17 mg/kg, respectively. Full substitution for Delta(9)-THC was also produced by a combination of JZL184 and PF-3845, but not by a combination of JZL184 and URB597 (i.e., 52% maximum). Cannabinoid receptor type 1 antagonist rimonabant attenuated the discriminative stimulus effects of Delta(9)-THC, SA-57, JZL195, and the combined effects of JZL184 and PF-3845. Full substitution for the Delta(9)-THC discriminative stimulus occurred only when both 2-AG and AEA were significantly elevated, and the patterns of increased endocannabinoid content were similar among brain regions. Overall, these results suggest that increasing both endogenous 2-AG and AEA produces qualitatively unique effects (i.e., the subjective effects of cannabis) that are not obtained from increasing either 2-AG or AEA separately.
ESTHER : Hruba_2015_J.Pharmacol.Exp.Ther_353_261
PubMedSearch : Hruba_2015_J.Pharmacol.Exp.Ther_353_261
PubMedID: 25711338

Title : Target-Based Screen Against a Periplasmic Serine Protease That Regulates Intrabacterial pH Homeostasis in Mycobacterium tuberculosis - Zhao_2015_ACS.Chem.Biol_10_364
Author(s) : Zhao N , Darby CM , Small J , Bachovchin DA , Jiang X , Burns-Huang KE , Botella H , Ehrt S , Boger DL , Anderson ED , Cravatt BF , Speers AE , Fernandez-Vega V , Hodder PS , Eberhart C , Rosen H , Spicer TP , Nathan CF
Ref : ACS Chemical Biology , 10 :364 , 2015
Abstract : Mycobacterium tuberculosis (Mtb) maintains its intrabacterial pH (pHIB) near neutrality in the acidic environment of phagosomes within activated macrophages. A previously reported genetic screen revealed that Mtb loses this ability when the mycobacterial acid resistance protease (marP) gene is disrupted. In the present study, a high throughput screen (HTS) of compounds against the protease domain of MarP identified benzoxazinones as inhibitors of MarP. A potent benzoxazinone, BO43 (6-chloro-2-(2'-methylphenyl)-4H-1,3-benzoxazin-4-one), acylated MarP and lowered Mtb's pHIB and survival during incubation at pH 4.5. BO43 had similar effects on MarP-deficient Mtb, suggesting the existence of additional target(s). Reaction of an alkynyl-benzoxazinone, BO43T, with Mycobacterium bovis variant bacille Calmette-Guerin (BCG) followed by click chemistry with azido-biotin identified both the MarP homologue and the high temperature requirement A1 (HtrA1) homologue, an essential protein. Thus, the chemical probe identified through a target-based screen not only reacted with its intended target in the intact cells but also implicated an additional enzyme that had eluded a genetic screen biased against essential genes.
ESTHER : Zhao_2015_ACS.Chem.Biol_10_364
PubMedSearch : Zhao_2015_ACS.Chem.Biol_10_364
PubMedID: 25457457

Title : Highly Selective, Reversible Inhibitor Identified by Comparative Chemoproteomics Modulates Diacylglycerol Lipase Activity in Neurons - Baggelaar_2015_J.Am.Chem.Soc_137_8851
Author(s) : Baggelaar MP , Chameau PJ , Kantae V , Hummel J , Hsu KL , Janssen F , van der Wel T , Soethoudt M , Deng H , den Dulk H , Allara M , Florea BI , Di Marzo V , Wadman WJ , Kruse CG , Overkleeft HS , Hankemeier T , Werkman TR , Cravatt BF , van der Stelt M
Ref : Journal of the American Chemical Society , 137 :8851 , 2015
Abstract : Diacylglycerol lipase (DAGL)-alpha and -beta are enzymes responsible for the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG). Selective and reversible inhibitors are required to study the function of DAGLs in neuronal cells in an acute and temporal fashion, but they are currently lacking. Here, we describe the identification of a highly selective DAGL inhibitor using structure-guided and a chemoproteomics strategy to characterize the selectivity of the inhibitor in complex proteomes. Key to the success of this approach is the use of comparative and competitive activity-based proteome profiling (ABPP), in which broad-spectrum and tailor-made activity-based probes are combined to report on the inhibition of a protein family in its native environment. Competitive ABPP with broad-spectrum fluorophosphonate-based probes and specific beta-lactone-based probes led to the discovery of alpha-ketoheterocycle LEI105 as a potent, highly selective, and reversible dual DAGL-alpha/DAGL-beta inhibitor. LEI105 did not affect other enzymes involved in endocannabinoid metabolism including abhydrolase domain-containing protein 6, abhydrolase domain-containing protein 12, monoacylglycerol lipase, and fatty acid amide hydrolase and did not display affinity for the cannabinoid CB1 receptor. Targeted lipidomics revealed that LEI105 concentration-dependently reduced 2-AG levels, but not anandamide levels, in Neuro2A cells. We show that cannabinoid CB1-receptor-mediated short-term synaptic plasticity in a mouse hippocampal slice model can be reduced by LEI105. Thus, we have developed a highly selective DAGL inhibitor and provide new pharmacological evidence to support the hypothesis that "on demand biosynthesis" of 2-AG is responsible for retrograde signaling.
ESTHER : Baggelaar_2015_J.Am.Chem.Soc_137_8851
PubMedSearch : Baggelaar_2015_J.Am.Chem.Soc_137_8851
PubMedID: 26083464
Gene_locus related to this paper: human-DAGLA , human-DAGLB

Title : Immunomodulatory lysophosphatidylserines are regulated by ABHD16A and ABHD12 interplay - Kamat_2015_Nat.Chem.Biol_11_164
Author(s) : Kamat SS , Camara K , Parsons WH , Chen DH , Dix MM , Bird TD , Howell AR , Cravatt BF
Ref : Nat Chemical Biology , 11 :164 , 2015
Abstract : Lysophosphatidylserines (lyso-PSs) are a class of signaling lipids that regulate immunological and neurological processes. The metabolism of lyso-PSs remains poorly understood in vivo. Recently, we determined that ABHD12 is a major brain lyso-PS lipase, implicating lyso-PSs in the neurological disease polyneuropathy, hearing loss, ataxia, retinitis pigmentosa and cataract (PHARC), which is caused by null mutations in the ABHD12 gene. Here, we couple activity-based profiling with pharmacological and genetic methods to annotate the poorly characterized enzyme ABHD16A as a phosphatidylserine (PS) lipase that generates lyso-PS in mammalian systems. We describe a small-molecule inhibitor of ABHD16A that depletes lyso-PSs from cells, including lymphoblasts derived from subjects with PHARC. In mouse macrophages, disruption of ABHD12 and ABHD16A respectively increases and decreases both lyso-PSs and lipopolysaccharide-induced cytokine production. Finally, Abhd16a(-/-) mice have decreased brain lyso-PSs, which runs counter to the elevation in lyso-PS in Abhd12(-/-) mice. Our findings illuminate an ABHD16A-ABHD12 axis that dynamically regulates lyso-PS metabolism in vivo, designating these enzymes as potential targets for treating neuroimmunological disorders.
ESTHER : Kamat_2015_Nat.Chem.Biol_11_164
PubMedSearch : Kamat_2015_Nat.Chem.Biol_11_164
PubMedID: 25580854
Gene_locus related to this paper: human-ABHD12 , human-ABHD16A , mouse-abd12 , mouse-Abhd16a

Title : Selective monoacylglycerol lipase inhibitors: antinociceptive versus cannabimimetic effects in mice - Ignatowska-Jankowska_2015_J.Pharmacol.Exp.Ther_353_424
Author(s) : Ignatowska-Jankowska B , Wilkerson JL , Mustafa M , Abdullah R , Niphakis M , Wiley JL , Cravatt BF , Lichtman AH
Ref : Journal of Pharmacology & Experimental Therapeutics , 353 :424 , 2015
Abstract : The endogenous cannabinoid 2-arachidonoylglycerol (2-AG) plays an important role in a variety of physiologic processes, but its rapid breakdown by monoacylglycerol lipase (MAGL) results in short-lived actions. Initial MAGL inhibitors were limited by poor selectivity and low potency. In this study, we tested JZL184 [4-nitrophenyl 4-[bis(2H-1,3-benzodioxol-5-yl)(hydroxy)methyl]piperidine-1-carboxylate] and MJN110 [2,5-dioxopyrrolidin-1-yl 4-(bis(4-chlorophenyl)methyl)piperazine-1-carboxylate], MAGL inhibitors that possess increased selectivity and potency, in mouse behavioral assays of neuropathic pain [chronic constriction injury (CCI) of the sciatic nerve], interoceptive cannabimimetic effects (drug-discrimination paradigm), and locomotor activity in an open field test. MJN110 (1.25 and 2.5 mg/kg) and JZL184 (16 and 40 mg/kg) significantly elevated 2-AG and decreased arachidonic acid but did not affect anandamide in whole brains. Both MAGL inhibitors significantly reduced CCI-induced mechanical allodynia with the following potencies [ED50 (95% confidence limit [CL]) values in mg/kg: MJN110 (0.43 [0.30-0.63]) > JZL184 (17.8 [11.6-27.4])] and also substituted for the potent cannabinoid receptor agonist CP55,940 [2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]-5-(2-methyloctan-2-yl)phe nol] in the drug-discrimination paradigm [ED50 (95% CL) values in mg/kg: MJN110 (0.84 [0.69-1.02]) > JZL184 (24.9 [14.6-42.5])]; however, these compounds elicited differential effects on locomotor behavior. Similar to cannabinoid 1 (CB1) receptor agonists, JZL184 produced hypomotility, whereas MJN110 increased locomotor behavior and did not produce catalepsy or hypothermia. Although both drugs substituted for CP55,940 in the drug discrimination assay, MJN110 was more potent in reversing allodynia in the CCI model than in producing CP55,940-like effects. Overall, these results suggest that MAGL inhibition may alleviate neuropathic pain, while displaying limited cannabimimetic effects compared with direct CB1 receptor agonists.
ESTHER : Ignatowska-Jankowska_2015_J.Pharmacol.Exp.Ther_353_424
PubMedSearch : Ignatowska-Jankowska_2015_J.Pharmacol.Exp.Ther_353_424
PubMedID: 25762694

Title : Comprehensive Analysis of Structure-Activity Relationships of alpha-Ketoheterocycles as sn-1-Diacylglycerol Lipase alpha Inhibitors - Janssen_2015_J.Med.Chem_58_9742
Author(s) : Janssen FJ , Baggelaar MP , Hummel JJ , Overkleeft HS , Cravatt BF , Boger DL , van der Stelt M
Ref : Journal of Medicinal Chemistry , 58 :9742 , 2015
Abstract : Diacylglycerol lipase alpha (DAGLalpha) is responsible for the formation of the endocannabinoid 2-arachidonoylglycerol (2-AG) in the central nervous system. DAGLalpha inhibitors are required to study the physiological role of 2-AG. Previously, we identified the alpha-ketoheterocycles as potent and highly selective DAGLalpha inhibitors. Here, we present the first comprehensive structure-activity relationship study of alpha-ketoheterocycles as DAGLalpha inhibitors. Our findings indicate that the active site of DAGLalpha is remarkably sensitive to the type of heterocyclic scaffold with oxazolo-4N-pyridines as the most active framework. We uncovered a fundamental substituent effect in which electron-withdrawing meta-oxazole substituents increased inhibitor potency. (C6-C9)-acyl chains with a distal phenyl group proved to be the most potent inhibitors. The integrated SAR data was consistent with the proposed binding pose in a DAGLalpha homology model. Altogether, our results may guide the design of future DAGLalpha inhibitors as leads for molecular therapies to treat neuroinflammation, obesity, and related metabolic disorders.
ESTHER : Janssen_2015_J.Med.Chem_58_9742
PubMedSearch : Janssen_2015_J.Med.Chem_58_9742
PubMedID: 26584396

Title : Monoacylglycerol Lipase Regulates Fever Response - Sanchez-Alavez_2015_PLoS.One_10_e0134437
Author(s) : Sanchez-Alavez M , Nguyen W , Mori S , Moroncini G , Viader A , Nomura DK , Cravatt BF , Conti B
Ref : PLoS ONE , 10 :e0134437 , 2015
Abstract : Cyclooxygenase inhibitors such as ibuprofen have been used for decades to control fever through reducing the levels of the pyrogenic lipid transmitter prostaglandin E2 (PGE2). Historically, phospholipases have been considered to be the primary generator of the arachidonic acid (AA) precursor pool for generating PGE2 and other eicosanoids. However, recent studies have demonstrated that monoacyglycerol lipase (MAGL), through hydrolysis of the endocannabinoid 2-arachidonoylglycerol, provides a major source of AA for PGE2 synthesis in the mammalian brain under basal and neuroinflammatory states. We show here that either genetic or pharmacological ablation of MAGL leads to significantly reduced fever responses in both centrally or peripherally-administered lipopolysaccharide or interleukin-1beta-induced fever models in mice. We also show that a cannabinoid CB1 receptor antagonist does not attenuate these anti-pyrogenic effects of MAGL inhibitors. Thus, much like traditional nonsteroidal anti-inflammatory drugs, MAGL inhibitors can control fever, but appear to do so through restricted control over prostaglandin production in the nervous system.
ESTHER : Sanchez-Alavez_2015_PLoS.One_10_e0134437
PubMedSearch : Sanchez-Alavez_2015_PLoS.One_10_e0134437
PubMedID: 26287872

Title : Combining cross-metathesis and activity-based protein profiling: New beta-lactone motifs for targeting serine hydrolases - Camara_2015_Bioorg.Med.Chem.Lett_25_317
Author(s) : Camara K , Kamat SS , Lasota CC , Cravatt BF , Howell AR
Ref : Bioorganic & Medicinal Chemistry Lett , 25 :317 , 2015
Abstract : beta-Lactones are a privileged structural motif as enzyme inhibitors and chemical probes, particularly for the inhibition of enzymes from the serine hydrolase class. Herein, we demonstrate that cross-metathesis (CM) of alpha-methylene-beta-lactones offers rapid access to structurally diverse, previously unexplored beta-lactones. Combining this approach with competitive activity-based protein profiling (ABPP) identified lead beta-lactone inhibitors/probes for several serine hydrolases, including disease-associated enzymes and enzymes of uncharacterized function. The structural diversity afforded by the alpha-methylene-beta-lactone scaffold thus expands the landscape of serine hydrolases that can be targeted by small-molecule inhibitors and should further the functional characterization of enzymes from this class through the optimization of target-selective probes.
ESTHER : Camara_2015_Bioorg.Med.Chem.Lett_25_317
PubMedSearch : Camara_2015_Bioorg.Med.Chem.Lett_25_317
PubMedID: 25541002

Title : ABHD4 regulates multiple classes of N-acyl phospholipids in the mammalian central nervous system - Lee_2015_Biochemistry_54_2539
Author(s) : Lee HC , Simon GM , Cravatt BF
Ref : Biochemistry , 54 :2539 , 2015
Abstract : N-Acyl phospholipids are atypical components of cell membranes that bear three acyl chains and serve as potential biosynthetic precursors for lipid mediators such as endocannabinoids. Biochemical studies have implicated ABHD4 as a brain N-acyl phosphatidylethanolamine (NAPE) lipase, but in vivo evidence for this functional assignment is lacking. Here, we describe ABHD4(-/-) mice and their characterization using untargeted lipidomics to discover that ABHD4 regulates multiple classes of brain N-acyl phospholipids. In addition to showing reductions in brain glycerophospho-NAEs (GP-NAEs) and plasmalogen-based lyso-NAPEs (lyso-pNAPEs), ABHD4(-/-) mice exhibited decreases in a distinct set of brain lipids that were structurally characterized as N-acyl lysophosphatidylserines (lyso-NAPSs). Biochemical assays confirmed that NAPS lipids are direct substrates of ABHD4. These findings, taken together, designate ABHD4 as a principal regulator of N-acyl phospholipid metabolism in the mammalian nervous system.
ESTHER : Lee_2015_Biochemistry_54_2539
PubMedSearch : Lee_2015_Biochemistry_54_2539
PubMedID: 25853435
Gene_locus related to this paper: human-ABHD4

Title : Selective N-Hydroxyhydantoin Carbamate Inhibitors of Mammalian Serine Hydrolases - Cognetta_2015_Chem.Biol_22_928
Author(s) : Cognetta AB, 3rd , Niphakis MJ , Lee HC , Martini ML , Hulce JJ , Cravatt BF
Ref : Chemical Biology , 22 :928 , 2015
Abstract : Serine hydrolase inhibitors, which facilitate enzyme function assignment and are used to treat a range of human disorders, often act by an irreversible mechanism that involves covalent modification of the serine hydrolase catalytic nucleophile. The portion of mammalian serine hydrolases for which selective inhibitors have been developed, however, remains small. Here, we show that N-hydroxyhydantoin (NHH) carbamates are a versatile class of irreversible serine hydrolase inhibitors that can be modified on both the staying (carbamylating) and leaving (NHH) groups to optimize potency and selectivity. Synthesis of a small library of NHH carbamates and screening by competitive activity-based protein profiling furnished selective, in vivo-active inhibitors and tailored activity-based probes for multiple mammalian serine hydrolases, including palmitoyl protein thioesterase 1, mutations of which cause the human disease infantile neuronal ceroid lipofuscinosis.
ESTHER : Cognetta_2015_Chem.Biol_22_928
PubMedSearch : Cognetta_2015_Chem.Biol_22_928
PubMedID: 26120000

Title : Full Fatty Acid Amide Hydrolase Inhibition Combined with Partial Monoacylglycerol Lipase Inhibition: Augmented and Sustained Antinociceptive Effects with Reduced Cannabimimetic Side Effects in Mice - Ghosh_2015_J.Pharmacol.Exp.Ther_354_111
Author(s) : Ghosh S , Kinsey SG , Liu QS , Hruba L , McMahon LR , Grim TW , Merritt CR , Wise LE , Abdullah RA , Selley DE , Sim-Selley LJ , Cravatt BF , Lichtman AH
Ref : Journal of Pharmacology & Experimental Therapeutics , 354 :111 , 2015
Abstract : Inhibition of fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL), the primary hydrolytic enzymes for the respective endocannabinoids N-arachidonoylethanolamine (AEA) and 2-arachidonylglycerol (2-AG), produces antinociception but with minimal cannabimimetic side effects. Although selective inhibitors of either enzyme often show partial efficacy in various nociceptive models, their combined blockade elicits augmented antinociceptive effects, but side effects emerge. Moreover, complete and prolonged MAGL blockade leads to cannabinoid receptor type 1 (CB1) receptor functional tolerance, which represents another challenge in this potential therapeutic strategy. Therefore, the present study tested whether full FAAH inhibition combined with partial MAGL inhibition would produce sustained antinociceptive effects with minimal cannabimimetic side effects. Accordingly, we tested a high dose of the FAAH inhibitor PF-3845 (N-3-pyridinyl-4-[[3-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenyl]methyl]-1-piper idinecarboxamide; 10 mg/kg) given in combination with a low dose of the MAGL inhibitor JZL184 [4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate] (4 mg/kg) in mouse models of inflammatory and neuropathic pain. This combination of inhibitors elicited profound increases in brain AEA levels (>10-fold) but only 2- to 3-fold increases in brain 2-AG levels. This combination produced significantly greater antinociceptive effects than single enzyme inhibition and did not elicit common cannabimimetic effects (e.g., catalepsy, hypomotility, hypothermia, and substitution for Delta(9)-tetrahydrocannabinol in the drug-discrimination assay), although these side effects emerged with high-dose JZL184 (i.e., 100 mg/kg). Finally, repeated administration of this combination did not lead to tolerance to its antiallodynic actions in the carrageenan assay or CB1 receptor functional tolerance. Thus, full FAAH inhibition combined with partial MAGL inhibition reduces neuropathic and inflammatory pain states with minimal cannabimimetic effects.
ESTHER : Ghosh_2015_J.Pharmacol.Exp.Ther_354_111
PubMedSearch : Ghosh_2015_J.Pharmacol.Exp.Ther_354_111
PubMedID: 25998048

Title : Blockade of 2-arachidonoylglycerol hydrolysis produces antidepressant-like effects and enhances adult hippocampal neurogenesis and synaptic plasticity - Zhang_2015_Hippocampus_25_16
Author(s) : Zhang Z , Wang W , Zhong P , Liu SJ , Long JZ , Zhao L , Gao HQ , Cravatt BF , Liu QS
Ref : Hippocampus , 25 :16 , 2015
Abstract : The endocannabinoid ligand 2-arachidonoylglycerol (2-AG) is inactivated primarily by monoacylglycerol lipase (MAGL). We have shown recently that chronic treatments with MAGL inhibitor JZL184 produce antidepressant- and anxiolytic-like effects in a chronic unpredictable stress (CUS) model of depression in mice. However, the underlying mechanisms remain poorly understood. Adult hippocampal neurogenesis has been implicated in animal models of anxiety and depression and behavioral effects of antidepressants. We tested whether CUS and chronic JZL184 treatments affected adult neurogenesis and synaptic plasticity in the dentate gyrus (DG) of mouse hippocampus. We report that CUS induced depressive-like behaviors and decreased the number of bromodeoxyuridine-labeled neural progenitor cells and doublecortin-positive immature neurons in the DG, while chronic JZL184 treatments prevented these behavioral and cellular deficits. We also investigated the effects of CUS and chronic JZL184 on a form long-term potentiation (LTP) in the DG known to be neurogenesis-dependent. CUS impaired LTP induction, whereas chronic JZL184 treatments restored LTP in CUS-exposed mice. These results suggest that enhanced adult neurogenesis and long-term synaptic plasticity in the DG of the hippocampus might contribute to antidepressant- and anxiolytic-like behavioral effects of JZL184.
ESTHER : Zhang_2015_Hippocampus_25_16
PubMedSearch : Zhang_2015_Hippocampus_25_16
PubMedID: 25131612
Gene_locus related to this paper: human-MGLL

Title : Inhibition of monoacylglycerol lipase reduces nicotine withdrawal - Muldoon_2015_Br.J.Pharmacol_172_869
Author(s) : Muldoon PP , Chen J , Harenza JL , Abdullah RA , Sim-Selley LJ , Cravatt BF , Miles MF , Chen X , Lichtman AH , Damaj MI
Ref : British Journal of Pharmacology , 172 :869 , 2015
Abstract : BACKGROUND AND PURPOSE: Abrupt discontinuation of nicotine, the main psychoactive component in tobacco, induces a withdrawal syndrome in nicotine-dependent animals, consisting of somatic and affective signs, avoidance of which contributes to drug maintenance. While blockade of fatty acid amide hydrolase, the primary catabolic enzyme of the endocannabinoid arachidonoylethanolamine (anandamide), exacerbates withdrawal responses in nicotine-dependent mice, the role of monoacylglycerol lipase (MAGL), the main hydrolytic enzyme of a second endocannabinoid 2-arachidonylglycerol (2-AG), in nicotine withdrawal remains unexplored. EXPERIMENTAL APPROACH: To evaluate the role of MAGL enzyme inhibition in nicotine withdrawal, we initially performed a genetic correlation approach using the BXD recombinant inbred mouse panel. We then assessed nicotine withdrawal intensity in the mouse after treatment with the selective MAGL inhibitor, JZL184, and after genetic deletion of the enzyme. Lastly, we assessed the association between genotypes and smoking withdrawal phenotypes in two human data sets. KEY
RESULTS: BXD mice displayed significant positive correlations between basal MAGL mRNA expression and nicotine withdrawal responses, consistent with the idea that increased 2-AG brain levels may attenuate withdrawal responses. Strikingly, the MAGL inhibitor, JZL184, dose-dependently reduced somatic and aversive withdrawal signs, which was blocked by rimonabant, indicating a CB1 receptor-dependent mechanism. MAGL-knockout mice also showed attenuated nicotine withdrawal. Lastly, genetic analyses in humans revealed associations of the MAGL gene with smoking withdrawal in humans. CONCLUSIONS AND IMPLICATIONS: Overall, our findings suggest that MAGL inhibition maybe a promising target for treatment of nicotine dependence.
ESTHER : Muldoon_2015_Br.J.Pharmacol_172_869
PubMedSearch : Muldoon_2015_Br.J.Pharmacol_172_869
PubMedID: 25258021
Gene_locus related to this paper: human-MGLL

Title : Effect of selective inhibition of monoacylglycerol lipase (MAGL) on acute nausea, anticipatory nausea, and vomiting in rats and Suncus murinus - Parker_2015_Psychopharmacology.(Berl)_232_583
Author(s) : Parker LA , Niphakis MJ , Downey R , Limebeer CL , Rock EM , Sticht MA , Morris H , Abdullah RA , Lichtman AH , Cravatt BF
Ref : Psychopharmacology (Berl) , 232 :583 , 2015
Abstract : RATIONALE: To determine the role of the endocannabinoid, 2-arachodonyl glycerol (2-AG), in the regulation of nausea and vomiting. OBJECTIVE: We evaluated the effectiveness of the potent selective monoacylglycerol lipase (MAGL) inhibitor, MJN110, which selectively elevates the endocannabinoid 2-AG, to suppress acute nausea and vomiting, as well as anticipatory nausea in rat and shrew models.
METHODS: The rat gaping models were used to evaluate the potential of MJN110 (5, 10, and 20 mg/kg, intraperitoneally [IP]) to suppress acute nausea produced by LiCl and of MJN110 (10 and 20 mg/kg, IP) to suppress anticipatory nausea elicited by a LiCl-paired context. The potential as well of MJN110 (10 and 20 mg/kg, IP) to suppress vomiting and contextually elicited gaping in the Suncus murinus was evaluated.
RESULTS: MJN110 suppressed acute nausea in rats, LiCl-induced vomiting in shrews and contextually-elicited anticipatory nausea in both rats (accompanied by elevation of 2-AG in the visceral insular cortex) and shrews. These effects were reversed by the CB1 antagonist/inverse agonist, SR141716. The MAGL inhibitor did not modify locomotion at any dose. An activity-based protein profiling analysis of samples of tissue collected from the visceral insular cortex in rats and whole brain tissues in shrews revealed that MJN110 selectively inhibited MAGL and the alternative 2-AG hydrolase, ABHD6.
CONCLUSIONS: MAGL inhibition by MJN110 which selectively elevates endogenous 2-AG has therapeutic potential in the treatment of acute nausea and vomiting as well as anticipatory nausea, a distressful symptom that is resistant to currently available treatments.
ESTHER : Parker_2015_Psychopharmacology.(Berl)_232_583
PubMedSearch : Parker_2015_Psychopharmacology.(Berl)_232_583
PubMedID: 25085768
Gene_locus related to this paper: human-MGLL

Title : In vivo characterization of the highly selective monoacylglycerol lipase inhibitor KML29: antinociceptive activity without cannabimimetic side effects - Ignatowska-Jankowska_2014_Br.J.Pharmacol_171_1392
Author(s) : Ignatowska-Jankowska BM , Ghosh S , Crowe MS , Kinsey SG , Niphakis MJ , Abdullah RA , Tao Q , ST ON , Walentiny DM , Wiley JL , Cravatt BF , Lichtman AH
Ref : British Journal of Pharmacology , 171 :1392 , 2014
Abstract : BACKGROUND AND PURPOSE: Since monoacylglycerol lipase (MAGL) has been firmly established as the predominant catabolic enzyme of the endocannabinoid 2-arachidonoylglycerol (2-AG), a great need has emerged for the development of highly selective MAGL inhibitors. Here, we tested the in vivo effects of one such compound, KML29 (1,1,1,3,3,3-hexafluoropropan-2-yl 4-(bis(benzo[d][1,3]dioxol-5-yl)(hydroxy)methyl)piperidine-1-carboxylate). EXPERIMENTAL APPROACH: In the present study, we tested KML29 in murine inflammatory (i.e. carrageenan) and sciatic nerve injury pain models, as well as the diclofenac-induced gastric haemorrhage model. KML29 was also evaluated for cannabimimetic effects, including measurements of locomotor activity, body temperature, catalepsy, and cannabinoid interoceptive effects in the drug discrimination paradigm. KEY
RESULTS: KML29 attenuated carrageenan-induced paw oedema and completely reversed carrageenan-induced mechanical allodynia. These effects underwent tolerance after repeated administration of high-dose KML29, which were accompanied by cannabinoid receptor 1 (CB1 ) receptor desensitization. Acute or repeated KML29 administration increased 2-AG levels and concomitantly reduced arachidonic acid levels, but without elevating anandamide (AEA) levels in the whole brain. Furthermore, KML29 partially reversed allodynia in the sciatic nerve injury model and completely prevented diclofenac-induced gastric haemorrhages. CB1 and CB2 receptors played differential roles in these pharmacological effects of KML29. In contrast, KML29 did not elicit cannabimimetic effects, including catalepsy, hypothermia and hypomotility. Although KML29 did not substitute for Delta(9) -tetrahydrocannabinol (THC) in C57BL/6J mice, it fully and dose-dependantly substituted for AEA in fatty acid amide hydrolase (FAAH) (-/-) mice, consistent with previous work showing that dual FAAH and MAGL inhibition produces THC-like subjective effects. CONCLUSIONS AND IMPLICATIONS: These results indicate that KML29, a highly selective MAGL inhibitor, reduces inflammatory and neuropathic nociceptive behaviour without occurrence of cannabimimetic side effects. LINKED ARTICLES: This article is part of a themed section on Cannabinoids 2013. To view the other articles in this section visit
ESTHER : Ignatowska-Jankowska_2014_Br.J.Pharmacol_171_1392
PubMedSearch : Ignatowska-Jankowska_2014_Br.J.Pharmacol_171_1392
PubMedID: 23848221
Gene_locus related to this paper: human-MGLL

Title : Serine hydrolase inhibitors block necrotic cell death by preventing calcium overload of the mitochondria and permeability transition pore formation - Yun_2014_J.Biol.Chem_289_1491
Author(s) : Yun B , Lee H , Ghosh M , Cravatt BF , Hsu KL , Bonventre JV , Ewing H , Gelb MH , Leslie CC
Ref : Journal of Biological Chemistry , 289 :1491 , 2014
Abstract : Perturbation of calcium signaling that occurs during cell injury and disease, promotes cell death. In mouse lung fibroblasts A23187 triggered mitochondrial permeability transition pore (MPTP) formation, lactate dehydrogenase (LDH) release, and necrotic cell death that were blocked by cyclosporin A (CsA) and EGTA. LDH release temporally correlated with arachidonic acid release but did not involve cytosolic phospholipase A2alpha (cPLA2alpha) or calcium-independent PLA2. Surprisingly, release of arachidonic acid and LDH from cPLA2alpha-deficient fibroblasts was inhibited by the cPLA2alpha inhibitor pyrrophenone, and another serine hydrolase inhibitor KT195, by preventing mitochondrial calcium uptake. Inhibitors of calcium/calmodulin-dependent protein kinase II, a mitochondrial Ca(2+) uniporter (MCU) regulator, also prevented MPTP formation and arachidonic acid release induced by A23187 and H2O2. Pyrrophenone blocked MCU-mediated mitochondrial calcium uptake in permeabilized fibroblasts but not in isolated mitochondria. Unlike pyrrophenone, the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol and CsA blocked cell death and arachidonic acid release not by preventing mitochondrial calcium uptake but by inhibiting MPTP formation. In fibroblasts stimulated with thapsigargin, which induces MPTP formation by a direct effect on mitochondria, LDH and arachidonic acid release were blocked by CsA and 1-oleoyl-2-acetyl-sn-glycerol but not by pyrrophenone or EGTA. Therefore serine hydrolase inhibitors prevent necrotic cell death by blocking mitochondrial calcium uptake but not the enzyme releasing fatty acids that occurs by a novel pathway during MPTP formation. This work reveals the potential for development of small molecule cell-permeable serine hydrolase inhibitors that block MCU-mediated mitochondrial calcium overload, MPTP formation, and necrotic cell death.
ESTHER : Yun_2014_J.Biol.Chem_289_1491
PubMedSearch : Yun_2014_J.Biol.Chem_289_1491
PubMedID: 24297180

Title : Identification of the major prostaglandin glycerol ester hydrolase in human cancer cells - Manna_2014_J.Biol.Chem_289_33741
Author(s) : Manna JD , Wepy JA , Hsu KL , Chang JW , Cravatt BF , Marnett LJ
Ref : Journal of Biological Chemistry , 289 :33741 , 2014
Abstract : Prostaglandin glycerol esters (PG-Gs) are produced as a result of the oxygenation of the endocannabinoid, 2-arachidonoylglycerol, by cyclooxygenase 2. Understanding the role that PG-Gs play in a biological setting has been difficult because of their sensitivity to enzymatic hydrolysis. By comparing PG-G hydrolysis across human cancer cell lines to serine hydrolase activities determined by activity-based protein profiling, we identified lysophospholipase A2 (LYPLA2) as a major enzyme responsible for PG-G hydrolysis. The principal role played by LYPLA2 in PGE2-G hydrolysis was confirmed by siRNA knockdown. Purified recombinant LYPLA2 hydrolyzed PG-Gs in the following order of activity: PGE2-G > PGF2alpha-G > PGD2-G; LYPLA2 hydrolyzed 1- but not 2-arachidonoylglycerol or arachidonoylethanolamide. Chemical inhibition of LYPLA2 in the mouse macrophage-like cell line, RAW264.7, elicited an increase in PG-G production. Our data indicate that LYPLA2 serves as a major PG-G hydrolase in human cells. Perturbation of this enzyme should enable selective modulation of PG-Gs without alterations in endocannabinoids, thereby providing a means to decipher the unique functions of PG-Gs in biology and disease.
ESTHER : Manna_2014_J.Biol.Chem_289_33741
PubMedSearch : Manna_2014_J.Biol.Chem_289_33741
PubMedID: 25301951
Gene_locus related to this paper: human-LYPLA2

Title : Application of activity-based protein profiling to study enzyme function in adipocytes - Galmozzi_2014_Methods.Enzymol_538_151
Author(s) : Galmozzi A , Dominguez E , Cravatt BF , Saez E
Ref : Methods Enzymol , 538 :151 , 2014
Abstract : Activity-based protein profiling (ABPP) is a chemical proteomics approach that utilizes small-molecule probes to determine the functional state of enzymes directly in native systems. ABPP probes selectively label active enzymes, but not their inactive forms, facilitating the characterization of changes in enzyme activity that occur without alterations in protein levels. ABPP can be a tool superior to conventional gene expression and proteomic profiling methods to discover new enzymes active in adipocytes and to detect differences in the activity of characterized enzymes that may be associated with disorders of adipose tissue function. ABPP probes have been developed that react selectively with most members of specific enzyme classes. Here, using as an example the serine hydrolase family that includes many enzymes with critical roles in adipocyte physiology, we describe methods to apply ABPP analysis to the study of adipocyte enzymatic pathways.
ESTHER : Galmozzi_2014_Methods.Enzymol_538_151
PubMedSearch : Galmozzi_2014_Methods.Enzymol_538_151
PubMedID: 24529438

Title : Design of activated serine-containing catalytic triads with atomic-level accuracy - Rajagopalan_2014_Nat.Chem.Biol_10_386
Author(s) : Rajagopalan S , Wang C , Yu K , Kuzin AP , Richter F , Lew S , Miklos AE , Matthews ML , Seetharaman J , Su M , Hunt JF , Cravatt BF , Baker D
Ref : Nat Chemical Biology , 10 :386 , 2014
Abstract : A challenge in the computational design of enzymes is that multiple properties, including substrate binding, transition state stabilization and product release, must be simultaneously optimized, and this has limited the absolute activity of successful designs. Here, we focus on a single critical property of many enzymes: the nucleophilicity of an active site residue that initiates catalysis. We design proteins with idealized serine-containing catalytic triads and assess their nucleophilicity directly in native biological systems using activity-based organophosphate probes. Crystal structures of the most successful designs show unprecedented agreement with computational models, including extensive hydrogen bonding networks between the catalytic triad (or quartet) residues, and mutagenesis experiments demonstrate that these networks are critical for serine activation and organophosphate reactivity. Following optimization by yeast display, the designs react with organophosphate probes at rates comparable to natural serine hydrolases. Co-crystal structures with diisopropyl fluorophosphate bound to the serine nucleophile suggest that the designs could provide the basis for a new class of organophosphate capture agents.
ESTHER : Rajagopalan_2014_Nat.Chem.Biol_10_386
PubMedSearch : Rajagopalan_2014_Nat.Chem.Biol_10_386
PubMedID: 24705591

Title : ThermoMouse: an in vivo model to identify modulators of UCP1 expression in brown adipose tissue - Galmozzi_2014_Cell.Rep_9_1584
Author(s) : Galmozzi A , Sonne SB , Altshuler-Keylin S , Hasegawa Y , Shinoda K , Luijten IH , Chang JW , Sharp LZ , Cravatt BF , Saez E , Kajimura S
Ref : Cell Rep , 9 :1584 , 2014
Abstract : Obesity develops when energy intake chronically exceeds energy expenditure. Because brown adipose tissue (BAT) dissipates energy in the form of heat, increasing energy expenditure by augmenting BAT-mediated thermogenesis may represent an approach to counter obesity and its complications. The ability of BAT to dissipate energy is dependent on expression of mitochondrial uncoupling protein 1 (UCP1). To facilitate the identification of pharmacological modulators of BAT UCP1 levels, which may have potential as antiobesity medications, we developed a transgenic model in which luciferase activity faithfully mimics endogenous UCP1 expression and its response to physiologic stimuli. Phenotypic screening of a library using cells derived from this model yielded a small molecule that increases UCP1 expression in brown fat cells and mice. Upon adrenergic stimulation, compound-treated mice showed increased energy expenditure. These tools offer an opportunity to identify pharmacologic modulators of UCP1 expression and uncover regulatory pathways that impact BAT-mediated thermogenesis.
ESTHER : Galmozzi_2014_Cell.Rep_9_1584
PubMedSearch : Galmozzi_2014_Cell.Rep_9_1584
PubMedID: 25466254

Title : ABHD6 Blockade Exerts Antiepileptic Activity in PTZ-Induced Seizures and in Spontaneous Seizures in R6\/2 Mice - Naydenov_2014_Neuron_83_361
Author(s) : Naydenov AV , Horne EA , Cheah CS , Swinney K , Hsu KL , Cao JK , Marrs WR , Blankman JL , Tu S , Cherry AE , Fung S , Wen A , Li W , Saporito MS , Selley DE , Cravatt BF , Oakley JC , Stella N
Ref : Neuron , 83 :361 , 2014
Abstract : The serine hydrolase alpha/beta-hydrolase domain 6 (ABHD6) hydrolyzes the most abundant endocannabinoid (eCB) in the brain, 2-arachidonoylglycerol (2-AG), and controls its availability at cannabinoid receptors. We show that ABHD6 inhibition decreases pentylenetetrazole (PTZ)-induced generalized tonic-clonic and myoclonic seizure incidence and severity. This effect is retained in Cnr1(-/-) or Cnr2(-/-) mice, but blocked by addition of a subconvulsive dose of picrotoxin, suggesting the involvement of GABAA receptors. ABHD6 inhibition also blocked spontaneous seizures in R6/2 mice, a genetic model of juvenile Huntington's disease known to exhibit dysregulated eCB signaling. ABHD6 blockade retained its antiepileptic activity over chronic dosing and was not associated with psychomotor or cognitive effects. While the etiology of seizures in R6/2 mice remains unsolved, involvement of the hippocampus is suggested by interictal epileptic discharges, increased expression of vGLUT1 but not vGAT, and reduced Neuropeptide Y (NPY) expression. We conclude that ABHD6 inhibition may represent a novel antiepileptic strategy.
ESTHER : Naydenov_2014_Neuron_83_361
PubMedSearch : Naydenov_2014_Neuron_83_361
PubMedID: 25033180
Gene_locus related to this paper: human-ABHD6 , mouse-ABHD6

Title : Endocannabinoids modulate cortical development by configuring Slit2\/Robo1 signalling - Alpar_2014_Nat.Commun_5_4421
Author(s) : Alpar A , Tortoriello G , Calvigioni D , Niphakis MJ , Milenkovic I , Bakker J , Cameron GA , Hanics J , Morris CV , Fuzik J , Kovacs GG , Cravatt BF , Parnavelas JG , Andrews WD , Hurd YL , Keimpema E , Harkany T
Ref : Nat Commun , 5 :4421 , 2014
Abstract : Local environmental cues are indispensable for axonal growth and guidance during brain circuit formation. Here, we combine genetic and pharmacological tools, as well as systems neuroanatomy in human fetuses and mouse models, to study the role of endocannabinoid and Slit/Robo signalling in axonal growth. We show that excess 2-arachidonoylglycerol, an endocannabinoid affecting directional axonal growth, triggers corpus callosum enlargement due to the errant CB1 cannabinoid receptor-containing corticofugal axon spreading. This phenotype mechanistically relies on the premature differentiation and end-feet proliferation of CB2R-expressing oligodendrocytes. We further show the dependence of both axonal Robo1 positioning and oligodendroglial Slit2 production on cell-type-specific cannabinoid receptor activation. Accordingly, Robo1 and/or Slit2 manipulation limits endocannabinoid modulation of axon guidance. We conclude that endocannabinoids can configure focal Slit2/Robo1 signalling to modulate directional axonal growth, which may provide a basis for understanding impaired brain wiring associated with metabolic deficits and prenatal drug exposure.
ESTHER : Alpar_2014_Nat.Commun_5_4421
PubMedSearch : Alpar_2014_Nat.Commun_5_4421
PubMedID: 25030704

Title : Remodeling natural products: chemistry and serine hydrolase activity of a rocaglate-derived beta-lactone - Lajkiewicz_2014_J.Am.Chem.Soc_136_2659
Author(s) : Lajkiewicz NJ , Cognetta AB, 3rd , Niphakis MJ , Cravatt BF , Porco JA, Jr.
Ref : Journal of the American Chemical Society , 136 :2659 , 2014
Abstract : Flavaglines are a class of natural products with potent insecticidal and anticancer activities. beta-Lactones are a privileged structural motif found in both therapeutic agents and chemical probes. Herein, we report the synthesis, unexpected light-driven di-epimerization, and activity-based protein profiling of a novel rocaglate-derived beta-lactone. In addition to in vitro inhibition of the serine hydrolases ABHD10 and ACOT1/2, the most potent beta-lactone enantiomer was also found to inhibit these enzymes, as well as the serine peptidases CTSA and SCPEP1, in PC3 cells.
ESTHER : Lajkiewicz_2014_J.Am.Chem.Soc_136_2659
PubMedSearch : Lajkiewicz_2014_J.Am.Chem.Soc_136_2659
PubMedID: 24447064
Gene_locus related to this paper: human-ABHD10 , human-ACOT1 , human-ACOT2 , human-CTSA , human-SCPEP1 , mouse-abhda

Title : Molecular medicine and neurodegenerative diseases -
Author(s) : Chang CJ , Cravatt BF , Johnson DS , Lim MH
Ref : Chem Soc Rev , 43 :6668 , 2014
PubMedID: 25105517

Title : Mapping the protein interaction landscape for fully functionalized small-molecule probes in human cells - Kambe_2014_J.Am.Chem.Soc_136_10777
Author(s) : Kambe T , Correia BE , Niphakis MJ , Cravatt BF
Ref : Journal of the American Chemical Society , 136 :10777 , 2014
Abstract : Phenotypic screening provides a means to discover small molecules that perturb cell biological processes. Discerning the proteins and biochemical pathways targeted by screening hits, however, remains technically challenging. We recently described the use of small molecules bearing photoreactive groups and latent affinity handles as fully functionalized probes for integrated phenotypic screening and target identification. The general utility of such probes, or, for that matter, any small-molecule screening library, depends on the scope of their protein interactions in cells, a parameter that remains largely unexplored. Here, we describe the synthesis of an ~60-member fully functionalized probe library, prepared from Ugi-azide condensation reactions to impart structural diversity and introduce diazirine and alkyne functionalities for target capture and enrichment, respectively. In-depth mass spectrometry-based analysis revealed a diverse array of probe targets in human cells, including enzymes, channels, adaptor and scaffolding proteins, and proteins of uncharacterized function. For many of these proteins, ligands have not yet been described. Most of the probe-protein interactions showed well-defined structure-activity relationships across the probe library and were blocked by small-molecule competitors in cells. These findings indicate that fully functionalized small molecules canvas diverse segments of the human proteome and hold promise as pharmacological probes of cell biology.
ESTHER : Kambe_2014_J.Am.Chem.Soc_136_10777
PubMedSearch : Kambe_2014_J.Am.Chem.Soc_136_10777
PubMedID: 25045785

Title : Enzyme inhibitor discovery by activity-based protein profiling - Niphakis_2014_Annu.Rev.Biochem_83_341
Author(s) : Niphakis MJ , Cravatt BF
Ref : Annual Review of Biochemistry , 83 :341 , 2014
Abstract : Eukaryotic and prokaryotic organisms possess huge numbers of uncharacterized enzymes. Selective inhibitors offer powerful probes for assigning functions to enzymes in native biological systems. Here, we discuss how the chemical proteomic platform activity-based protein profiling (ABPP) can be implemented to discover selective and in vivo-active inhibitors for enzymes. We further describe how these inhibitors have been used to delineate the biochemical and cellular functions of enzymes, leading to the discovery of metabolic and signaling pathways that make important contributions to human physiology and disease. These studies demonstrate the value of selective chemical probes as drivers of biological inquiry.
ESTHER : Niphakis_2014_Annu.Rev.Biochem_83_341
PubMedSearch : Niphakis_2014_Annu.Rev.Biochem_83_341
PubMedID: 24905785

Title : Integrated phenotypic and activity-based profiling links Ces3 to obesity and diabetes - Dominguez_2014_Nat.Chem.Biol_10_113
Author(s) : Dominguez E , Galmozzi A , Chang JW , Hsu KL , Pawlak J , Li W , Godio C , Thomas J , Partida D , Niessen S , O'Brien PE , Russell AP , Watt MJ , Nomura DK , Cravatt BF , Saez E
Ref : Nat Chemical Biology , 10 :113 , 2014
Abstract : Phenotypic screening is making a comeback in drug discovery as the maturation of chemical proteomics methods has facilitated target identification for bioactive small molecules. A limitation of these approaches is that time-consuming genetic methods or other means are often required to determine the biologically relevant target (or targets) from among multiple protein-compound interactions that are typically detected. Here, we have combined phenotypic screening of a directed small-molecule library with competitive activity-based protein profiling to map and functionally characterize the targets of screening hits. Using this approach, we identify carboxylesterase 3 (Ces3, also known as Ces1d) as a primary molecular target of bioactive compounds that promote lipid storage in adipocytes. We further show that Ces3 activity is markedly elevated during adipocyte differentiation. Treatment of two mouse models of obesity-diabetes with a Ces3 inhibitor ameliorates multiple features of metabolic syndrome, illustrating the power of the described strategy to accelerate the identification and pharmacologic validation of new therapeutic targets.
ESTHER : Dominguez_2014_Nat.Chem.Biol_10_113
PubMedSearch : Dominguez_2014_Nat.Chem.Biol_10_113
PubMedID: 24362705
Gene_locus related to this paper: human-CES3 , mouse-Ces1d

Title : Monoacylglycerol lipase inhibition blocks chronic stress-induced depressive-like behaviors via activation of mTOR signaling - Zhong_2014_Neuropsychopharmacology_39_1763
Author(s) : Zhong P , Wang W , Pan B , Liu X , Zhang Z , Long JZ , Zhang HT , Cravatt BF , Liu QS
Ref : Neuropsychopharmacology , 39 :1763 , 2014
Abstract : The endocannabinoid (eCB) system regulates mood, emotion, and stress coping, and dysregulation of the eCB system is critically involved in pathophysiology of depression. The eCB ligand 2-arachidonoylglycerol (2-AG) is inactivated by monoacylglycerol lipase (MAGL). Using chronic unpredictable mild stress (CUS) as a mouse model of depression, we examined how 2-AG signaling in the hippocampus was altered in depressive-like states and how this alteration contributed to depressive-like behavior. We report that CUS led to impairment of depolarization-induced suppression of inhibition (DSI) in mouse hippocampal CA1 pyramidal neurons, and this deficiency in 2-AG-mediated retrograde synaptic depression was rescued by MAGL inhibitor JZL184. CUS induced depressive-like behaviors and decreased mammalian target of rapamycin (mTOR) activation in the hippocampus, and these biochemical and behavioral abnormalities were ameliorated by chronic JZL184 treatments. The effects of JZL184 were mediated by cannabinoid CB1 receptors. Genetic deletion of mTOR with adeno-associated viral (AAV) vector carrying the Cre recombinase in the hippocampus of mTORf/f mice recapitulated depressive-like behaviors induced by CUS and abrogated the antidepressant-like effects of chronic JZL184 treatments. Our results suggest that CUS decreases eCB-mTOR signaling in the hippocampus, leading to depressive-like behaviors, whereas MAGL inhibitor JZL184 produces antidepressant-like effects through enhancement of eCB-mTOR signaling.
ESTHER : Zhong_2014_Neuropsychopharmacology_39_1763
PubMedSearch : Zhong_2014_Neuropsychopharmacology_39_1763
PubMedID: 24476943

Title : Prolonged monoacylglycerol lipase blockade causes equivalent cannabinoid receptor type 1 receptor-mediated adaptations in fatty acid amide hydrolase wild-type and knockout mice - Schlosburg_2014_J.Pharmacol.Exp.Ther_350_196
Author(s) : Schlosburg JE , Kinsey SG , Ignatowska-Jankowska B , Ramesh D , Abdullah RA , Tao Q , Booker L , Long JZ , Selley DE , Cravatt BF , Lichtman AH
Ref : Journal of Pharmacology & Experimental Therapeutics , 350 :196 , 2014
Abstract : Complementary genetic and pharmacological approaches to inhibit monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), the primary hydrolytic enzymes of the respective endogenous cannabinoids 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine, enable the exploration of potential therapeutic applications and physiologic roles of these enzymes. Complete and simultaneous inhibition of both FAAH and MAGL produces greatly enhanced cannabimimetic responses, including increased antinociception, and other cannabimimetic effects, far beyond those seen with inhibition of either enzyme alone. While cannabinoid receptor type 1 (CB1) function is maintained following chronic FAAH inactivation, prolonged excessive elevation of brain 2-AG levels, via MAGL inhibition, elicits both behavioral and molecular signs of cannabinoid tolerance and dependence. Here, we evaluated the consequences of a high dose of the MAGL inhibitor JZL184 [4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate; 40 mg/kg] given acutely or for 6 days in FAAH(-/-) and (+/+) mice. While acute administration of JZL184 to FAAH(-/-) mice enhanced the magnitude of a subset of cannabimimetic responses, repeated JZL184 treatment led to tolerance to its antinociceptive effects, cross-tolerance to the pharmacological effects of Delta(9)-tetrahydrocannabinol, decreases in CB1 receptor agonist-stimulated guanosine 5'-O-(3-[(35)S]thio)triphosphate binding, and dependence as indicated by rimonabant-precipitated withdrawal behaviors, regardless of genotype. Together, these data suggest that simultaneous elevation of both endocannabinoids elicits enhanced cannabimimetic activity but MAGL inhibition drives CB1 receptor functional tolerance and cannabinoid dependence.
ESTHER : Schlosburg_2014_J.Pharmacol.Exp.Ther_350_196
PubMedSearch : Schlosburg_2014_J.Pharmacol.Exp.Ther_350_196
PubMedID: 24849924
Gene_locus related to this paper: human-MGLL

Title : Repeated low-dose administration of the monoacylglycerol lipase inhibitor JZL184 retains cannabinoid receptor type 1-mediated antinociceptive and gastroprotective effects - Kinsey_2013_J.Pharmacol.Exp.Ther_345_492
Author(s) : Kinsey SG , Wise LE , Ramesh D , Abdullah R , Selley DE , Cravatt BF , Lichtman AH
Ref : Journal of Pharmacology & Experimental Therapeutics , 345 :492 , 2013
Abstract : The monoacylglycerol lipase (MAGL) inhibitor 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184) produces antinociceptive and anti-inflammatory effects. However, repeated administration of high-dose JZL184 (40 mg/kg) causes dependence, antinociceptive tolerance, cross-tolerance to the pharmacological effects of cannabinoid receptor agonists, and cannabinoid receptor type 1 (CB1) downregulation and desensitization. This functional CB1 receptor tolerance poses a hurdle in the development of MAGL inhibitors for therapeutic use. Consequently, the present study tested whether repeated administration of low-dose JZL184 maintains its antinociceptive actions in the chronic constriction injury of the sciatic nerve neuropathic pain model and protective effects in a model of nonsteroidal anti-inflammatory drug-induced gastric hemorrhages. Mice given daily injections of high-dose JZL184 (>/=16 mg/kg) for 6 days displayed decreased CB1 receptor density and function in the brain, as assessed in [(3)H]SR141716A binding and CP55,940 [(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol]-stimulated guanosine 5'-O-(3-[(35)S]thio)triphosphate binding assays, respectively. In contrast, normal CB1 receptor expression and function were maintained following repeated administration of low-dose JZL184 (</=8 mg/kg). Likewise, the antinociceptive and gastroprotective effects of high-dose JZL184 underwent tolerance following repeated administration, but these effects were maintained following repeated low-dose JZL184 treatment. Consistent with these observations, repeated high-dose JZL184, but not repeated low-dose JZL184, elicited cross-tolerance to the common pharmacological effects of Delta(9)-tetrahydrocannabinol. This same pattern of effects was found in a rimonabant [(5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyraz ole-3-carboxamide)]-precipitated withdrawal model of cannabinoid dependence. Taken together, these results indicate that prolonged, partial MAGL inhibition maintains potentially beneficial antinociceptive and anti-inflammatory effects, without producing functional CB1 receptor tachyphylaxis/tolerance or cannabinoid dependence.
ESTHER : Kinsey_2013_J.Pharmacol.Exp.Ther_345_492
PubMedSearch : Kinsey_2013_J.Pharmacol.Exp.Ther_345_492
PubMedID: 23412396
Gene_locus related to this paper: human-MGLL

Title : Characterization of a Selective, Reversible Inhibitor of Lysophospholipase 2 (LYPLA2) - Adibekian_2013_Probe.Report__4
Author(s) : Adibekian A , Martin BR , Chang JW , Hsu KL , Tsuboi K , Bachovchin DA , Speers AE , Brown SJ , Spicer T , Fernandez-Vega V , Ferguson J , Cravatt BF , Hodder P , Rosen H
Ref : Probe Report , : , 2013
Abstract : Protein palmitoylation is an essential post-translational modification necessary for trafficking and localization of regulatory proteins that play key roles in cell growth and signaling. Multiple oncogenes, including HRAS and SRC, require palmitoylation for malignant transformation. Lysophospholipase 1 (LYPLA1) has been identified as a candidate protein palmitoyl thioesterase responsible for HRAS depalmitoylation in mammalian cells. LYPLA1 has a close homolog, LYPLA2 (65% sequence identity), whose substrate specificity and biochemical roles are, as yet, uncharacterized. Seeking chemical tools to investigate biochemical pathway involvement and potential roles in cancer pathogenesis of these enzymes, we conducted a fluorescence polarization-based competitive activity-based protein profiling (fluopol-ABPP) HTS campaign to identify inhibitors of LYPLA1 and LYPLA2. HTS identified a lead triazole urea micromolar inhibitor, which we optimized as dual LYPLA1/LYPLA2 inhibitor ML211, and reversible compounds ML348 and ML349 that act as selective LYPLA1 and LYPLA2 inhibitors, respectively. Using an advanced competitive ABPP strategy employing ABPP probes with controlled reactivity rates, we successfully confirmed potent and selective target engagement of these reversible compounds in living systems as detailed here for ML349 and in the accompanying ML348 Probe Report. Together, these compounds should greatly aid investigations into the biological function of LYPLA1 and LYPLA2.
ESTHER : Adibekian_2013_Probe.Report__4
PubMedSearch : Adibekian_2013_Probe.Report__4
PubMedID: 24624468
Gene_locus related to this paper: human-LYPLA2

Title : Characterization of a Selective, Reversible Inhibitor of Lysophospholipase 1 (LYPLA1) - Adibekian_2013_Probe.Report__3
Author(s) : Adibekian A , Martin BR , Chang JW , Hsu KL , Tsuboi K , Bachovchin DA , Speers AE , Brown SJ , Spicer T , Fernandez-Vega V , Ferguson J , Cravatt BF , Hodder P , Rosen H
Ref : Probe Report , : , 2013
Abstract : Protein palmitoylation is an essential post-translational modification necessary for trafficking and localization of regulatory proteins that play key roles in cell growth and signaling. Multiple oncogenes, including HRAS and SRC, require palmitoylation for malignant transformation. Lysophospholipase 1 (LYPLA1) has been identified as a candidate protein palmitoyl thioesterase responsible for HRAS depalmitoylation in mammalian cells. Seeking chemical tools to investigate biochemical pathway involvement and potential roles in cancer pathogenesis, we conducted a fluorescence polarization-based competitive activity-based protein profiling (fluopol-ABPP) HTS campaign to identify inhibitors of LYPLA1 and the structurally related LYPLA2. HTS identified a lead triazole urea micromolar inhibitor, which we optimized as dual LYPLA1/LYPLA2 inhibitor ML211, and reversible compounds ML348 and ML349 that act as selective LYPLA1 and LYPLA2 inhibitors, respectively. Using an advanced competitive ABPP strategy employing ABPP probes with controlled reactivity rates, we successfully confirmed potent and selective target engagement of these reversible compounds in living systems as detailed here for ML348 and in the accompanying ML349 Probe Report. Together, these compounds should greatly aid investigations into the biological function of LYPLA1 and LYPLA2
ESTHER : Adibekian_2013_Probe.Report__3
PubMedSearch : Adibekian_2013_Probe.Report__3
PubMedID: 24624465

Title : Proteome-wide mapping of cholesterol-interacting proteins in mammalian cells - Hulce_2013_Nat.Methods_10_259
Author(s) : Hulce JJ , Cognetta AB, 3rd , Niphakis MJ , Tully SE , Cravatt BF
Ref : Nat Methods , 10 :259 , 2013
Abstract : Cholesterol is an essential structural component of cellular membranes and serves as a precursor for several classes of signaling molecules. Cholesterol exerts its effects and is, itself, regulated in large part by engagement in specific interactions with proteins. The full complement of sterol-binding proteins that exist in mammalian cells, however, remains unknown. Here we describe a chemoproteomic strategy that uses clickable, photoreactive sterol probes in combination with quantitative mass spectrometry to globally map cholesterol-protein interactions directly in living cells. We identified over 250 cholesterol-binding proteins, including receptors, channels and enzymes involved in many established and previously unreported interactions. Prominent among the newly identified interacting proteins were enzymes that regulate sugars, glycerolipids and cholesterol itself as well as proteins involved in vesicular transport and protein glycosylation and degradation, pointing to key nodes in biochemical pathways that may couple sterol concentrations to the control of other metabolites and protein localization and modification.
ESTHER : Hulce_2013_Nat.Methods_10_259
PubMedSearch : Hulce_2013_Nat.Methods_10_259
PubMedID: 23396283

Title : Diced electrophoresis gel assay for screening enzymes with specified activities - Komatsu_2013_J.Am.Chem.Soc_135_6002
Author(s) : Komatsu T , Hanaoka K , Adibekian A , Yoshioka K , Terai T , Ueno T , Kawaguchi M , Cravatt BF , Nagano T
Ref : Journal of the American Chemical Society , 135 :6002 , 2013
Abstract : We have established the diced electrophoresis gel (DEG) assay as a proteome-wide screening tool to identify enzymes with activities of interest using turnover-based fluorescent substrates. The method utilizes the combination of native polyacrylamide gel electrophoresis (PAGE) with a multiwell-plate-based fluorometric assay to find protein spots with the specified activity. By developing fluorescent substrates that mimic the structure of neutrophil chemoattractants, we could identify enzymes involved in metabolic inactivation of the chemoattractants.
ESTHER : Komatsu_2013_J.Am.Chem.Soc_135_6002
PubMedSearch : Komatsu_2013_J.Am.Chem.Soc_135_6002
PubMedID: 23581642

Title : A novel fluorophosphonate inhibitor of the biosynthesis of the endocannabinoid 2-arachidonoylglycerol with potential anti-obesity effects - Bisogno_2013_Br.J.Pharmacol_169_784
Author(s) : Bisogno T , Mahadevan A , Coccurello R , Chang JW , Allara M , Chen Y , Giacovazzo G , Lichtman A , Cravatt BF , Moles A , Di Marzo V
Ref : British Journal of Pharmacology , 169 :784 , 2013
Abstract : BACKGROUND AND PURPOSE: The development of potent and selective inhibitors of the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG) via DAG lipases (DAGL) alpha and beta is just starting to be considered as a novel and promising source of pharmaceuticals for the treatment of disorders that might benefit from a reduction in endocannabinoid tone, such as hyperphagia in obese subjects. EXPERIMENTAL APPROACH: Three new fluorophosphonate compounds O-7458, O-7459 and O-7460 were synthesized and characterized in various enzymatic assays. The effects of O-7460 on high-fat diet intake were tested in mice. KEY
RESULTS: Of the new compounds, O-7460 exhibited the highest potency (IC50 = 690 nM) against the human recombinant DAGLalpha, and selectivity (IC50 > 10 muM) towards COS-7 cell and human monoacylglycerol lipase (MAGL), and rat brain fatty acid amide hydrolase. Competitive activity-based protein profiling confirmed that O-7460 inhibits mouse brain MAGL only at concentrations >/=10 muM, and showed that this compound has only one major 'off-target', that is, the serine hydrolase KIAA1363. O-7460 did not exhibit measurable affinity for human recombinant CB1 or CB2 cannabinoid receptors (Ki > 10 muM). In mouse neuroblastoma N18TG2 cells stimulated with ionomycin, O-7460 (10 muM) reduced 2-AG levels. When administered to mice, O-7460 dose-dependently (0-12 , i.p.) inhibited the intake of a high-fat diet over a 14 h observation period, and, subsequently, slightly but significantly reduced body weight. CONCLUSIONS AND IMPLICATIONS: O-7460 might be considered a useful pharmacological tool to investigate further the role played by 2-AG both in vitro and in vivo under physiological as well as pathological conditions. LINKED ARTICLES: This article is part of a themed section on Cannabinoids. To view the other articles in this section visit http:\/\/ & http:\/\/
ESTHER : Bisogno_2013_Br.J.Pharmacol_169_784
PubMedSearch : Bisogno_2013_Br.J.Pharmacol_169_784
PubMedID: 23072382
Gene_locus related to this paper: human-DAGLA , human-DAGLB

Title : Chemoproteomic discovery of AADACL1 as a regulator of human platelet activation - Holly_2013_Chem.Biol_20_1125
Author(s) : Holly SP , Chang JW , Li W , Niessen S , Phillips RM , Piatt R , Black JL , Smith MC , Boulaftali Y , Weyrich AS , Bergmeier W , Cravatt BF , Parise LV
Ref : Chemical Biology , 20 :1125 , 2013
Abstract : A comprehensive knowledge of the platelet proteome is necessary for understanding thrombosis and for envisioning antiplatelet therapies. To discover other biochemical pathways in human platelets, we screened platelets with a carbamate library designed to interrogate the serine hydrolase subproteome and used competitive activity-based protein profiling to map the targets of active carbamates. We identified an inhibitor that targets arylacetamide deacetylase-like 1 (AADACL1), a lipid deacetylase originally identified in invasive cancers. Using this compound, along with highly selective second-generation inhibitors of AADACL1, metabolomics, and RNA interference, we show that AADACL1 regulates platelet aggregation, thrombus growth, RAP1 and PKC activation, lipid metabolism, and fibrinogen binding to platelets and megakaryocytes. These data provide evidence that AADACL1 regulates platelet and megakaryocyte activation and highlight the value of this chemoproteomic strategy for target discovery in platelets.
ESTHER : Holly_2013_Chem.Biol_20_1125
PubMedSearch : Holly_2013_Chem.Biol_20_1125
PubMedID: 23993462
Gene_locus related to this paper: human-NCEH1

Title : Discovery and optimization of piperidyl-1,2,3-triazole ureas as potent, selective, and in vivo-active inhibitors of alpha\/beta-hydrolase domain containing 6 (ABHD6) - Hsu_2013_J.Med.Chem_56_8270
Author(s) : Hsu KL , Tsuboi K , Chang JW , Whitby LR , Speers AE , Pugh H , Cravatt BF
Ref : Journal of Medicinal Chemistry , 56 :8270 , 2013
Abstract : alpha/beta-Hydrolase domain containing 6 (ABHD6) is a transmembrane serine hydrolase that hydrolyzes the endogenous cannabinoid 2-arachidonoylglycerol (2-AG) to regulate certain forms of cannabinoid receptor-dependent signaling in the nervous system. The full spectrum of ABHD6 metabolic activities and functions is currently unknown and would benefit from selective, in vivo-active inhibitors. Here, we report the development and characterization of an advanced series of irreversible (2-substituted)-piperidyl-1,2,3-triazole urea inhibitors of ABHD6, including compounds KT182 and KT203, which show exceptional potency and selectivity in cells (<5 nM) and, at equivalent doses in mice (1 mg kg(-1)), act as systemic and peripherally restricted ABHD6 inhibitors, respectively. We also describe an orally bioavailable ABHD6 inhibitor, KT185, that displays excellent selectivity against other brain and liver serine hydrolases in vivo. We thus describe several chemical probes for biological studies of ABHD6, including brain-penetrant and peripherally restricted inhibitors that should prove of value for interrogating ABHD6 function in animal models.
ESTHER : Hsu_2013_J.Med.Chem_56_8270
PubMedSearch : Hsu_2013_J.Med.Chem_56_8270
PubMedID: 24152295

Title : The serine hydrolase ABHD6 Is a critical regulator of the metabolic syndrome - Thomas_2013_Cell.Rep_5_508
Author(s) : Thomas G , Betters JL , Lord CC , Brown AL , Marshall S , Ferguson D , Sawyer J , Davis MA , Melchior JT , Blume LC , Howlett AC , Ivanova PT , Milne SB , Myers DS , Mrak I , Leber V , Heier C , Taschler U , Blankman JL , Cravatt BF , Lee RG , Crooke RM , Graham MJ , Zimmermann R , Brown HA , Brown JM
Ref : Cell Rep , 5 :508 , 2013
Abstract : The serine hydrolase alpha/beta hydrolase domain 6 (ABHD6) has recently been implicated as a key lipase for the endocannabinoid 2-arachidonylglycerol (2-AG) in the brain. However, the biochemical and physiological function for ABHD6 outside of the central nervous system has not been established. To address this, we utilized targeted antisense oligonucleotides (ASOs) to selectively knock down ABHD6 in peripheral tissues in order to identify in vivo substrates and understand ABHD6's role in energy metabolism. Here, we show that selective knockdown of ABHD6 in metabolic tissues protects mice from high-fat-diet-induced obesity, hepatic steatosis, and systemic insulin resistance. Using combined in vivo lipidomic identification and in vitro enzymology approaches, we show that ABHD6 can hydrolyze several lipid substrates, positioning ABHD6 at the interface of glycerophospholipid metabolism and lipid signal transduction. Collectively, these data suggest that ABHD6 inhibitors may serve as therapeutics for obesity, nonalcoholic fatty liver disease, and type II diabetes.
ESTHER : Thomas_2013_Cell.Rep_5_508
PubMedSearch : Thomas_2013_Cell.Rep_5_508
PubMedID: 24095738
Gene_locus related to this paper: human-ABHD6

Title : Development and optimization of piperidyl-1,2,3-triazole ureas as selective chemical probes of endocannabinoid biosynthesis - Hsu_2013_J.Med.Chem_56_8257
Author(s) : Hsu KL , Tsuboi K , Whitby LR , Speers AE , Pugh H , Inloes J , Cravatt BF
Ref : Journal of Medicinal Chemistry , 56 :8257 , 2013
Abstract : We have previously shown that 1,2,3-triazole ureas (1,2,3-TUs) act as versatile class of irreversible serine hydrolase inhibitors that can be tuned to create selective probes for diverse members of this large enzyme class, including diacylglycerol lipase-beta (DAGLbeta), a principal biosynthetic enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG). Here, we provide a detailed account of the discovery, synthesis, and structure-activity relationship (SAR) of (2-substituted)-piperidyl-1,2,3-TUs that selectively inactivate DAGLbeta in living systems. Key to success was the use of activity-based protein profiling (ABPP) with broad-spectrum and tailored activity-based probes to guide our medicinal chemistry efforts. We also describe an expanded repertoire of DAGL-tailored activity-based probes that includes biotinylated and alkyne agents for enzyme enrichment coupled with mass spectrometry-based proteomics and assessment of proteome-wide selectivity. Our findings highlight the broad utility of 1,2,3-TUs for serine hydrolase inhibitor development and their application to create selective probes of endocannabinoid biosynthetic pathways.
ESTHER : Hsu_2013_J.Med.Chem_56_8257
PubMedSearch : Hsu_2013_J.Med.Chem_56_8257
PubMedID: 24152245
Gene_locus related to this paper: human-DAGLB

Title : Selective inhibitors and tailored activity probes for lipoprotein-associated phospholipase A(2) - Nagano_2013_Bioorg.Med.Chem.Lett_23_839
Author(s) : Nagano JM , Hsu KL , Whitby LR , Niphakis MJ , Speers AE , Brown SJ , Spicer T , Fernandez-Vega V , Ferguson J , Hodder P , Srinivasan P , Gonzalez TD , Rosen H , Bahnson BJ , Cravatt BF
Ref : Bioorganic & Medicinal Chemistry Lett , 23 :839 , 2013
Abstract : Lipoprotein-associated phospholipase A(2) (Lp-PLA(2) or PLA(2)G7) binds to low-density lipoprotein (LDL) particles, where it is thought to hydrolyze oxidatively truncated phospholipids. Lp-PLA(2) has also been implicated as a pro-tumorigenic enzyme in human prostate cancer. Several inhibitors of Lp-PLA(2) have been described, including darapladib, which is currently in phase 3 clinical development for the treatment of atherosclerosis. The selectivity that darapladib and other Lp-PLA(2) inhibitors display across the larger serine hydrolase family has not, however, been reported. Here, we describe the use of both general and tailored activity-based probes for profiling Lp-PLA(2) and inhibitors of this enzyme in native biological systems. We show that both darapladib and a novel class of structurally distinct carbamate inhibitors inactivate Lp-PLA(2) in mouse tissues and human cell lines with high selectivity. Our findings thus identify both inhibitors and chemoproteomic probes that are suitable for investigating Lp-PLA(2) function in biological systems.
ESTHER : Nagano_2013_Bioorg.Med.Chem.Lett_23_839
PubMedSearch : Nagano_2013_Bioorg.Med.Chem.Lett_23_839
PubMedID: 23260346

Title : Proteome-wide reactivity profiling identifies diverse carbamate chemotypes tuned for serine hydrolase inhibition - Chang_2013_ACS.Chem.Biol_8_1590
Author(s) : Chang JW , Cognetta AB, 3rd , Niphakis MJ , Cravatt BF
Ref : ACS Chemical Biology , 8 :1590 , 2013
Abstract : Serine hydrolases are one of the largest and most diverse enzyme classes in Nature. Inhibitors of serine hydrolases are used to treat many diseases, including obesity, diabetes, cognitive dementia, and bacterial and viral infections. Nonetheless, the majority of the 200+ serine hydrolases in mammals still lack selective inhibitors for their functional characterization. We and others have shown that activated carbamates, through covalent reaction with the conserved serine nucleophile of serine hydrolases, can serve as useful inhibitors for members of this enzyme family. The extent to which carbamates, however, cross-react with other protein classes remains mostly unexplored. Here, we address this problem by investigating the proteome-wide reactivity of a diverse set of activated carbamates in vitro and in vivo, using a combination of competitive and click chemistry (CC)-activity-based protein profiling (ABPP). We identify multiple classes of carbamates, including O-aryl, O-hexafluoroisopropyl (HFIP), and O-N-hydroxysuccinimidyl (NHS) carbamates that react selectively with serine hydrolases across entire mouse tissue proteomes in vivo. We exploit the proteome-wide specificity of HFIP carbamates to create in situ imaging probes for the endocannabinoid hydrolases monoacylglycerol lipase (MAGL) and alpha-beta hydrolase-6 (ABHD6). These findings, taken together, designate the carbamate as a privileged reactive group for serine hydrolases that can accommodate diverse structural modifications to produce inhibitors that display exceptional potency and selectivity across the mammalian proteome.
ESTHER : Chang_2013_ACS.Chem.Biol_8_1590
PubMedSearch : Chang_2013_ACS.Chem.Biol_8_1590
PubMedID: 23701408

Title : Two Novel Mutations in ABHD12: Expansion of the Mutation Spectrum in PHARC and Assessment of Their Functional Effects - Chen_2013_Hum.Mutat_34_1672
Author(s) : Chen DH , Naydenov A , Blankman JL , Mefford HC , Davis M , Sul Y , Barloon AS , Bonkowski E , Wolff J , Matsushita M , Smith C , Cravatt BF , Mackie K , Raskind WH , Stella N , Bird TD
Ref : Hum Mutat , 34 :1672 , 2013
Abstract : PHARC (polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataracts) is a recently described autosomal-recessive neurodegenerative disease caused by mutations in the alpha-beta-hydrolase domain-containing 12 gene (ABHD12). Only five homozygous ABHD12 mutations have been reported and the pathogenesis of PHARC remains unclear. We evaluated a woman who manifested short stature as well as the typical features of PHARC. Sequence analysis of ABHD12 revealed a novel heterozygous c.1129A>T (p.Lys377*) mutation. Targeted comparative genomic hybridization detected a 59-kb deletion that encompasses exon 1 of ABHD12 and exons 1-4 of an adjacent gene, GINS1, and includes the promoters of both genes. The heterozygous deletion was also carried by the patient's asymptomatic mother. Quantitative reverse transcription-PCR demonstrated approximately 50% decreased expression of ABHD12 RNA in lymphoblastoid cell lines from both individuals. Activity-based protein profiling of serine hydrolases revealed absence of ABHD12 hydrolase activity in the patient and 50% reduction in her mother. This is the first report of compound heterozygosity in PHARC and the first study to describe how a mutation might affect ABHD12 expression and function. The possible involvement of haploinsufficiency for GINS1, a DNA replication complex protein, in the short stature of the patient and her mother requires further studies.
ESTHER : Chen_2013_Hum.Mutat_34_1672
PubMedSearch : Chen_2013_Hum.Mutat_34_1672
PubMedID: 24027063
Gene_locus related to this paper: human-ABHD12

Title : ABHD12 controls brain lysophosphatidylserine pathways that are deregulated in a murine model of the neurodegenerative disease PHARC - Blankman_2013_Proc.Natl.Acad.Sci.U.S.A_110_1500
Author(s) : Blankman JL , Long JZ , Trauger SA , Siuzdak G , Cravatt BF
Ref : Proc Natl Acad Sci U S A , 110 :1500 , 2013
Abstract : Advances in human genetics are leading to the discovery of new disease-causing mutations at a remarkable rate. Many such mutations, however, occur in genes that encode for proteins of unknown function, which limits our molecular understanding of, and ability to devise treatments for, human disease. Here, we use untargeted metabolomics combined with a genetic mouse model to determine that the poorly characterized serine hydrolase alpha/beta-hydrolase domain-containing (ABHD)12, mutations in which cause the human neurodegenerative disorder PHARC (polyneuropathy, hearing loss, ataxia, retinosis pigmentosa, and cataract), is a principal lysophosphatidylserine (LPS) lipase in the mammalian brain. ABHD12(-/-) mice display massive increases in a rare set of very long chain LPS lipids that have been previously reported as Toll-like receptor 2 activators. We confirm that recombinant ABHD12 protein exhibits robust LPS lipase activity, which is also substantially reduced in ABHD12(-/-) brain tissue. Notably, elevations in brain LPS lipids in ABHD12(-/-) mice occur early in life (2-6 mo) and are followed by age-dependent increases in microglial activation and auditory and motor defects that resemble the behavioral phenotypes of human PHARC patients. Taken together, our data provide a molecular model for PHARC, where disruption of ABHD12 causes deregulated LPS metabolism and the accumulation of proinflammatory lipids that promote microglial and neurobehavioral abnormalities.
ESTHER : Blankman_2013_Proc.Natl.Acad.Sci.U.S.A_110_1500
PubMedSearch : Blankman_2013_Proc.Natl.Acad.Sci.U.S.A_110_1500
PubMedID: 23297193

Title : Evaluation of NHS carbamates as a potent and selective class of endocannabinoid hydrolase inhibitors - Niphakis_2013_ACS.Chem.Neurosci_4_1322
Author(s) : Niphakis MJ , Cognetta AB, 3rd , Chang JW , Buczynski MW , Parsons LH , Byrne F , Burston JJ , Chapman V , Cravatt BF
Ref : ACS Chem Neurosci , 4 :1322 , 2013
Abstract : Monoacylglycerol lipase (MAGL) is a principal metabolic enzyme responsible for hydrolyzing the endogenous cannabinoid (endocannabinoid) 2-arachidonoylglycerol (2-AG). Selective inhibitors of MAGL offer valuable probes to further understand the enzyme's function in biological systems and may lead to drugs for treating a variety of diseases, including psychiatric disorders, neuroinflammation, and pain. N-Hydroxysuccinimidyl (NHS) carbamates have recently been identified as a promising class of serine hydrolase inhibitors that shows minimal cross-reactivity with other proteins in the proteome. Here, we explore NHS carbamates more broadly and demonstrate their potential as inhibitors of endocannabinoid hydrolases and additional enzymes from the serine hydrolase class. We extensively characterize an NHS carbamate 1a (MJN110) as a potent, selective, and in-vivo-active MAGL inhibitor. Finally, we demonstrate that MJN110 alleviates mechanical allodynia in a rat model of diabetic neuropathy, marking NHS carbamates as a promising class of MAGL inhibitors.
ESTHER : Niphakis_2013_ACS.Chem.Neurosci_4_1322
PubMedSearch : Niphakis_2013_ACS.Chem.Neurosci_4_1322
PubMedID: 23731016
Gene_locus related to this paper: human-MGLL

Title : Dual inhibition of endocannabinoid catabolic enzymes produces enhanced antiwithdrawal effects in morphine-dependent mice - Ramesh_2013_Neuropsychopharmacology_38_1039
Author(s) : Ramesh D , Gamage TF , Vanuytsel T , Owens RA , Abdullah RA , Niphakis MJ , Shea-Donohue T , Cravatt BF , Lichtman AH
Ref : Neuropsychopharmacology , 38 :1039 , 2013
Abstract : Inhibition of the endocannabinoid catabolic enzymes, monoacylglycerol lipase (MAGL) or fatty acid amide hydrolase (FAAH) attenuates naloxone-precipitated opioid withdrawal signs in mice via activation of CB1 receptors. Complete FAAH inhibition blocks only a subset of withdrawal signs, whereas complete MAGL inhibition elicits enhanced antiwithdrawal efficacy, but is accompanied with some cannabimimetic side effects. Thus, the primary objective of the present study was to determine whether combined, full FAAH inhibition and partial MAGL represents an optimal strategy to reduce opioid withdrawal. To test this hypothesis, we examined whether combined administration of high-dose of the FAAH inhibitor PF-3845 and low-dose of the MAGL inhibitor JZL184, as well as the novel dual FAAH-MAGL inhibitor SA-57, which is 100-fold more potent in inhibiting FAAH than MAGL, would prevent spontaneous withdrawal in morphine-dependent mice, a model with greater face validity than precipitating withdrawal with mu-opioid receptor antagonists. Strikingly, a combination of low-dose JZL184 and high-dose PF-3845 as well as the dual inhibitor SA-57 reduced all abrupt withdrawal signs (ie, platform jumping, paw flutters, head shakes, diarrhea, and total body weight loss), but did not elicit any cannabimimetic side effects. In addition, JZL184 or PF-3845 blocked naloxone-precipitated hypersecretion in morphine-dependent small intestinal tissue. Collectively, these results are the first to show that endocannabinoid catabolic enzyme inhibitors reduce abrupt withdrawal in morpine-dependent mice and are effective in a novel in vitro model of opioid withdrawal. More generally, these findings support the idea that joint MAGL and FAAH inhibition represents a promising approach for the treatment of opioid dependence.
ESTHER : Ramesh_2013_Neuropsychopharmacology_38_1039
PubMedSearch : Ramesh_2013_Neuropsychopharmacology_38_1039
PubMedID: 23303065
Gene_locus related to this paper: human-MGLL

Title : Monoacylglycerol lipase controls endocannabinoid and eicosanoid signaling and hepatic injury in mice - Cao_2013_Gastroenterology_144_808
Author(s) : Cao Z , Mulvihill MM , Mukhopadhyay P , Xu H , Erdelyi K , Hao E , Holovac E , Hasko G , Cravatt BF , Nomura DK , Pacher P
Ref : Gastroenterology , 144 :808 , 2013
Abstract : BACKGROUND & AIMS: The endocannabinoid and eicosanoid lipid signaling pathways have important roles in inflammatory syndromes. Monoacylglycerol lipase (MAGL) links these pathways, hydrolyzing the endocannabinoid 2-arachidonoylglycerol to generate the arachidonic acid precursor pool for prostaglandin production. We investigated whether blocking MAGL protects against inflammation and damage from hepatic ischemia/reperfusion (I/R) and other insults.
METHODS: We analyzed the effects of hepatic I/R in mice given the selective MAGL inhibitor JZL184, in Mgll(-/-) mice, fatty acid amide hydrolase(-/-) mice, and in cannabinoid receptor type 1(-/-) (CB1-/-) and cannabinoid receptor type 2(-/-) (CB2-/-). Liver tissues were collected and analyzed, along with cultured hepatocytes and Kupffer cells. We measured endocannabinoids, eicosanoids, and markers of inflammation, oxidative stress, and cell death using molecular biology, biochemistry, and mass spectrometry analyses.
RESULTS: Wild-type mice given JZL184 and Mgll(-/-) mice were protected from hepatic I/R injury by a mechanism that involved increased endocannabinoid signaling via CB2 and reduced production of eicosanoids in the liver. JZL184 suppressed the inflammation and oxidative stress that mediate hepatic I/R injury. Hepatocytes were the major source of hepatic MAGL activity and endocannabinoid and eicosanoid production. JZL184 also protected from induction of liver injury by D-(+)-galactosamine and lipopolysaccharides or CCl4.
CONCLUSIONS: MAGL modulates hepatic injury via endocannabinoid and eicosanoid signaling; blockade of this pathway protects mice from liver injury. MAGL inhibitors might be developed to treat conditions that expose the liver to oxidative stress and inflammatory damage.
ESTHER : Cao_2013_Gastroenterology_144_808
PubMedSearch : Cao_2013_Gastroenterology_144_808
PubMedID: 23295443
Gene_locus related to this paper: human-MGLL

Title : Chemical probes of endocannabinoid metabolism - Blankman_2013_Pharmacol.Rev_65_849
Author(s) : Blankman JL , Cravatt BF
Ref : Pharmacol Rev , 65 :849 , 2013
Abstract : The endocannabinoid signaling system regulates diverse physiologic processes and has attracted considerable attention as a potential pharmaceutical target for treating diseases, such as pain, anxiety/depression, and metabolic disorders. The principal ligands of the endocannabinoid system are the lipid transmitters N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG), which activate the two major cannabinoid receptors, CB1 and CB2. Anandamide and 2-AG signaling pathways in the nervous system are terminated by enzymatic hydrolysis mediated primarily by the serine hydrolases fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. In this review, we will discuss the development of FAAH and MAGL inhibitors and their pharmacological application to investigate the function of anandamide and 2-AG signaling pathways in preclinical models of neurobehavioral processes, such as pain, anxiety, and addiction. We will place emphasis on how these studies are beginning to discern the different roles played by anandamide and 2-AG in the nervous system and the resulting implications for advancing endocannabinoid hydrolase inhibitors as next-generation therapeutics.
ESTHER : Blankman_2013_Pharmacol.Rev_65_849
PubMedSearch : Blankman_2013_Pharmacol.Rev_65_849
PubMedID: 23512546
Gene_locus related to this paper: human-MGLL

Title : The monoacylglycerol lipase inhibitor JZL184 suppresses inflammatory pain in the mouse carrageenan model - Ghosh_2013_Life.Sci_92_498
Author(s) : Ghosh S , Wise LE , Chen Y , Gujjar R , Mahadevan A , Cravatt BF , Lichtman AH
Ref : Life Sciences , 92 :498 , 2013
Abstract : AIM: The present study tested whether the selective monoacylglycerol lipase (MAGL) inhibitor JZL184 would reduce allodynia and paw edema in the carrageenan test. MAIN
METHODS: The anti-edematous and anti-allodynic effects of JZL184 were compared to those of PF-3845, an inhibitor of fatty acid amide hydrolase (FAAH), and diclofenac, a non-selective cyclooxygenase inhibitor. Cannabinoid receptor involvement in the anti-edematous and anti-allodynic effects of JZL184 was evaluated by administration of the respective CB1 and CB2 receptor antagonists rimonabant and SR144528 as well as with CB1(-/-) and CB2(-/-) mice. JZL184 (1.6, 4, 16, or 40mg/kg) was administered for six days to assess tolerance. KEY FINDINGS: JZL184 administered before or after carrageenan significantly attenuated carrageenan-induced paw edema and mechanical allodynia. Complementary genetic and pharmacological approaches revealed that the anti-allodynic effects of JZL184 required both CB1 and CB2 receptors, but only CB2 receptors mediated its anti-edematous actions. Importantly, both the anti-edematous and anti-allodynic effects underwent tolerance following repeated injections of high dose JZL184 (16 or 40mg/kg), but repeated administration of low dose JZL184 (4mg/kg) retained efficacy. SIGNIFICANCE: These results suggest that the MAGL inhibitor JZL184 reduces inflammatory nociception through the activation of both CB1 and CB2 receptors, with no evidence of tolerance following repeated administration of low doses.
ESTHER : Ghosh_2013_Life.Sci_92_498
PubMedSearch : Ghosh_2013_Life.Sci_92_498
PubMedID: 22749865
Gene_locus related to this paper: human-MGLL

Title : Confirming target engagement for reversible inhibitors in vivo by kinetically tuned activity-based probes - Adibekian_2012_J.Am.Chem.Soc_134_10345
Author(s) : Adibekian A , Martin BR , Chang JW , Hsu KL , Tsuboi K , Bachovchin DA , Speers AE , Brown SJ , Spicer T , Fernandez-Vega V , Ferguson J , Hodder PS , Rosen H , Cravatt BF
Ref : J Am Chem Soc , 134 :10345 , 2012
Abstract : The development of small-molecule inhibitors for perturbing enzyme function requires assays to confirm that the inhibitors interact with their enzymatic targets in vivo. Determining target engagement in vivo can be particularly challenging for poorly characterized enzymes that lack known biomarkers (e.g., endogenous substrates and products) to report on their inhibition. Here, we describe a competitive activity-based protein profiling (ABPP) method for measuring the binding of reversible inhibitors to enzymes in animal models. Key to the success of this approach is the use of activity-based probes that show tempered rates of reactivity with enzymes, such that competition for target engagement with reversible inhibitors can be measured in vivo. We apply the competitive ABPP strategy to evaluate a newly described class of piperazine amide reversible inhibitors for the serine hydrolases LYPLA1 and LYPLA2, two enzymes for which selective, in vivo active inhibitors are lacking. Competitive ABPP identified individual piperazine amides that selectively inhibit LYPLA1 or LYPLA2 in mice. In summary, competitive ABPP adapted to operate with moderately reactive probes can assess the target engagement of reversible inhibitors in animal models to facilitate the discovery of small-molecule probes for characterizing enzyme function in vivo.
ESTHER : Adibekian_2012_J.Am.Chem.Soc_134_10345
PubMedSearch : Adibekian_2012_J.Am.Chem.Soc_134_10345
PubMedID: 22690931

Title : The pharmacological landscape and therapeutic potential of serine hydrolases - Bachovchin_2012_Nat.Rev.Drug.Discov_11_52
Author(s) : Bachovchin DA , Cravatt BF
Ref : Nat Rev Drug Discov , 11 :52 , 2012
Abstract : Serine hydrolases perform crucial roles in many biological processes, and several of these enzymes are targets of approved drugs for indications such as type 2 diabetes, Alzheimer's disease and infectious diseases. Despite this, most of the human serine hydrolases (of which there are more than 200) remain poorly characterized with respect to their physiological substrates and functions, and the vast majority lack selective, in vivo-active inhibitors. Here, we review the current state of pharmacology for mammalian serine hydrolases, including marketed drugs, compounds that are under clinical investigation and selective inhibitors emerging from academic probe development efforts. We also highlight recent methodological advances that have accelerated the rate of inhibitor discovery and optimization for serine hydrolases, which we anticipate will aid in their biological characterization and, in some cases, therapeutic validation.
ESTHER : Bachovchin_2012_Nat.Rev.Drug.Discov_11_52
PubMedSearch : Bachovchin_2012_Nat.Rev.Drug.Discov_11_52
PubMedID: 22212679

Title : Selective inhibition of plant serine hydrolases by agrochemicals revealed by competitive ABPP - Kaschani_2012_Bioorg.Med.Chem_20_597
Author(s) : Kaschani F , Nickel S , Pandey B , Cravatt BF , Kaiser M , van der Hoorn RA
Ref : Bioorganic & Medicinal Chemistry , 20 :597 , 2012
Abstract : Organophosphate and -phosphonates and their thio derivatives are often used in agroindustry as herbicides and insecticides, but their potential off-targets in the plant are poorly investigated. Here, we use competitive activity-based protein profiling (ABPP) of serine hydrolases (SHs) to detect targets of these agrochemicals and other compounds in Arabidopsis thaliana. Using broad-range and specific probes, and by overexpression of various SHs in planta, we are able to confirm eight SH-compound interactions, including selective inhibition of carboxylesterase CXE12, prolyloligopeptidase, methylesterase MES2 and tripeptidyl peptidase TPP2. These observations can be used for the design of novel probes and selective inhibitors and may help to assess physiological effects of agrochemicals on crop plants.
ESTHER : Kaschani_2012_Bioorg.Med.Chem_20_597
PubMedSearch : Kaschani_2012_Bioorg.Med.Chem_20_597
PubMedID: 21764588

Title : DAGLbeta inhibition perturbs a lipid network involved in macrophage inflammatory responses - Hsu_2012_Nat.Chem.Biol_8_999
Author(s) : Hsu KL , Tsuboi K , Adibekian A , Pugh H , Masuda K , Cravatt BF
Ref : Nat Chemical Biology , 8 :999 , 2012
Abstract : The endocannabinoid 2-arachidonoylglycerol (2-AG) is biosynthesized by diacylglycerol lipases DAGLalpha and DAGLbeta. Chemical probes to perturb DAGLs are needed to characterize endocannabinoid function in biological processes. Here we report a series of 1,2,3-triazole urea inhibitors, along with paired negative-control and activity-based probes, for the functional analysis of DAGLbeta in living systems. Optimized inhibitors showed high selectivity for DAGLbeta over other serine hydrolases, including DAGLalpha ( approximately 60-fold selectivity), and the limited off-targets, such as ABHD6, were also inhibited by the negative-control probe. Using these agents and Daglb(-/-) mice, we show that DAGLbeta inactivation lowers 2-AG, as well as arachidonic acid and eicosanoids, in mouse peritoneal macrophages in a manner that is distinct and complementary to disruption of cytosolic phospholipase-A2. We observed a corresponding reduction in lipopolysaccharide-induced tumor necrosis factor-alpha release. These findings indicate that DAGLbeta is a key metabolic hub within a lipid network that regulates proinflammatory responses in macrophages.
ESTHER : Hsu_2012_Nat.Chem.Biol_8_999
PubMedSearch : Hsu_2012_Nat.Chem.Biol_8_999
PubMedID: 23103940
Gene_locus related to this paper: human-DAGLA , human-DAGLB

Title : Global profiling of dynamic protein palmitoylation - Martin_2012_Nat.Methods_9_84
Author(s) : Martin BR , Wang C , Adibekian A , Tully SE , Cravatt BF
Ref : Nat Methods , 9 :84 , 2012
Abstract : The reversible thioester linkage of palmitic acid on cysteines, known as protein S-palmitoylation, facilitates the membrane association and proper subcellular localization of proteins. Here we report the metabolic incorporation of the palmitic acid analog 17-octadecynoic acid (17-ODYA) in combination with stable-isotope labeling with amino acids in cell culture (SILAC) and pulse-chase methods to generate a global quantitative map of dynamic protein palmitoylation events in cells. We distinguished stably palmitoylated proteins from those that turn over rapidly. Treatment with a serine lipase-selective inhibitor identified a pool of dynamically palmitoylated proteins regulated by palmitoyl-protein thioesterases. This subset was enriched in oncoproteins and other proteins linked to aberrant cell growth, migration and cancer. Our method provides a straightforward way to characterize global palmitoylation dynamics in cells and confirms enzyme-mediated depalmitoylation as a critical regulatory mechanism for a specific subset of rapidly cycling palmitoylated proteins.
ESTHER : Martin_2012_Nat.Methods_9_84
PubMedSearch : Martin_2012_Nat.Methods_9_84
PubMedID: 22056678

Title : Optimization and characterization of a triazole urea inhibitor for diacylglycerol lipase beta (DAGL-beta) - Hsu_2012_Probe.Report__
Author(s) : Hsu KL , Tsuboi K , Speers AE , Brown SJ , Spicer T , Fernandez-Vega V , Ferguson J , Cravatt BF , Hodder P , Rosen H
Ref : Probe Report , : , 2012
Abstract : Endocannabinoids (ECs) are a unique group of lipids that function as chemical messengers in the nervous system. The two principle ECs thus far identified in mammals are N-arachidonoyl-ethanolamine (anandamide) and 2-arachidonoyl-glycerol (2-AG). These compounds have been implicated in various physiological and pathological functions including appetite, pain, sensation, memory, and addiction. Because ECs are synthesized and released on demand and then rapidly degraded to terminate signaling, the metabolic pathways that govern EC turnover directly influence the magnitude and duration of neuronal signaling events. There is strong evidence that two serine hydrolases, diacylglycerol lipase-alpha and -beta (DAGL-alpha and -beta) function as 2-AG synthetic enzymes both in vitro and in vivo. However, because constitutive gene disruption, the only currently available means to investigate DAGL-alpha/beta biology due to a lack of selective chemical inhibitors, can result in compensatory effects and network-wide changes, there is still uncertainty surrounding the extent to which DAGL-alpha/beta contribute to 2-AG-mediated signaling. In an effort to provide chemical tools for manipulation of DAGL-beta activity, we initiated a competitive activity-based protein profiling (ABPP) screen of triazole urea compounds to identify selective enzyme inhibitors. This campaign, made possible by previous inhibitor development efforts for LYPLA1/2 (ML211), PAFAH2 (ML225), and ABHD11 (ML226) based on the triazole urea scaffold, yielded the medchem optimized probe ML294 (SID 125269120). ML294 is highly potent against its target enzyme (IC50 = 56 nM in vitro; 12 nM in situ), and is active in vivo, showing both oral bioavailability and blood-brain barrier penetration. Out of more than 20 serine hydrolases (SHs) profiled by gel-based competitive ABPP, ML294 is observed to have one anti-target, alpha/beta hydrolase domain-containing protein 6 (ABHD6). Otherwise, ML294 is at least 35-fold selective for all other brain SHs (approximately 20) assessed by gel-based competitive ABPP and 7-fold selective vs. its closest homolog, DAGL-alpha. To control for ABHD6-directed activity in biological studies, we also developed a structurally related ABHD6-selective control "anti-probe", ML295, also based on the triazole urea scaffold. The complete properties, characterization, and synthesis of ML294 are detailed in this report, and full details of ABHD6 inhibitors are detailed in the Probe Report for ML295 and ML296.
ESTHER : Hsu_2012_Probe.Report__
PubMedSearch : Hsu_2012_Probe.Report__
PubMedID: 23658950
Gene_locus related to this paper: human-ABHD6

Title : An activity-based imaging probe for the integral membrane hydrolase KIAA1363 -
Author(s) : Chang JW , Moellering RE , Cravatt BF
Ref : Angew Chem Int Ed Engl , 51 :966 , 2012
PubMedID: 22162147
Gene_locus related to this paper: human-NCEH1

Title : Inhibition of monoacylglycerol lipase attenuates vomiting in Suncus murinus and 2-arachidonoyl glycerol attenuates nausea in rats - Sticht_2012_Br.J.Pharmacol_165_2425
Author(s) : Sticht MA , Long JZ , Rock EM , Limebeer CL , Mechoulam R , Cravatt BF , Parker LA
Ref : British Journal of Pharmacology , 165 :2425 , 2012
Abstract : BACKGROUND AND PURPOSE: To evaluate the role of 2-arachidonoyl glycerol (2AG) in the regulation of nausea and vomiting using animal models of vomiting and of nausea-like behaviour (conditioned gaping). EXPERIMENTAL APPROACH: Vomiting was assessed in shrews (Suncus murinus), pretreated with JZL184, a selective monoacylglycerol lipase (MAGL) inhibitor which elevates endogenous 2AG levels, 1 h before administering the emetogenic compound, LiCl. Regulation of nausea-like behaviour in rats by exogenous 2AG or its metabolite arachidonic acid (AA) was assessed, using the conditioned gaping model. The role of cannabinoid CB(1) receptors, CB(2) receptors and cyclooxygenase (COX) inhibition in suppression of vomiting or nausea-like behaviour was assessed. KEY
RESULTS: JZL184 dose-dependently suppressed vomiting in shrews, an effect prevented by pretreatment with the CB(1) receptor inverse agonist/antagonist, AM251. In shrew brain tissue, JZL184 inhibited MAGL activity in vivo. In rats, 2AG suppressed LiCl-induced conditioned gaping but this effect was not prevented by AM251 or the CB(2) receptor antagonist, AM630. Instead, the COX inhibitor, indomethacin, prevented suppression of conditioned gaping by 2AG or AA. However, when rats were pretreated with a high dose of JZL184 (40 ), suppression of gaping by 2AG was partially reversed by AM251. Suppression of conditioned gaping was not due to interference with learning because the same dose of 2AG did not modify the strength of conditioned freezing to a shock-paired tone. CONCLUSIONS AND IMPLICATIONS: Our results suggest that manipulations that elevate 2AG may have anti-emetic or anti-nausea potential. LINKED ARTICLES: This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit To view Part I of Cannabinoids in Biology and Medicine visit
ESTHER : Sticht_2012_Br.J.Pharmacol_165_2425
PubMedSearch : Sticht_2012_Br.J.Pharmacol_165_2425
PubMedID: 21470205

Title : Competitive activity-based protein profiling identifies aza-beta-lactams as a versatile chemotype for serine hydrolase inhibition - Zuhl_2012_J.Am.Chem.Soc_134_5068
Author(s) : Zuhl AM , Mohr JT , Bachovchin DA , Niessen S , Hsu KL , Berlin JM , Dochnahl M , Lopez-Alberca MP , Fu GC , Cravatt BF
Ref : J Am Chem Soc , 134 :5068 , 2012
Abstract : Serine hydrolases are one of the largest and most diverse enzyme classes in Nature. Most serine hydrolases lack selective inhibitors, which are valuable probes for assigning functions to these enzymes. We recently discovered a set of aza-beta-lactams (ABLs) that act as potent and selective inhibitors of the mammalian serine hydrolase protein-phosphatase methylesterase-1 (PME-1). The ABLs inactivate PME-1 by covalent acylation of the enzyme's serine nucleophile, suggesting that they could offer a general scaffold for serine hydrolase inhibitor discovery. Here, we have tested this hypothesis by screening ABLs more broadly against cell and tissue proteomes by competitive activity-based protein profiling (ABPP), leading to the discovery of lead inhibitors for several serine hydrolases, including the uncharacterized enzyme alpha,beta-hydrolase domain-containing 10 (ABHD10). ABPP-guided medicinal chemistry yielded a compound ABL303 that potently (IC(50) approximately 30 nM) and selectively inactivated ABHD10 in vitro and in living cells. A comparison of optimized inhibitors for PME-1 and ABHD10 indicates that modest structural changes that alter steric bulk can tailor the ABL to selectively react with distinct, distantly related serine hydrolases. Our findings, taken together, designate the ABL as a versatile reactive group for creating first-in-class serine hydrolase inhibitors.
ESTHER : Zuhl_2012_J.Am.Chem.Soc_134_5068
PubMedSearch : Zuhl_2012_J.Am.Chem.Soc_134_5068
PubMedID: 22400490
Gene_locus related to this paper: human-ABHD10

Title : Dual fatty acid amide hydrolase and monoacylglycerol lipase blockade produces THC-like Morris water maze deficits in mice - Wise_2012_ACS.Chem.Neurosci_3_369
Author(s) : Wise LE , Long KA , Abdullah RA , Long JZ , Cravatt BF , Lichtman AH
Ref : ACS Chem Neurosci , 3 :369 , 2012
Abstract : Acute administration of Delta(9)-tetrahydrocannabinol (THC) or exposure to marijuana smoke impairs short-term spatial memory in water maze tasks through a CB(1) receptor mechanism of action. N-Arachidonoylethanolamine (anandamide; AEA) and 2-arachidonoylglycerol (2-AG) are endogenous cannabinoids that are predominantly metabolized by the respective enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). Although the MAGL inhibitor JZL184 enhances short-term synaptic plasticity, it has yet to be evaluated in the Morris water maze. Previous research demonstrated that simultaneous, complete blockade of FAAH and MAGL produces full blown THC-like effects. Thus, in the following studies we tested whether dual blockade of FAAH and MAGL would impair learning in a repeated acquisition Morris water maze task. Mice treated with the dual FAAH/MAGL inhibitor JZL195 (20 mg/kg) as well as JZL184-treated FAAH -/- mice displayed robust deficits in Morris water maze performance that were similar in magnitude to THC-treated mice. While 20 or 40 mg/kg impaired water maze performance in FAAH -/- mice, only the high dose of JZL184 disrupted performance in FAAH +/+ mice. The memory impairing effects of JZL184 were blocked by the CB(1) receptor antagonist rimonabant. Neither JZL184 nor JZL195 impaired performance in a cued version of the water maze task, arguing against the notion that sensorimotor or motivational deficits accounted for the impaired acquisition performance. JZL184 increased 2-AG levels in the hippocampus, prefrontal cortex, and cerebellum to a similar degree in FAAH -/- and +/+ mice. FAAH -/- mice, regardless of drug treatment, possessed elevated AEA levels in each brain region assessed. The results of this study reveal that concomitant increases in AEA and 2-AG disrupt short-term spatial memory performance in a manner similar to that of THC.
ESTHER : Wise_2012_ACS.Chem.Neurosci_3_369
PubMedSearch : Wise_2012_ACS.Chem.Neurosci_3_369
PubMedID: 22860205
Gene_locus related to this paper: human-MGLL

Title : Elucidation and chemical modulation of sulfolipid-1 biosynthesis in Mycobacterium tuberculosis - Seeliger_2012_J.Biol.Chem_287_7990
Author(s) : Seeliger JC , Holsclaw CM , Schelle MW , Botyanszki Z , Gilmore SA , Tully SE , Niederweis M , Cravatt BF , Leary JA , Bertozzi CR
Ref : Journal of Biological Chemistry , 287 :7990 , 2012
Abstract : Mycobacterium tuberculosis possesses unique cell-surface lipids that have been implicated in virulence. One of the most abundant is sulfolipid-1 (SL-1), a tetraacyl-sulfotrehalose glycolipid. Although the early steps in SL-1 biosynthesis are known, the machinery underlying the final acylation reactions is not understood. We provide genetic and biochemical evidence for the activities of two proteins, Chp1 and Sap (corresponding to gene loci rv3822 and rv3821), that complete this pathway. The membrane-associated acyltransferase Chp1 accepts a synthetic diacyl sulfolipid and transfers an acyl group regioselectively from one donor substrate molecule to a second acceptor molecule in two successive reactions to yield a tetraacylated product. Chp1 is fully active in vitro, but in M. tuberculosis, its function is potentiated by the previously identified sulfolipid transporter MmpL8. We also show that the integral membrane protein Sap and MmpL8 are both essential for sulfolipid transport. Finally, the lipase inhibitor tetrahydrolipstatin disrupts Chp1 activity in M. tuberculosis, suggesting an avenue for perturbing SL-1 biosynthesis in vivo. These data complete the SL-1 biosynthetic pathway and corroborate a model in which lipid biosynthesis and transmembrane transport are coupled at the membrane-cytosol interface through the activity of multiple proteins, possibly as a macromolecular complex.
ESTHER : Seeliger_2012_J.Biol.Chem_287_7990
PubMedSearch : Seeliger_2012_J.Biol.Chem_287_7990
PubMedID: 22194604
Gene_locus related to this paper: myctu-Rv3822

Title : O-hydroxyacetamide carbamates as a highly potent and selective class of endocannabinoid hydrolase inhibitors - Niphakis_2012_ACS.Chem.Neurosci_3_418
Author(s) : Niphakis MJ , Johnson DS , Ballard TE , Stiff C , Cravatt BF
Ref : ACS Chem Neurosci , 3 :418 , 2012
Abstract : The two major endocannabinoid transmitters, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are degraded by distinct enzymes in the nervous system, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. FAAH and MAGL inhibitors cause elevations in brain AEA and 2-AG levels, respectively, and reduce pain, anxiety, and depression in rodents without causing the full spectrum of psychotropic behavioral effects observed with direct cannabinoid receptor-1 (CB1) agonists. These findings have inspired the development of several classes of endocannabinoid hydrolase inhibitors, most of which have been optimized to show specificity for either FAAH or MAGL or, in certain cases, equipotent activity for both enzymes. Here, we investigate an unusual class of O-hydroxyacetamide carbamate inhibitors and find that individual compounds from this class can serve as selective FAAH or dual FAAH/MAGL inhibitors in vivo across a dose range (0.125-12.5 mg kg(-1)) suitable for behavioral studies. Competitive and click chemistry activity-based protein profiling confirmed that the O-hydroxyacetamide carbamate SA-57 is remarkably selective for FAAH and MAGL in vivo, targeting only one other enzyme in brain, the additional 2-AG hydrolase ABHD6. These data designate O-hydroxyacetamide carbamates as a versatile chemotype for creating endocannabinoid hydrolase inhibitors that display excellent in vivo activity and tunable selectivity for FAAH-anandamide versus MAGL (and ABHD6)-2-AG pathways.
ESTHER : Niphakis_2012_ACS.Chem.Neurosci_3_418
PubMedSearch : Niphakis_2012_ACS.Chem.Neurosci_3_418
PubMedID: 22860211
Gene_locus related to this paper: human-MGLL

Title : Rapid development of a potent photo-triggered inhibitor of the serine hydrolase RBBP9 - Liu_2012_Chembiochem_13_2082
Author(s) : Liu X , Dix M , Speers AE , Bachovchin DA , Zuhl AM , Cravatt BF , Kodadek TJ
Ref : Chembiochem , 13 :2082 , 2012
Abstract : The serine hydrolases constitute a large class of enzymes that play important roles in physiology. There is great interest in the development of potent and selective pharmacological inhibitors of these proteins. Traditional active-site inhibitors often have limited selectivity within this superfamily and are tedious and expensive to discover. Using the serine hydrolase RBBP9 as a model target, we designed a rapid and relatively inexpensive route to highly selective peptoid-based inhibitors that can be activated by visible light. This technology provides rapid access to photo-activated tool compounds capable of selectively blocking the function of particular serine hydrolases.
ESTHER : Liu_2012_Chembiochem_13_2082
PubMedSearch : Liu_2012_Chembiochem_13_2082
PubMedID: 22907802
Gene_locus related to this paper: human-RBBP9

Title : Characterization of the effects of reuptake and hydrolysis inhibition on interstitial endocannabinoid levels in the brain: an in vivo microdialysis study - Wiskerke_2012_ACS.Chem.Neurosci_3_407
Author(s) : Wiskerke J , Irimia C , Cravatt BF , De Vries TJ , Schoffelmeer AN , Pattij T , Parsons LH
Ref : ACS Chem Neurosci , 3 :407 , 2012
Abstract : The present experiments employed in vivo microdialysis to characterize the effects of commonly used endocannabinoid clearance inhibitors on basal and depolarization-induced alterations in interstitial endocannabinoid levels in the nucleus accumbens of rat brain. Compounds targeting the putative endocannabinoid transporter and hydrolytic enzymes (FAAH and MAGL) were compared. The transporter inhibitor AM404 modestly enhanced depolarization-induced increases in 2-arachidonoyl glycerol (2-AG) levels but did not alter levels of N-arachidonoyl-ethanolamide (anandamide, AEA). The transport inhibitor UCM707 did not alter dialysate levels of either endocannabinoid. The FAAH inhibitors URB597 and PF-3845 robustly increased AEA levels during depolarization without altering 2-AG levels. The MAGL inhibitor URB602 significantly enhanced depolarization-induced increases in 2-AG, but did not alter AEA levels. In contrast, the MAGL inhibitor JZL184 did not alter 2-AG or AEA levels under any condition tested. Finally, the dual FAAH/MAGL inhibitor JZL195 significantly enhanced depolarization-induced increases in both AEA and 2-AG levels. In contrast to the present observations in rats, prior work in mice has demonstrated a robust JZL184-induced enhancement of depolarization-induced increases in dialysate 2-AG. Thus, to further investigate species differences, additional tests with JZL184, PF-3845, and JZL195 were performed in mice. Consistent with prior reports, JZL184 significantly enhanced depolarization-induced increases in 2-AG without altering AEA levels. PF-3845 and JZL195 produced profiles in mouse dialysates comparable to those observed in rats. These findings confirm that interstitial endocannabinoid levels in the brain can be selectively manipulated by endocannabinoid clearance inhibitors. While PF-3845 and JZL195 produce similar effects in both rats and mice, substantial species differences in JZL184 efficacy are evident, which is consistent with previous studies.
ESTHER : Wiskerke_2012_ACS.Chem.Neurosci_3_407
PubMedSearch : Wiskerke_2012_ACS.Chem.Neurosci_3_407
PubMedID: 22860210

Title : Highly selective inhibitors of monoacylglycerol lipase bearing a reactive group that is bioisosteric with endocannabinoid substrates - Chang_2012_Chem.Biol_19_579
Author(s) : Chang JW , Niphakis MJ , Lum KM , Cognetta AB, 3rd , Wang C , Matthews ML , Niessen S , Buczynski MW , Parsons LH , Cravatt BF
Ref : Chemical Biology , 19 :579 , 2012
Abstract : The endocannabinoids 2-arachidonoyl glycerol (2-AG) and N-arachidonoyl ethanolamine (anandamide) are principally degraded by monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), respectively. The recent discovery of O-aryl carbamates such as JZL184 as selective MAGL inhibitors has enabled functional investigation of 2-AG signaling pathways in vivo. Nonetheless, JZL184 and other reported MAGL inhibitors still display low-level cross-reactivity with FAAH and peripheral carboxylesterases, which can complicate their use in certain biological studies. Here, we report a distinct class of O-hexafluoroisopropyl (HFIP) carbamates that inhibits MAGL in vitro and in vivo with excellent potency and greatly improved selectivity, including showing no detectable cross-reactivity with FAAH. These findings designate HFIP carbamates as a versatile chemotype for inhibiting MAGL and should encourage the pursuit of other serine hydrolase inhibitors that bear reactive groups resembling the structures of natural substrates.
ESTHER : Chang_2012_Chem.Biol_19_579
PubMedSearch : Chang_2012_Chem.Biol_19_579
PubMedID: 22542104
Gene_locus related to this paper: human-MGLL

Title : Alterations of endocannabinoid signaling, synaptic plasticity, learning, and memory in monoacylglycerol lipase knock-out mice - Pan_2011_J.Neurosci_31_13420
Author(s) : Pan B , Wang W , Zhong P , Blankman JL , Cravatt BF , Liu QS
Ref : Journal of Neuroscience , 31 :13420 , 2011
Abstract : Endocannabinoid (eCB) signaling is tightly regulated by eCB biosynthetic and degradative enzymes. The eCB 2-arachidonoylglycerol (2-AG) is hydrolyzed primarily by monoacylglycerol lipase (MAGL). Here, we investigated whether eCB signaling, synaptic function, and learning behavior were altered in MAGL knock-out mice. We report that MAGL(-)/(-) mice exhibited prolonged depolarization-induced suppression of inhibition (DSI) in hippocampal CA1 pyramidal neurons, providing genetic evidence that the inactivation of 2-AG by MAGL determines the time course of the eCB-mediated retrograde synaptic depression. CB(1) receptor antagonists enhanced basal IPSCs in CA1 pyramidal neurons in MAGL(-)/(-) mice, while the magnitude of DSI or CB(1) receptor agonist-induced depression of IPSCs was decreased in MAGL(-)/(-) mice. These results suggest that 2-AG elevations in MAGL(-)/(-) mice cause tonic activation and partial desensitization of CB(1) receptors. Genetic deletion of MAGL selectively enhanced theta burst stimulation (TBS)-induced long-term potentiation (LTP) in the CA1 region of hippocampal slices but had no significant effect on LTP induced by high-frequency stimulation or long-term depression induced by low-frequency stimulation. The enhancement of TBS-LTP in MAGL(-)/(-) mice appears to be mediated by 2-AG-induced suppression of GABA(A) receptor-mediated inhibition. MAGL(-)/(-) mice exhibited enhanced learning as shown by improved performance in novel object recognition and Morris water maze. These results indicate that genetic deletion of MAGL causes profound changes in eCB signaling, long-term synaptic plasticity, and learning behavior.
ESTHER : Pan_2011_J.Neurosci_31_13420
PubMedSearch : Pan_2011_J.Neurosci_31_13420
PubMedID: 21940435
Gene_locus related to this paper: human-MGLL , mouse-MGLL

Title : Genetic deletion of monoacylglycerol lipase alters endocannabinoid-mediated retrograde synaptic depression in the cerebellum - Zhong_2011_J.Physiol_589_4847
Author(s) : Zhong P , Pan B , Gao XP , Blankman JL , Cravatt BF , Liu QS
Ref : Journal de Physiologie , 589 :4847 , 2011
Abstract : The endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) is hydrolysed primarily by monoacylglycerol lipase (MAGL). Here, we investigated whether eCB-mediated retrograde synaptic depression in cerebellar slices was altered in MAGL knockout (MAGL(-/-)) mice. Depolarization-induced suppression of excitation (DSE) and metabotropic glutamate receptor (mGluR1)-mediated synaptic depression are mediated by 2-AG-induced activation of CB(1) receptors. We show that genetic deletion of MAGL prolonged DSE at parallel fibre (PF) or climbing fibre (CF) to Purkinje cell (PC) synapses. Likewise, mGluR1-mediated synaptic depression, induced either by high-frequency stimulation of PF or mGluR1 agonist DHPG, was prolonged in MAGL(-/-) mice. About 15% of 2-AG in the brain is hydrolysed by serine hydrolase alpha-beta-hydrolase domain 6 and 12 (ABHD6 and ABHD12). However, the selective ABHD6 inhibitor WWL123 had no significant effect on cerebellar DSE in MAGL(+/+) and (-/-) mice. The CB(1) receptor antagonist SR141716 significantly increased the amplitude of basal excitatory postsynaptic currents (EPSCs) in MAGL(-/-) mice but not in MAGL(+/+) mice. Conversely, the CB(1) agonist WIN55212 induced less depression of basal EPSCs in MAGL(-/-) mice than in MAGL(+/+) mice. These results provide genetic evidence that inactivation of 2-AG by MAGL determines the time course of eCB-mediated retrograde synaptic depression and that genetic deletion of MAGL causes tonic activation and consequential desensitization of CB(1) receptors.
ESTHER : Zhong_2011_J.Physiol_589_4847
PubMedSearch : Zhong_2011_J.Physiol_589_4847
PubMedID: 21911610
Gene_locus related to this paper: human-MGLL , mouse-MGLL

Title : Inhibition of endocannabinoid catabolic enzymes elicits anxiolytic-like effects in the marble burying assay - Kinsey_2011_Pharmacol.Biochem.Behav_98_21
Author(s) : Kinsey SG , O'Neal ST , Long JZ , Cravatt BF , Lichtman AH
Ref : Pharmacol Biochem Behav , 98 :21 , 2011
Abstract : Cannabinoids have long been shown to have a range of potential therapeutic effects, including antiemetic actions, analgesia, and anxiolysis. However, psychomimetic and memory disruptive side effects, as well as the potential for abuse and dependence, have restricted their clinical development. Endogenous cannabinoids (i.e., endocannabinoids; eCBs), such as anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are produced throughout the limbic system and other brain regions associated with emotionality and are believed to modulate behavioral responses to stress-related conditions. AEA and 2-AG are rapidly metabolized by the respective enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). Accordingly, inhibition of each enzyme increases brain levels of the appropriate eCB. Although FAAH inhibition has been established to decrease anxiety-like behavior, the role of 2-AG has been difficult to ascertain until the recent synthesis of JZL184, a potent and selective MAGL inhibitor. In the present study, we investigated the effects of inhibiting FAAH or MAGL on anxiety-like behavior in marble burying, a model of repetitive, compulsive behaviors germane to anxiety disorders such as obsessive-compulsive disorder. The FAAH inhibitor PF-3845, the MAGL inhibitor JZL184, and the benzodiazepine diazepam decreased marble burying at doses that did not affect locomotor activity. In contrast, Delta9-tetrahydrocannabinol (THC), the primary psychoactive constituent of marijuana, did not consistently reduce marble burying without also eliciting profound decreases in locomotor behavior. The CB1 cannabinoid receptor antagonist rimonabant blocked the reduction in marble burying caused by FAAH and MAGL inhibitors, but not by diazepam, indicating a CB1 receptor mechanism of action. These data indicate that elevation of AEA or 2-AG reduces marble burying behavior and suggest that their catabolic enzymes represent potential targets for the development of new classes of pharmacotherapeutics to treat anxiety-related disorders.
ESTHER : Kinsey_2011_Pharmacol.Biochem.Behav_98_21
PubMedSearch : Kinsey_2011_Pharmacol.Biochem.Behav_98_21
PubMedID: 21145341

Title : Inhibition of monoacylglycerol lipase attenuates nonsteroidal anti-inflammatory drug-induced gastric hemorrhages in mice - Kinsey_2011_J.Pharmacol.Exp.Ther_338_795
Author(s) : Kinsey SG , Nomura DK , O'Neal ST , Long JZ , Mahadevan A , Cravatt BF , Grider JR , Lichtman AH
Ref : Journal of Pharmacology & Experimental Therapeutics , 338 :795 , 2011
Abstract : Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used analgesics, but can cause gastric and esophageal hemorrhages, erosion, and ulceration. The endogenous cannabinoid (endocannabinoid; eCB) system possesses several potential targets to reduce gastric inflammatory states, including cannabinoid receptor type 1 (CB(1)), cannabinoid receptor type 2 (CB(2)), and enzymes that regulate the eCB ligands 2-arachidonoylglycerol (2-AG) and N-arachidonoyl ethanolamine (anandamide; AEA). In the presented study, we tested whether 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184), a selective inhibitor of the primary catabolic enzyme of 2-AG, monoacylglycerol lipase (MAGL), would protect against NSAID-induced gastric damage. Food-deprived mice administered the nonselective cyclooxygenase inhibitor diclofenac sodium displayed gastric hemorrhages and increases in proinflammatory cytokines. JZL184, the proton pump inhibitor omeprazole (positive control), or the primary constituent of marijuana, delta(9)-tetrahydrocannabinol (THC), significantly prevented diclofenac-induced gastric hemorrhages. JZL184 also increased stomach levels of 2-AG, but had no effect on AEA, arachidonic acid, or the prostaglandins E(2) and D(2). MAGL inhibition fully blocked diclofenac-induced increases in gastric levels of proinflammatory cytokines interleukin (IL)-1beta, IL-6, tumor necrosis factor alpha, and granulocyte colony-stimulating factor, as well as IL-10. Pharmacological inhibition or genetic deletion of CB(1) or CB(2) revealed that the gastroprotective effects of JZL184 and THC were mediated via CB(1). The antihemorrhagic effects of JZL184 persisted with repeated administration, indicating a lack of tolerance. These data indicate that increasing 2-AG protects against gastric damage induced by NSAIDs, and its primary catabolic enzyme MAGL offers a promising target for the development of analgesic therapeutics possessing gastroprotective properties.
ESTHER : Kinsey_2011_J.Pharmacol.Exp.Ther_338_795
PubMedSearch : Kinsey_2011_J.Pharmacol.Exp.Ther_338_795
PubMedID: 21659471

Title : Endocannabinoid hydrolysis generates brain prostaglandins that promote neuroinflammation - Nomura_2011_Science_334_809
Author(s) : Nomura DK , Morrison BE , Blankman JL , Long JZ , Kinsey SG , Marcondes MC , Ward AM , Hahn YK , Lichtman AH , Conti B , Cravatt BF
Ref : Science , 334 :809 , 2011
Abstract : Phospholipase A(2)(PLA(2)) enzymes are considered the primary source of arachidonic acid for cyclooxygenase (COX)-mediated biosynthesis of prostaglandins. Here, we show that a distinct pathway exists in brain, where monoacylglycerol lipase (MAGL) hydrolyzes the endocannabinoid 2-arachidonoylglycerol to generate a major arachidonate precursor pool for neuroinflammatory prostaglandins. MAGL-disrupted animals show neuroprotection in a parkinsonian mouse model. These animals are spared the hemorrhaging caused by COX inhibitors in the gut, where prostaglandins are instead regulated by cytosolic PLA(2). These findings identify MAGL as a distinct metabolic node that couples endocannabinoid to prostaglandin signaling networks in the nervous system and suggest that inhibition of this enzyme may be a new and potentially safer way to suppress the proinflammatory cascades that underlie neurodegenerative disorders.
ESTHER : Nomura_2011_Science_334_809
PubMedSearch : Nomura_2011_Science_334_809
PubMedID: 22021672

Title : Optimization and characterization of a triazole urea inhibitor for alpha\/beta hydrolase domain-containing protein 11 (ABHD11): anti-probe for LYPLA1\/LYPLA2 dual inhibitor ML211 - Adibekian_2011_Probe.Report__1
Author(s) : Adibekian A , Hsu KL , Speers AE , Brown SJ , Spicer T , Fernandez-Vega V , Ferguson J , Cravatt BF , Hodder P , Rosen H
Ref : Probe Report , : , 2011
Abstract : Protein palmitoylation is an essential post-translational modification necessary for trafficking and localization of regulatory proteins that play key roles in cell growth and signaling. Multiple oncogenes, including HRAS and SRC, require palmitoylation for malignant transformation. We and others have previously identified lysophospholipase 1 (LYPLA1) as a candidate protein palmitoyl thioesterase responsible for HRAS depalmitoylation in mammalian cells. Seeking chemical tools to investigate biochemical pathway involvement and potential roles in cancer pathogenesis, we conducted a fluorescence polarization-based competitive activity-based protein profiling (FluoPol ABPP) high throughput screening (HTS) campaign to identify inhibitors of LYPLA1 and the structurally related LYPLA2. HTS identified a micromolar triazole urea inhibitor, that we successfully optimized via several rounds of SAR-by-synthesis as ML211 (SID 99445338), a low nanomolar dual inhibitor of LYPLA1 and LYPLA2. The reported probe operates by a covalent mechanism of action and is active both in vitro and in situ. Out of more than 20 serine hydrolases (SHs) profiled by gel-based competitive activity-based protein profiling (ABPP), ML211 was observed to have one anti-target, alpha/beta hydrolase domain-containing protein 11 (ABHD11). Fortuitously, one of the triazole urea library members synthesized during the course of probe optimization was found to be a potent and selective inhibitor of ABHD11, a poorly characterized SH that is hemizygously deleted in Williams-Beuren syndrome [4], and was presented as a control anti-probe (SID 99445332) in the ML211 Probe Report. The optimized ABHD11 probe ML226 is a potent inhibitor of ABHD11, with an IC50 of 15 nM, and exhibits at least 100-fold selectivity for all other SHs (~20) assessed by gel-based competitive ABPP. The probe is also active in situ, completely and selectively inhibiting ABHD11 at sub-nanomolar concentrations. As with ML211, the probe operates by a covalent mechanism of action, carbamoylating the active site serine of ABHD11. The complete properties, characterization, and synthesis of ML226 are detailed in this Probe Report.
ESTHER : Adibekian_2011_Probe.Report__1
PubMedSearch : Adibekian_2011_Probe.Report__1
PubMedID: 23658953

Title : Blockade of endocannabinoid hydrolytic enzymes attenuates precipitated opioid withdrawal symptoms in mice - Ramesh_2011_J.Pharmacol.Exp.Ther_339_173
Author(s) : Ramesh D , Ross GR , Schlosburg JE , Owens RA , Abdullah RA , Kinsey SG , Long JZ , Nomura DK , Sim-Selley LJ , Cravatt BF , Akbarali HI , Lichtman AH
Ref : Journal of Pharmacology & Experimental Therapeutics , 339 :173 , 2011
Abstract : Delta(9)-Tetrahydrocannbinol (THC), the primary active constituent of Cannabis sativa, has long been known to reduce opioid withdrawal symptoms. Although THC produces most of its pharmacological actions through the activation of CB(1) and CB(2) cannabinoid receptors, the role these receptors play in reducing the variety of opioid withdrawal symptoms remains unknown. The endogenous cannabinoids, N-arachidonoylethanolamine (anandamide; AEA) and 2-arachidonylglycerol (2-AG), activate both cannabinoid receptors but are rapidly metabolized by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. The objective of this study was to test whether increasing AEA or 2-AG, via inhibition of their respective hydrolytic enzymes, reduces naloxone-precipitated morphine withdrawal symptoms in in vivo and in vitro models of opioid dependence. Morphine-dependent mice challenged with naloxone reliably displayed a profound withdrawal syndrome, consisting of jumping, paw tremors, diarrhea, and weight loss. THC and the MAGL inhibitor 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184) dose dependently reduced the intensity of most measures through the activation of CB(1) receptors. JZL184 also attenuated spontaneous withdrawal signs in morphine-dependent mice. The FAAH inhibitor N-(pyridin-3-yl)-4-(3-(5-(trifluoromethyl)pyridin-2-yloxy)benzyl)-piperdine-1-car boxamide (PF-3845) reduced the intensity of naloxone-precipitated jumps and paw flutters through the activation of CB(1) receptors but did not ameliorate incidence of diarrhea or weight loss. In the final series of experiments, we investigated whether JZL184 or PF-3845 would attenuate naloxone-precipitated contractions in morphine-dependent ilea. Both enzyme inhibitors attenuated the intensity of naloxone-induced contractions, although this model does not account mechanistically for the autonomic withdrawal responses (i.e., diarrhea) observed in vivo. These results indicate that endocannabinoid catabolic enzymes are promising targets to treat opioid dependence.
ESTHER : Ramesh_2011_J.Pharmacol.Exp.Ther_339_173
PubMedSearch : Ramesh_2011_J.Pharmacol.Exp.Ther_339_173
PubMedID: 21719468

Title : COX-2 and fatty acid amide hydrolase can regulate the time course of depolarization-induced suppression of excitation - Straiker_2011_Br.J.Pharmacol_164_1672
Author(s) : Straiker A , Wager-Miller J , Hu SS , Blankman JL , Cravatt BF , Mackie K
Ref : British Journal of Pharmacology , 164 :1672 , 2011
Abstract : BACKGROUND AND PURPOSE: Depolarization-induced suppression of inhibition (DSI) and excitation (DSE) are two forms of cannabinoid CB(1) receptor-mediated inhibition of synaptic transmission, whose durations are regulated by endocannabinoid (eCB) degradation. We have recently shown that in cultured hippocampal neurons monoacylglycerol lipase (MGL) controls the duration of DSE, while DSI duration is determined by both MGL and COX-2. This latter result suggests that DSE might be attenuated, and excitatory transmission enhanced, during inflammation and in other settings where COX-2 expression is up-regulated. EXPERIMENTAL APPROACH: To investigate whether it is possible to control the duration of eCB-mediated synaptic plasticity by varied expression of eCB-degrading enzymes, we transfected excitatory autaptic hippocampal neurons with putative 2-AG metabolizing enzymes: COX-2, fatty acid amide hydrolase (FAAH), alpha/beta hydrolase domain 6 (ABHD6), alpha/beta hydrolase domain 12 (ABHD12) or MGL. KEY RESULTS: We found that overexpression of either COX-2 or FAAH shortens the duration of DSE while ABHD6 or ABHD12 do not. In contrast, genetic deletion (MGL(-/-)) and overexpression of MGL both radically altered eCB-mediated synaptic plasticity. CONCLUSIONS AND IMPLICATIONS: We conclude that both FAAH and COX-2 can be trafficked to neuronal sites where they are able to degrade eCBs to modulate DSE duration and, by extension, net endocannabinoid signalling at a given synapse. The results for COX-2, which is often up-regulated under pathological conditions, are of particular note in that they offer a mechanism by which up-regulated COX-2 may promote neuronal excitation by suppressing DSE while enhancing conversion of 2-AG to PGE(2) -glycerol ester under pathological conditions.
ESTHER : Straiker_2011_Br.J.Pharmacol_164_1672
PubMedSearch : Straiker_2011_Br.J.Pharmacol_164_1672
PubMedID: 21564090

Title : A potent and selective inhibitor of KIAA1363\/AADACL1 that impairs prostate cancer pathogenesis - Chang_2011_Chem.Biol_18_476
Author(s) : Chang JW , Nomura DK , Cravatt BF
Ref : Chemical Biology , 18 :476 , 2011
Abstract : Cancer cells show alterations in metabolism that support malignancy and disease progression. Prominent among these metabolic changes is elevations in neutral ether lipids (NELs). We have previously shown that the hydrolytic enzyme KIAA1363 (or AADACL1) is highly elevated in aggressive cancer cells, where it plays a key role in generating the monoalkylglycerol ether (MAGE) class of NELs. Here, we use activity-based protein profiling-guided medicinal chemistry to discover a highly potent and selective inhibitor of KIAA1363, the carbamate JW480. We show that JW480, and an shRNA probe that targets KIAA1363, reduce MAGEs and impair the migration, invasion, survival, and in vivo tumor growth of human prostate cancer cell lines. These findings indicate that the KIAA1363-MAGE pathway is important for prostate cancer pathogenesis and designate JW480 as a versatile pharmacological probe for disrupting this pro-tumorigenic metabolic pathway.
ESTHER : Chang_2011_Chem.Biol_18_476
PubMedSearch : Chang_2011_Chem.Biol_18_476
PubMedID: 21513884
Gene_locus related to this paper: human-NCEH1

Title : Discovery of PF-04457845: A Highly Potent, Orally Bioavailable, and Selective Urea FAAH Inhibitor - Johnson_2011_ACS.Med.Chem.Lett_2_91
Author(s) : Johnson DS , Stiff C , Lazerwith SE , Kesten SR , Fay LK , Morris M , Beidler D , Liimatta MB , Smith SE , Dudley DT , Sadagopan N , Bhattachar SN , Kesten SJ , Nomanbhoy TK , Cravatt BF , Ahn K
Ref : ACS Med Chem Lett , 2 :91 , 2011
Abstract : Fatty acid amide hydrolase (FAAH) is an integral membrane serine hydrolase that degrades the fatty acid amide family of signaling lipids, including the endocannabinoid anandamide. Genetic or pharmacological inactivation of FAAH leads to analgesic and anti-inflammatory phenotypes in rodents without showing the undesirable side effects observed with direct cannabinoid receptor agonists, indicating that FAAH may represent an attractive therapeutic target for the treatment of inflammatory pain and other nervous system disorders. Herein, we report the discovery and characterization of a highly efficacious and selective FAAH inhibitor PF-04457845 (23). Compound 23 inhibits FAAH by a covalent, irreversible mechanism involving carbamylation of the active-site serine nucleophile of FAAH with high in vitro potency (k(inact)/K(i) and IC(50) values of 40300 M(-1) s(-1) and 7.2 nM, respectively, for human FAAH). Compound 23 has exquisite selectivity for FAAH relative to other members of the serine hydrolase superfamily as demonstrated by competitive activity-based protein profiling. Oral administration of 23 at 0.1 mg/kg results in efficacy comparable to that of naproxen at 10 mg/kg in a rat model of inflammatory pain. Compound 23 is being evaluated in human clinical trials.
ESTHER : Johnson_2011_ACS.Med.Chem.Lett_2_91
PubMedSearch : Johnson_2011_ACS.Med.Chem.Lett_2_91
PubMedID: 21666860

Title : Metabolomics annotates ABHD3 as a physiologic regulator of medium-chain phospholipids - Long_2011_Nat.Chem.Biol_7_763
Author(s) : Long JZ , Cisar JS , Milliken D , Niessen S , Wang C , Trauger SA , Siuzdak G , Cravatt BF
Ref : Nat Chemical Biology , 7 :763 , 2011
Abstract : All organisms, including humans, possess a huge number of uncharacterized enzymes. Here we describe a general cell-based screen for enzyme substrate discovery by untargeted metabolomics and its application to identify the protein alpha/beta-hydrolase domain-containing 3 (ABHD3) as a lipase that selectively cleaves medium-chain and oxidatively truncated phospholipids. Abhd3(-/-) mice possess elevated myristoyl (C14)-phospholipids, including the bioactive lipid C14-lysophosphatidylcholine, confirming the physiological relevance of our substrate assignments.
ESTHER : Long_2011_Nat.Chem.Biol_7_763
PubMedSearch : Long_2011_Nat.Chem.Biol_7_763
PubMedID: 21926997
Gene_locus related to this paper: human-ABHD3 , mouse-abhd3

Title : Click-generated triazole ureas as ultrapotent in vivo-active serine hydrolase inhibitors - Adibekian_2011_Nat.Chem.Biol_7_469
Author(s) : Adibekian A , Martin BR , Wang C , Hsu KL , Bachovchin DA , Niessen S , Hoover H , Cravatt BF
Ref : Nat Chemical Biology , 7 :469 , 2011
Abstract : Serine hydrolases are a diverse enzyme class representing approximately 1% of all human proteins. The biological functions of most serine hydrolases remain poorly characterized owing to a lack of selective inhibitors to probe their activity in living systems. Here we show that a substantial number of serine hydrolases can be irreversibly inactivated by 1,2,3-triazole ureas, which show negligible cross-reactivity with other protein classes. Rapid lead optimization by click chemistry-enabled synthesis and competitive activity-based profiling identified 1,2,3-triazole ureas that selectively inhibit enzymes from diverse branches of the serine hydrolase class, including peptidases (acyl-peptide hydrolase, or APEH), lipases (platelet-activating factor acetylhydrolase-2, or PAFAH2) and uncharacterized hydrolases (alpha,beta-hydrolase-11, or ABHD11), with exceptional potency in cells (sub-nanomolar) and mice (<1 mg kg(-1)). We show that APEH inhibition leads to accumulation of N-acetylated proteins and promotes proliferation in T cells. These data indicate 1,2,3-triazole ureas are a pharmacologically privileged chemotype for serine hydrolase inhibition, combining broad activity across the serine hydrolase class with tunable selectivity for individual enzymes.
ESTHER : Adibekian_2011_Nat.Chem.Biol_7_469
PubMedSearch : Adibekian_2011_Nat.Chem.Biol_7_469
PubMedID: 21572424

Title : Optimization and characterization of a triazole urea inhibitor for platelet-activating factor acetylhydrolase type 2 (PAFAH2) - Adibekian_2011_Probe.Report__2
Author(s) : Adibekian A , Hsu KL , Speers AE , Monillas ES , Brown SJ , Spicer T , Fernandez-Vega V , Ferguson J , Bahnson BJ , Cravatt BF , Hodder P , Rosen H
Ref : Probe Report , : , 2011
Abstract : Oxidative stress has been implicated as an underlying inflammatory factor in several disease pathologies, including cancer, atherosclerosis, aging, and various neurodegenerative disorders. Phospholipids in particular are susceptible to oxidative damage, and it is thought that the cytosolic enzyme type II platelet-activating factor acetylhydrolase (PAFAH2) may facilitate turnover of oxidized phospholipids via hydrolysis of their oxidatively truncated acyl chains. In support of this theory, over-expression of PAFAH2 has been shown to reduce oxidative stress-induced cell death. However, no selective inhibitors of PAFAH2 are known for investigation of PAFAH2 biology. We initiated a fluorescence polarization activity-based protein profiling (FluoPol-ABPP) HTS campaign to identify potential inhibitors of PAFAH2 (AIDs 492956 and 493030). The assay also served as a counterscreen for inhibitor discovery for the related enzyme, plasma PAFAH (pPAFAH; AIDs 463082, 463230). Interestingly, the triazole urea SID 7974398a top lead in the lysophospholipase (LYPLA1) inhibitor screen from which we derived a dual inhibitor of LYPLA1/LYPLA2 (ML211) and inhibitor of ABHD11 (ML226) was also a top hit in the PAFAH2 HTS assay. Given that triazole ureas were previously found to have tunable potency and selectivity, low cytotoxicity, and good activity in situ, we endeavored to derive a PAFAH2-selective probe from the triazole urea scaffold. The medchem optimized probe (ML225, SID 103913572) is highly potent against its target enzyme (IC50 = 3 nM), and is active in situ at sub-nanomolar concentrations. ML225 is at least 333-fold selective for all other serine hydrolases (~20) assessed by gel-based competitive activity-based protein profiling, and is selective for other PAFAH enzymes. ML225 inhibits PAFAH2 by carbamoylating the active site serine. The complete properties, characterization, and synthesis of ML225 are detailed in this Probe Report.
ESTHER : Adibekian_2011_Probe.Report__2
PubMedSearch : Adibekian_2011_Probe.Report__2
PubMedID: 23658960

Title : A substrate-free activity-based protein profiling screen for the discovery of selective PREPL inhibitors - Lone_2011_J.Am.Chem.Soc_133_11665
Author(s) : Lone AM , Bachovchin DA , Westwood DB , Speers AE , Spicer TP , Fernandez-Vega V , Chase P , Hodder PS , Rosen H , Cravatt BF , Saghatelian A
Ref : Journal of the American Chemical Society , 133 :11665 , 2011
Abstract : Peptidases play vital roles in physiology through the biosynthesis, degradation, and regulation of peptides. Prolyl endopeptidase-like (PREPL) is a newly described member of the prolyl peptidase family, with significant homology to mammalian prolyl endopeptidase and the bacterial peptidase oligopeptidase B. The biochemistry and biology of PREPL are of fundamental interest due to this enzyme's homology to the biomedically important prolyl peptidases and its localization in the central nervous system. Furthermore, genetic studies of patients suffering from hypotonia-cystinuria syndrome (HCS) have revealed a deletion of a portion of the genome that includes the PREPL gene. HCS symptoms thought to be caused by lack of PREPL include neuromuscular and mild cognitive deficits. A number of complementary approaches, ranging from biochemistry to genetics, will be required to understand the biochemical, cellular, physiological, and pathological mechanisms regulated by PREPL. We are particularly interested in investigating physiological substrates and pathways controlled by PREPL. Here, we use a fluorescence polarization activity-based protein profiling (fluopol-ABPP) assay to discover selective small-molecule inhibitors of PREPL. Fluopol-ABPP is a substrate-free approach that is ideally suited for studying serine hydrolases for which no substrates are known, such as PREPL. After screening over 300,000 compounds using fluopol-ABPP, we employed a number of secondary assays to confirm assay hits and characterize a group of 3-oxo-1-phenyl-2,3,5,6,7,8-hexahydroisoquinoline-4-carbonitrile and 1-alkyl-3-oxo-3,5,6,7-tetrahydro-2H-cyclopenta[c]pyridine-4-carbonitrile PREPL inhibitors that are able to block PREPL activity in cells. Moreover, when administered to mice, 1-isobutyl-3-oxo-3,5,6,7-tetrahydro-2H-cyclopenta[c]pyridine-4-carbonitrile distributes to the brain, indicating that it may be useful for in vivo studies. The application of fluopol-ABPP has led to the first reported PREPL inhibitors, and these inhibitors will be of great value in studying the biochemistry of PREPL and in eventually understanding the link between PREPL and HCS.
ESTHER : Lone_2011_J.Am.Chem.Soc_133_11665
PubMedSearch : Lone_2011_J.Am.Chem.Soc_133_11665
PubMedID: 21692504
Gene_locus related to this paper: human-PREPL

Title : Academic cross-fertilization by public screening yields a remarkable class of protein phosphatase methylesterase-1 inhibitors - Bachovchin_2011_Proc.Natl.Acad.Sci.U.S.A_108_6811
Author(s) : Bachovchin DA , Mohr JT , Speers AE , Wang C , Berlin JM , Spicer TP , Fernandez-Vega V , Chase P , Hodder PS , Schurer SC , Nomura DK , Rosen H , Fu GC , Cravatt BF
Ref : Proc Natl Acad Sci U S A , 108 :6811 , 2011
Abstract : National Institutes of Health (NIH)-sponsored screening centers provide academic researchers with a special opportunity to pursue small-molecule probes for protein targets that are outside the current interest of, or beyond the standard technologies employed by, the pharmaceutical industry. Here, we describe the outcome of an inhibitor screen for one such target, the enzyme protein phosphatase methylesterase-1 (PME-1), which regulates the methylesterification state of protein phosphatase 2A (PP2A) and is implicated in cancer and neurodegeneration. Inhibitors of PME-1 have not yet been described, which we attribute, at least in part, to a dearth of substrate assays compatible with high-throughput screening. We show that PME-1 is assayable by fluorescence polarization-activity-based protein profiling (fluopol-ABPP) and use this platform to screen the 300,000+ member NIH small-molecule library. This screen identified an unusual class of compounds, the aza-beta-lactams (ABLs), as potent (IC(50) values of approximately 10 nM), covalent PME-1 inhibitors. Interestingly, ABLs did not derive from a commercial vendor but rather an academic contribution to the public library. We show using competitive-ABPP that ABLs are exquisitely selective for PME-1 in living cells and mice, where enzyme inactivation leads to substantial reductions in demethylated PP2A. In summary, we have combined advanced synthetic and chemoproteomic methods to discover a class of ABL inhibitors that can be used to selectively perturb PME-1 activity in diverse biological systems. More generally, these results illustrate how public screening centers can serve as hubs to create spontaneous collaborative opportunities between synthetic chemistry and chemical biology labs interested in creating first-in-class pharmacological probes for challenging protein targets.
ESTHER : Bachovchin_2011_Proc.Natl.Acad.Sci.U.S.A_108_6811
PubMedSearch : Bachovchin_2011_Proc.Natl.Acad.Sci.U.S.A_108_6811
PubMedID: 21398589
Gene_locus related to this paper: human-PPME1

Title : The metabolic serine hydrolases and their functions in mammalian physiology and disease -
Author(s) : Long JZ , Cravatt BF
Ref : Chem Rev , 111 :6022 , 2011
PubMedID: 21696217

Title : Optimization and characterization of a triazole urea dual inhibitor for lysophospholipase 1 (LYPLA1) and lysophospholipase 2 (LYPLA2) - Adibekian_2011_Probe.Report__5
Author(s) : Adibekian A , Martin BR , Speers AE , Brown SJ , Spicer T , Fernandez-Vega V , Ferguson J , Cravatt BF , Hodder P , Rosen H
Ref : Probe Report , : , 2011
Abstract : Protein palmitoylation is an essential post-translational modification necessary for trafficking and localization of regulatory proteins that play key roles in cell growth and signaling. Multiple oncogenes, including HRAS and SRC, require palmitoylation for malignant transformation. We and others have previously identified lysophospholipase 1 (LYPLA1) as a candidate protein palmitoyl thioesterase responsible for HRAS depalmitoylation in mammalian cells. Seeking chemical tools to investigate biochemical pathway involvement and potential roles in cancer pathogenesis, we conducted a fluorescence polarization-based competitive activity-based protein profiling (FluoPol ABPP) high throughput screening (HTS) campaign to identify inhibitors of LYPLA1 and the structurally related LYPLA2. HTS identified a micromolar triazole urea inhibitor, which we successfully optimized via several rounds of structure activity relationship (SAR)-by-synthesis to produce ML211 (SID 99445338), a low nanomolar dual inhibitor of LYPLA1 and LYPLA2. The reported probe operates by a covalent mechanism of action and is active both in vitro and in situ. Out of more than 20 serine hydrolases (SHs) profiled by gel-based competitive ABPP, ML211 is observed to have one anti-target, alpha/beta hydrolase domain-containing protein 11 (ABHD11). However, during our SAR campaign, we fortuitously discovered a selective ABHD11 inhibitor from among the synthetic triazole urea library compounds. This compound, ML226, is presented as an anti-probe for control studies.
ESTHER : Adibekian_2011_Probe.Report__5
PubMedSearch : Adibekian_2011_Probe.Report__5
PubMedID: 23658947

Title : Probe Report for RBBP9 Inhibitors - Probe 1 - Brown_2010_Probe.Report__
Author(s) : Brown SJ , Bachovchin DA , Cravatt BF , Fernandez-Vega V , Spicer T , Mercer BA , Hodder P , Rosen HR
Ref : Probe Report , : , 2010
Abstract : The retinoblastoma (RB) tumor suppressor protein controls cell cycle progression by regulating the activity of the transcription factor E2F, which in turn activates genes essential for DNA replication. Thus, factors that bind and regulate RB activity provide for valuable targets for preventing tumorigenesis. The enzyme, RB binding protein 9 (RBBP9), is widely expressed in a number of different tissues and is upregulated in certain tumors. As a result, the identification of compounds that selectively inhibit RBBP9 activity would serve as potentially valuable probes for the study of apoptosis, cell cycle, and tumorigenesis. The probe ML081 (CID- 6603320; emetine hydrochloride) represents the first non-covalent, selective RBBP9 inhibitor, and will be useful for exploring the enzymatic functions of RBBP9 in biological systems. Moreover, the tight structure-activity relationship of the emetine-RBBP9 interaction suggests that only minor modifications to the emetine structure will improve its activity. As a result, future studies will involve semi-synthetic addition of small moieties to the emetine and cephaeline scaffolds.
ESTHER : Brown_2010_Probe.Report__
PubMedSearch : Brown_2010_Probe.Report__
PubMedID: 21433353
Gene_locus related to this paper: human-RBBP9

Title : Activity-based proteomics of enzyme superfamilies: serine hydrolases as a case study - Simon_2010_J.Biol.Chem_285_11051
Author(s) : Simon GM , Cravatt BF
Ref : Journal of Biological Chemistry , 285 :11051 , 2010
Abstract : Genome sequencing projects have uncovered thousands of uncharacterized enzymes in eukaryotic and prokaryotic organisms. Deciphering the physiological functions of enzymes requires tools to profile and perturb their activities in native biological systems. Activity-based protein profiling has emerged as a powerful chemoproteomic strategy to achieve these objectives through the use of chemical probes that target large swaths of enzymes that share active-site features. Here, we review activity-based protein profiling and its implementation to annotate the enzymatic proteome, with particular attention given to probes that target serine hydrolases, a diverse superfamily of enzymes replete with many uncharacterized members.
ESTHER : Simon_2010_J.Biol.Chem_285_11051
PubMedSearch : Simon_2010_J.Biol.Chem_285_11051
PubMedID: 20147750

Title : Monoacylglycerol lipase regulates a fatty acid network that promotes cancer pathogenesis - Nomura_2010_Cell_140_49
Author(s) : Nomura DK , Long JZ , Niessen S , Hoover HS , Ng SW , Cravatt BF
Ref : Cell , 140 :49 , 2010
Abstract : Tumor cells display progressive changes in metabolism that correlate with malignancy, including development of a lipogenic phenotype. How stored fats are liberated and remodeled to support cancer pathogenesis, however, remains unknown. Here, we show that the enzyme monoacylglycerol lipase (MAGL) is highly expressed in aggressive human cancer cells and primary tumors, where it regulates a fatty acid network enriched in oncogenic signaling lipids that promotes migration, invasion, survival, and in vivo tumor growth. Overexpression of MAGL in nonaggressive cancer cells recapitulates this fatty acid network and increases their pathogenicity-phenotypes that are reversed by an MAGL inhibitor. Impairments in MAGL-dependent tumor growth are rescued by a high-fat diet, indicating that exogenous sources of fatty acids can contribute to malignancy in cancers lacking MAGL activity. Together, these findings reveal how cancer cells can co-opt a lipolytic enzyme to translate their lipogenic state into an array of protumorigenic signals. PAPERFLICK:
ESTHER : Nomura_2010_Cell_140_49
PubMedSearch : Nomura_2010_Cell_140_49
PubMedID: 20079333
Gene_locus related to this paper: human-MGLL , mouse-MGLL

Title : Probe Development Efforts to Identify Novel Inhibitors of Protein Phosphatase Methylesterase-1 (PME-1) - Bachovchin_2010_Probe.Report__1
Author(s) : Bachovchin DA , Speers AE , Brown SJ , Spicer TP , Fernandez V , Ferguson J , Mohr JT , Murphy J , Fu GC , Cravatt BF , Hodder PS , Rosen H
Ref : Probe Report , : , 2010
Abstract : Reversible protein phosphorylation networks play essential roles in most cellular processes. While over 500 kinases catalyze protein phosphorylation, only two enzymes, PP1 and PP2A, are responsible for more than 90% of all serine/threonine phosphatase activity. Phosphatases, unlike kinases, achieve substrate specificity through complex subunit assembly and post-translational modifications rather than number. Mutations in several of the PP2A subunits have been identified in human cancers, suggesting that PP2A may act as a tumor suppressor. Adding further complexity, several residues of the catalytic subunit of PP2A can be reversibly phosphorylated, and the C-terminal leucine residue can be reversibly methylated. Protein phosphatase methylesterase-1 (PME-1) is specifically responsible for demethylation of the carboxyl terminus. Methylesterification is thought to control the binding of different subunits to PP2A, but little is known about physiological significance of this post-translational modification in vivo. Recently, PME-1 has been identified as a protector of sustained ERK pathway activity in malignant gliomas. PME-1 knockout mice generated by targeted gene disruption result in perinatal lethality, underscoring the importance of PME-1 but hindering biological studies. The Scripps Research Institute Molecular Screening Center (SRIMSC), part of the Molecular Libraries Probe Production Centers Network (MLPCN), identified a potent and selective PME-1 inhibitor probe, ML174, by high-throughput screening using fluorescence polarization-activity-based protein profiling (FluoPol-ABPP). ML174, with an IC50 of 10 nM, is based on the aza-beta-lactam scaffold and is selective for PME-1 among serine hydrolases in human cell line proteomes as assessed by gel-based competitive-activity-based protein profiling. Among more than 30 serine hydrolase anti-targets, ML174 is selective at 1 muM. Additionally, ML174 was shown in situ to be highly active against PME-1 and to result in 85% reduction of demethylated PP2A. We previously reported a modestly potent 500 nM inhibitor that was selective for PME-1, the first reported selective PME-1 inhibitor. ML174 is 50 times more potent and from an entirely different structural and mechanistic class of inhibitors. Due to its much higher potency, ML174 has greater potential for use in long time-course in situ studies, and is a much better candidate for in vivo applications.
ESTHER : Bachovchin_2010_Probe.Report__1
PubMedSearch : Bachovchin_2010_Probe.Report__1
PubMedID: 22834039

Title : Probe Report for PME-1 Inhibitors - Bachovchin_2010_Probe.Report__2
Author(s) : Bachovchin DA , Speers AE , Zuhl AM , Brown SJ , Cravatt BF , Fernandez V , Spicer T , Mercer BA , Ferguson J , Hodder P , Rosen HR
Ref : Probe Report , : , 2010
Abstract : Recent findings have identified protein phosphatase methylesterase-1 (PME-1) as a protector of sustained ERK pathway activity in malignant gliomas. PME-1 is a protein methylesterase that functions in the regulation of protein phosphatase 2A (PP2A) by reversible methylation. Biochemical elucidation of PME-1 would thus greatly benefit from the development of potent and selective chemical inhibitors. The probe compound ML136 (CID-44607965), containing a sulfonyl acrylonitrile core, represents the first potent, selective inhibitor of PME-1. Moreover, the probe does not appear to exhibit cytotoxicity. Thus, ML136 should serve as a useful tool for in vitro and in situ research assays in which it is desirable to specifically block PME-1 activity.
ESTHER : Bachovchin_2010_Probe.Report__2
PubMedSearch : Bachovchin_2010_Probe.Report__2
PubMedID: 21433379

Title : Chronic monoacylglycerol lipase blockade causes functional antagonism of the endocannabinoid system - Schlosburg_2010_Nat.Neurosci_13_1113
Author(s) : Schlosburg JE , Blankman JL , Long JZ , Nomura DK , Pan B , Kinsey SG , Nguyen PT , Ramesh D , Booker L , Burston JJ , Thomas EA , Selley DE , Sim-Selley LJ , Liu QS , Lichtman AH , Cravatt BF
Ref : Nat Neurosci , 13 :1113 , 2010
Abstract : Prolonged exposure to drugs of abuse, such as cannabinoids and opioids, leads to pharmacological tolerance and receptor desensitization in the nervous system. We found that a similar form of functional antagonism was produced by sustained inactivation of monoacylglycerol lipase (MAGL), the principal degradative enzyme for the endocannabinoid 2-arachidonoylglycerol. After repeated administration, the MAGL inhibitor JZL184 lost its analgesic activity and produced cross-tolerance to cannabinoid receptor (CB1) agonists in mice, effects that were phenocopied by genetic disruption of Mgll (encoding MAGL). Chronic MAGL blockade also caused physical dependence, impaired endocannabinoid-dependent synaptic plasticity and desensitized brain CB1 receptors. These data contrast with blockade of fatty acid amide hydrolase, an enzyme that degrades the other major endocannabinoid anandamide, which produced sustained analgesia without impairing CB1 receptors. Thus, individual endocannabinoids generate distinct analgesic profiles that are either sustained or transitory and associated with agonism and functional antagonism of the brain cannabinoid system, respectively.
ESTHER : Schlosburg_2010_Nat.Neurosci_13_1113
PubMedSearch : Schlosburg_2010_Nat.Neurosci_13_1113
PubMedID: 20729846

Title : The serine hydrolase ABHD6 controls the accumulation and efficacy of 2-AG at cannabinoid receptors - Marrs_2010_Nat.Neurosci_13_951
Author(s) : Marrs WR , Blankman JL , Horne EA , Thomazeau A , Lin YH , Coy J , Bodor AL , Muccioli GG , Hu SS , Woodruff G , Fung S , Lafourcade M , Alexander JP , Long JZ , Li W , Xu C , Moller T , Mackie K , Manzoni OJ , Cravatt BF , Stella N
Ref : Nat Neurosci , 13 :951 , 2010
Abstract : The endocannabinoid 2-arachidonoylglycerol (2-AG) regulates neurotransmission and neuroinflammation by activating CB1 cannabinoid receptors on neurons and CB2 cannabinoid receptors on microglia. Enzymes that hydrolyze 2-AG, such as monoacylglycerol lipase, regulate the accumulation and efficacy of 2-AG at cannabinoid receptors. We found that the recently described serine hydrolase alpha-beta-hydrolase domain 6 (ABHD6) also controls the accumulation and efficacy of 2-AG at cannabinoid receptors. In cells from the BV-2 microglia cell line, ABHD6 knockdown reduced hydrolysis of 2-AG and increased the efficacy with which 2-AG can stimulate CB2-mediated cell migration. ABHD6 was expressed by neurons in primary culture and its inhibition led to activity-dependent accumulation of 2-AG. In adult mouse cortex, ABHD6 was located postsynaptically and its selective inhibition allowed the induction of CB1-dependent long-term depression by otherwise subthreshold stimulation. Our results indicate that ABHD6 is a rate-limiting step of 2-AG signaling and is therefore a bona fide member of the endocannabinoid signaling system.
ESTHER : Marrs_2010_Nat.Neurosci_13_951
PubMedSearch : Marrs_2010_Nat.Neurosci_13_951
PubMedID: 20657592
Gene_locus related to this paper: human-ABHD6

Title : Superfamily-wide portrait of serine hydrolase inhibition achieved by library-versus-library screening - Bachovchin_2010_Proc.Natl.Acad.Sci.U.S.A_107_20941
Author(s) : Bachovchin DA , Ji T , Li W , Simon GM , Blankman JL , Adibekian A , Hoover H , Niessen S , Cravatt BF
Ref : Proc Natl Acad Sci U S A , 107 :20941 , 2010
Abstract : Serine hydrolases (SHs) are one of the largest and most diverse enzyme classes in mammals. They play fundamental roles in virtually all physiological processes and are targeted by drugs to treat diseases such as diabetes, obesity, and neurodegenerative disorders. Despite this, we lack biological understanding for most of the 110+ predicted mammalian metabolic SHs, in large part because of a dearth of assays to assess their biochemical activities and a lack of selective inhibitors to probe their function in living systems. We show here that the vast majority (> 80%) of mammalian metabolic SHs can be labeled in proteomes by a single, active site-directed fluorophosphonate probe. We exploit this universal activity-based assay in a library-versus-library format to screen 70+ SHs against 140+ structurally diverse carbamates. Lead inhibitors were discovered for approximately 40% of the screened enzymes, including many poorly characterized SHs. Global profiles identified carbamate inhibitors that discriminate among highly sequence-related SHs and, conversely, enzymes that share inhibitor sensitivity profiles despite lacking sequence homology. These findings indicate that sequence relatedness is not a strong predictor of shared pharmacology within the SH superfamily. Finally, we show that lead carbamate inhibitors can be optimized into pharmacological probes that inactivate individual SHs with high specificity in vivo.
ESTHER : Bachovchin_2010_Proc.Natl.Acad.Sci.U.S.A_107_20941
PubMedSearch : Bachovchin_2010_Proc.Natl.Acad.Sci.U.S.A_107_20941
PubMedID: 21084632

Title : Characterization of tunable piperidine and piperazine carbamates as inhibitors of endocannabinoid hydrolases - Long_2010_J.Med.Chem_53_1830
Author(s) : Long JZ , Jin X , Adibekian A , Li W , Cravatt BF
Ref : Journal of Medicinal Chemistry , 53 :1830 , 2010
Abstract : Monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) are two enzymes from the serine hydrolase superfamily that degrade the endocannabinoids 2-arachidonoylglycerol and anandamide, respectively. We have recently discovered that MAGL and FAAH are both inhibited by carbamates bearing an N-piperidine/piperazine group. Piperidine/piperazine carbamates show excellent in vivo activity, raising brain endocannabinoid levels and producing CB1-dependent behavioral effects in mice, suggesting that they represent a promising class of inhibitors for studying the endogenous functions of MAGL and FAAH. Herein, we disclose a full account of the syntheses, structure-activity relationships, and inhibitory activities of piperidine/piperazine carbamates against members of the serine hydrolase family. These scaffolds can be tuned for MAGL-selective or dual MAGL-FAAH inhibition by the attachment of an appropriately substituted bisarylcarbinol or aryloxybenzyl moiety, respectively, on the piperidine/piperazine ring. Modifications to the piperidine/piperazine ring ablated inhibitory activity, suggesting a strict requirement for a six-membered ring to maintain potency.
ESTHER : Long_2010_J.Med.Chem_53_1830
PubMedSearch : Long_2010_J.Med.Chem_53_1830
PubMedID: 20099888

Title : Endocannabinoid overload - Lichtman_2010_Mol.Pharmacol_78_993
Author(s) : Lichtman AH , Blankman JL , Cravatt BF
Ref : Molecular Pharmacology , 78 :993 , 2010
Abstract : The signaling capacity of endogenous cannabinoids ("endocannabinoids") is tightly regulated by degradative enzymes. This Perspective highlights a research article in this issue (p. 996) in which the authors show that genetic disruption of monoacylglycerol lipase (MAGL), the principal degradative enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG), causes marked elevations in 2-AG levels that lead to desensitization of brain cannabinoid receptors. These findings highlight the central role that MAGL plays in endocannabinoid metabolism in vivo and reveal that excessive 2-AG signaling can lead to functional antagonism of the brain cannabinoid system.
ESTHER : Lichtman_2010_Mol.Pharmacol_78_993
PubMedSearch : Lichtman_2010_Mol.Pharmacol_78_993
PubMedID: 20952498

Title : Fatty acid amide hydrolase and monoacylglycerol lipase inhibitors produce anti-allodynic effects in mice through distinct cannabinoid receptor mechanisms - Kinsey_2010_J.Pain_11_1420
Author(s) : Kinsey SG , Long JZ , Cravatt BF , Lichtman AH
Ref : J Pain , 11 :1420 , 2010
Abstract : UNLABELLED: The endocannabinoids anandamide and 2-arachidonoylglycerol are predominantly regulated by the respective catabolic enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). Inhibition of these enzymes elevates endocannabinoid levels and attenuates neuropathic pain. In the present study, CB(1) and CB(2) receptor-deficient mice were subjected to chronic constriction injury (CCI) of the sciatic nerve to examine the relative contribution of each receptor for the anti-allodynic effects of the FAAH inhibitor, PF-3845, and the MAGL inhibitor, JZL184. CCI caused marked hypersensitivity to mechanical and cold stimuli, which was not altered by deletion of either the CB(1) or CB(2) receptor, but was attenuated by gabapentin, as well as by each enzyme inhibitor. Whereas PF-3845 lacked anti-allodynic efficacy in both knockout lines, JZL184 did not produce anti-allodynic effects in CB(1) (-/-) mice, but retained its anti-allodynic effects in CB(2) (-/-) mice. These data indicate that FAAH and MAGL inhibitors reduce nerve injury-related hyperalgesic states through distinct cannabinoid receptor mechanisms of action. In conclusion, although endogenous cannabinoids do not appear to play a tonic role in long-term expression of neuropathic pain states, both FAAH and MAGL represent potential therapeutic targets for the development of pharmacological agents to treat chronic pain resulting from nerve injury. PERSPECTIVE: This article presents data addressing the cannabinoid receptor mechanisms underlying the anti-allodynic actions of endocannabinoid catabolic enzyme inhibitors in the mouse sciatic nerve ligation model. Fatty acid amide hydrolase and monoacylglycerol lipase inhibitors reduced allodynia through distinct cannabinoid receptor mechanisms. These enzymes offer potential targets to treat neuropathic pain.
ESTHER : Kinsey_2010_J.Pain_11_1420
PubMedSearch : Kinsey_2010_J.Pain_11_1420
PubMedID: 20554481

Title : Activity-based protein profiling for biochemical pathway discovery in cancer - Nomura_2010_Nat.Rev.Cancer_10_630
Author(s) : Nomura DK , Dix MM , Cravatt BF
Ref : Nat Rev Cancer , 10 :630 , 2010
Abstract : Large-scale profiling methods have uncovered numerous gene and protein expression changes that correlate with tumorigenesis. However, determining the relevance of these expression changes and which biochemical pathways they affect has been hindered by our incomplete understanding of the proteome and its myriad functions and modes of regulation. Activity-based profiling platforms enable both the discovery of cancer-relevant enzymes and selective pharmacological probes to perturb and characterize these proteins in tumour cells. When integrated with other large-scale profiling methods, activity-based proteomics can provide insight into the metabolic and signalling pathways that support cancer pathogenesis and illuminate new strategies for disease diagnosis and treatment.
ESTHER : Nomura_2010_Nat.Rev.Cancer_10_630
PubMedSearch : Nomura_2010_Nat.Rev.Cancer_10_630
PubMedID: 20703252

Title : RBBP9: a tumor-associated serine hydrolase activity required for pancreatic neoplasia - Shields_2010_Proc.Natl.Acad.Sci.U.S.A_107_2189
Author(s) : Shields DJ , Niessen S , Murphy EA , Mielgo A , Desgrosellier JS , Lau SK , Barnes LA , Lesperance J , Bouvet M , Tarin D , Cravatt BF , Cheresh DA
Ref : Proc Natl Acad Sci U S A , 107 :2189 , 2010
Abstract : Pancreatic cancer is one of the most lethal malignancies. To discover functionally relevant modulators of pancreatic neoplasia, we performed activity-based proteomic profiling on primary human ductal adenocarcinomas. Here, we identify retinoblastoma-binding protein 9 (RBBP9) as a tumor-associated serine hydrolase that displays elevated activity in pancreatic carcinomas. Whereas RBBP9 is expressed in normal and malignant tissues at similar levels, its elevated activity in tumor cells promotes anchorage-independent growth in vitro as well as pancreatic carcinogenesis in vivo. At the molecular level, RBBP9 activity overcomes TGF-beta-mediated antiproliferative signaling by reducing Smad2/3 phosphorylation, a previously unknown role for a serine hydrolase in cancer biology. Conversely, loss of endogenous RBBP9 or expression of mutationally inactive RBBP9 leads to elevated Smad2/3 phosphorylation, implicating this serine hydrolase as an essential suppressor of TGF-beta signaling. Finally, RBBP9-mediated suppression of TGF-beta signaling is required for E-cadherin expression as loss of the serine hydrolase activity leads to a reduction in E-cadherin levels and a concomitant decrease in the integrity of tumor cell-cell junctions. These data not only define a previously uncharacterized serine hydrolase activity associated with epithelial neoplasia, but also demonstrate the potential benefit of functional proteomics in the identification of new therapeutic targets.
ESTHER : Shields_2010_Proc.Natl.Acad.Sci.U.S.A_107_2189
PubMedSearch : Shields_2010_Proc.Natl.Acad.Sci.U.S.A_107_2189
PubMedID: 20080647
Gene_locus related to this paper: human-RBBP9

Title : Oxime esters as selective, covalent inhibitors of the serine hydrolase retinoblastoma-binding protein 9 (RBBP9) - Bachovchin_2010_Bioorg.Med.Chem.Lett_20_2254
Author(s) : Bachovchin DA , Wolfe MR , Masuda K , Brown SJ , Spicer TP , Fernandez-Vega V , Chase P , Hodder PS , Rosen H , Cravatt BF
Ref : Bioorganic & Medicinal Chemistry Lett , 20 :2254 , 2010
Abstract : We recently described a fluorescence polarization platform for competitive activity-based protein profiling (fluopol-ABPP) that enables high-throughput inhibitor screening for enzymes with poorly characterized biochemical activity. Here, we report the discovery of a class of oxime ester inhibitors for the unannotated serine hydrolase RBBP9 from a full-deck (200,000+ compound) fluopol-ABPP screen conducted in collaboration with the Molecular Libraries Screening Center Network (MLSCN). We show that these compounds covalently inhibit RBBP9 by modifying enzyme's active site serine nucleophile and, based on competitive ABPP in cell and tissue proteomes, are selective for RBBP9 relative to other mammalian serine hydrolases.
ESTHER : Bachovchin_2010_Bioorg.Med.Chem.Lett_20_2254
PubMedSearch : Bachovchin_2010_Bioorg.Med.Chem.Lett_20_2254
PubMedID: 20207142
Gene_locus related to this paper: human-RBBP9

Title : Probe Report for RBBP9 Inhibitors - Probe 2 - Bachovchin_2010_Probe.Report.1__
Author(s) : Bachovchin DA , Speers AE , Brown SJ , Cravatt BF , Spicer T , Mercer BA , Ferguson J , Hodder P , Rosen HR
Ref : Probe Report , : , 2010
Abstract : The retinoblastoma (RB) tumor suppressor protein controls cell cycle progression by regulating the activity of the transcription factor E2F, which activates genes essential for DNA replication. Thus, factors that bind and regulate RB activity are considered valuable targets for preventing tumorigenesis. The enzyme RB binding protein 9 (RBBP9) is widely expressed in different tissues and upregulated in certain tumors. As a result, the identification of compounds that selectively inhibit RBBP9 activity would serve as potentially valuable probes for the study of apoptosis, cell cycle, and tumorigenesis. We previously reported a modestly potent, RBBP9 reversible inhibitor, ML081 (CID-6603320). However, ML081 exhibits high cytotoxicity. We, therefore, have now identified a newer probe, ML114 (CID-5934766), which is 10-fold more potent than ML081, exhibits no cytotoxicity, and is from an entirely different structural and mechanistic class of compounds that covalently inhibit RBBP9. This new probe will be useful for in vitro assays in which it is desirable to specifically block RBBP9 activity for primary research purposes.
ESTHER : Bachovchin_2010_Probe.Report.1__
PubMedSearch : Bachovchin_2010_Probe.Report.1__
PubMedID: 21433382
Gene_locus related to this paper: human-RBBP9

Title : Monoacylglycerol lipase limits the duration of endocannabinoid-mediated depolarization-induced suppression of excitation in autaptic hippocampal neurons - Straiker_2009_Mol.Pharmacol_76_1220
Author(s) : Straiker A , Hu SS , Long JZ , Arnold A , Wager-Miller J , Cravatt BF , Mackie K
Ref : Molecular Pharmacology , 76 :1220 , 2009
Abstract : Depolarization-induced suppression of excitation (DSE) is a major form of cannabinoid-mediated short-term retrograde neuronal plasticity and is found in numerous brain regions. Autaptically cultured murine hippocampal neurons are an architecturally simple model for the study of cannabinoid signaling, including DSE. The transient nature of DSE--tens of seconds--is probably determined by the regulated hydrolysis of the endocannabinoid 2-arachidonoyl glycerol (2-AG). No less than five candidate enzymes have been considered to serve this role: fatty acid amide hydrolase (FAAH), cyclooxygenase-2 (COX-2), monoacylglycerol lipase (MGL), and alpha/beta-hydrolase domain (ABHD) 6 and 12. We previously found that FAAH and COX-2 do not have a role in determining the duration of autaptic DSE. In the current study, we found that two structurally distinct inhibitors of MGL [N-arachidonoyl maleimide and 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184)] prolong DSE in autaptic hippocampal neurons, whereas inhibition of ABHD6 by N-methyl-N-[[3-(4-pyridinyl)phenyl]methyl]-4'-(aminocarbonyl)[1,1'-biphenyl]-4-yl ester, carbamic acid (WWL70) had no effect. In addition, we developed antibodies against MGL and ABHD6 and determined their expression in autaptic cultures. MGL is chiefly expressed at presynaptic terminals, optimally positioned to break down 2-AG that has engaged presynaptic CB(1) receptors. ABHD6 is expressed in two distinct locations on autaptic islands, including a prominent localization in some dendrites. In summary, we provide strong pharmacological and anatomical evidence that MGL regulates DSE in autaptic hippocampal neurons and, taken together with other studies, emphasizes that endocannabinoid signaling is terminated in temporally diverse ways.
ESTHER : Straiker_2009_Mol.Pharmacol_76_1220
PubMedSearch : Straiker_2009_Mol.Pharmacol_76_1220
PubMedID: 19767452
Gene_locus related to this paper: human-MGLL

Title : Blockade of endocannabinoid-degrading enzymes attenuates neuropathic pain - Kinsey_2009_J.Pharmacol.Exp.Ther_330_902
Author(s) : Kinsey SG , Long JZ , O'Neal ST , Abdullah RA , Poklis JL , Boger DL , Cravatt BF , Lichtman AH
Ref : Journal of Pharmacology & Experimental Therapeutics , 330 :902 , 2009
Abstract : Direct-acting cannabinoid receptor agonists are well known to reduce hyperalgesic responses and allodynia after nerve injury, although their psychoactive side effects have damped enthusiasm for their therapeutic development. Alternatively, inhibiting fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), the principal enzymes responsible for the degradation of the respective endogenous cannabinoids, anandamide (AEA) and 2-arachydonylglycerol (2-AG), reduce nociception in a variety of nociceptive assays, with no or minimal behavioral effects. In the present study we tested whether inhibition of these enzymes attenuates mechanical allodynia, and acetone-induced cold allodynia in mice subjected to chronic constriction injury of the sciatic nerve. Acute administration of the irreversible FAAH inhibitor, cyclohexylcarbamic acid 3'-carbamoylbiphenyl-3-yl ester (URB597), or the reversible FAAH inhibitor, 1-oxo-1-[5-(2-pyridyl)-2-yl]-7-phenylheptane (OL-135), decreased allodynia in both tests. This attenuation was completely blocked by pretreatment with either CB(1) or CB(2) receptor antagonists, but not by the TRPV1 receptor antagonist, capsazepine, or the opioid receptor antagonist, naltrexone. The novel MAGL inhibitor, 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184) also attenuated mechanical and cold allodynia via a CB(1), but not a CB(2), receptor mechanism of action. Whereas URB597 did not elicit antiallodynic effects in FAAH(-/-) mice, the effects of JZL184 were FAAH-independent. Finally, URB597 increased brain and spinal cord AEA levels, whereas JZL184 increased 2-AG levels in these tissues, but no differences in either endo-cannabinoid were found between nerve-injured and control mice. These data indicate that inhibition of FAAH and MAGL reduces neuropathic pain through distinct receptor mechanisms of action and present viable targets for the development of analgesic therapeutics.
ESTHER : Kinsey_2009_J.Pharmacol.Exp.Ther_330_902
PubMedSearch : Kinsey_2009_J.Pharmacol.Exp.Ther_330_902
PubMedID: 19502530

Title : Dual blockade of FAAH and MAGL identifies behavioral processes regulated by endocannabinoid crosstalk in vivo - Long_2009_Proc.Natl.Acad.Sci.U.S.A_106_20270
Author(s) : Long JZ , Nomura DK , Vann RE , Walentiny DM , Booker L , Jin X , Burston JJ , Sim-Selley LJ , Lichtman AH , Wiley JL , Cravatt BF
Ref : Proc Natl Acad Sci U S A , 106 :20270 , 2009
Abstract : Delta(9)-tetrahydrocannabinol (THC), the psychoactive component of marijuana, and other direct cannabinoid receptor (CB1) agonists produce a number of neurobehavioral effects in mammals that range from the beneficial (analgesia) to the untoward (abuse potential). Why, however, this full spectrum of activities is not observed upon pharmacological inhibition or genetic deletion of either fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL), enzymes that regulate the two major endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG), respectively, has remained unclear. Here, we describe a selective and efficacious dual FAAH/MAGL inhibitor, JZL195, and show that this agent exhibits broad activity in the tetrad test for CB1 agonism, causing analgesia, hypomotilty, and catalepsy. Comparison of JZL195 to specific FAAH and MAGL inhibitors identified behavioral processes that were regulated by a single endocannabinoid pathway (e.g., hypomotility by the 2-AG/MAGL pathway) and, interestingly, those where disruption of both FAAH and MAGL produced additive effects that were reversed by a CB1 antagonist. Falling into this latter category was drug discrimination behavior, where dual FAAH/MAGL blockade, but not disruption of either FAAH or MAGL alone, produced THC-like responses that were reversed by a CB1 antagonist. These data indicate that AEA and 2-AG signaling pathways interact to regulate specific behavioral processes in vivo, including those relevant to drug abuse, thus providing a potential mechanistic basis for the distinct pharmacological profiles of direct CB1 agonists and inhibitors of individual endocannabinoid degradative enzymes.
ESTHER : Long_2009_Proc.Natl.Acad.Sci.U.S.A_106_20270
PubMedSearch : Long_2009_Proc.Natl.Acad.Sci.U.S.A_106_20270
PubMedID: 19918051

Title : Inhibitors of endocannabinoid-metabolizing enzymes reduce precipitated withdrawal responses in THC-dependent mice - Schlosburg_2009_AAPS.J_11_342
Author(s) : Schlosburg JE , Carlson BL , Ramesh D , Abdullah RA , Long JZ , Cravatt BF , Lichtman AH
Ref : AAPS J , 11 :342 , 2009
Abstract : Abstinence symptoms in cannabis-dependent individuals are believed to contribute to the maintenance of regular marijuana use. However, there are currently no medications approved by the FDA to treat cannabis-related disorders. The only treatment currently shown consistently to alleviate cannabinoid withdrawal in both animals and humans is substitution therapy using the psychoactive constituent of marijuana, Delta(9)-tetrahydrocannabinol (THC). However, new genetic and pharmacological tools are available to increase endocannabinoid levels by targeting fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL), the enzymes responsible for the degradation of the endogenous cannabinoid ligands anandamide and 2-arachidonoylglycerol, respectively. In the present study, we investigated whether increasing endogenous cannabinoids levels, through the use of FAAH (-/-) mice as well as the FAAH inhibitor URB597 or the MAGL inhibitor JZL184, would reduce the intensity of withdrawal signs precipitated by the CB(1) receptor antagonist rimonabant in THC-dependent mice. Strikingly, acute administration of either URB597 or JZL184 significantly attenuated rimonabant-precipitated withdrawal signs in THC-dependent mice. In contrast, FAAH (-/-) mice showed identical withdrawal responses as wild-type mice under a variety of conditions, suggesting that the absence of this enzyme across the development of dependence and during rimonabant challenge does not affect withdrawal responses. Of importance, subchronic administration of URB597 did not lead to cannabinoid dependence and neither URB597 nor JZL184 impaired rotarod motor coordination. These results support the concept of targeting endocannabinoid metabolizing enzymes as a promising treatment for cannabis withdrawal.
ESTHER : Schlosburg_2009_AAPS.J_11_342
PubMedSearch : Schlosburg_2009_AAPS.J_11_342
PubMedID: 19430909

Title : Diversity of serine hydrolase activities of unchallenged and botrytis-infected Arabidopsis thaliana - Kaschani_2009_Mol.Cell.Proteomics_8_1082
Author(s) : Kaschani F , Gu C , Niessen S , Hoover H , Cravatt BF , van der Hoorn RA
Ref : Mol Cell Proteomics , 8 :1082 , 2009
Abstract : Activity-based protein profiling is a powerful method to display enzyme activities in proteomes and provides crucial information on enzyme activity rather than protein or transcript abundance. We applied activity-based protein profiling using fluorophosphonate-based probes to display the activities of Ser hydrolases in the model plant Arabidopsis thaliana. Multidimensional protein identification technology and in-gel analysis of fluorophosphonate-labeled leaf extracts revealed over 50 Ser hydrolases, including dozens of proteases, esterases, and lipases, representing over 10 different enzyme families. Except for some well characterized Ser hydrolases like subtilases TPP2 and ARA12, prolyl oligopeptidase acylamino acid-releasing enzyme, serine carboxypeptidase-like SNG1 and BRS1, carboxylesterase-like CXE12, methylesterases MES2 and MES3, and S-formylglutathione hydrolase, the majority of these serine hydrolases have not been described before. We studied transiently expressed SNG1 and investigated plants infected with the fungal pathogen Botrytis cinerea. Besides the down-regulation of several Arabidopsis Ser hydrolase activities during Botrytis infection, we detected the activities of Botrytis-derived cutinases and lipases, which are thought to contribute to pathogenicity.
ESTHER : Kaschani_2009_Mol.Cell.Proteomics_8_1082
PubMedSearch : Kaschani_2009_Mol.Cell.Proteomics_8_1082
PubMedID: 19136719

Title : Large-scale profiling of protein palmitoylation in mammalian cells - Martin_2009_Nat.Methods_6_135
Author(s) : Martin BR , Cravatt BF
Ref : Nat Methods , 6 :135 , 2009
Abstract : S-palmitoylation is a pervasive post-translational modification required for the trafficking, compartmentalization and membrane tethering of many proteins. We demonstrate that the commercially available compound 17-octadecynoic acid (17-ODYA) can serve as a bioorthogonal, click chemistry probe for in situ labeling, identification and verification of palmitoylated proteins in human cells. We identified approximately 125 predicted palmitoylated proteins, including G proteins, receptors and a family of uncharacterized hydrolases whose plasma membrane localization depends on palmitoylation.
ESTHER : Martin_2009_Nat.Methods_6_135
PubMedSearch : Martin_2009_Nat.Methods_6_135
PubMedID: 19137006
Gene_locus related to this paper: human-ABHD17A , human-ABHD17B , human-ABHD17C

Title : Blockade of 2-arachidonoylglycerol hydrolysis by selective monoacylglycerol lipase inhibitor 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184) Enhances retrograde endocannabinoid signaling - Pan_2009_J.Pharmacol.Exp.Ther_331_591
Author(s) : Pan B , Wang W , Long JZ , Sun D , Hillard CJ , Cravatt BF , Liu QS
Ref : Journal of Pharmacology & Experimental Therapeutics , 331 :591 , 2009
Abstract : Endocannabinoid (eCB) signaling mediates depolarization-induced suppression of excitation (DSE) and inhibition (DSI), two prominent forms of retrograde synaptic depression. N-Arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG), two known eCBs, are degraded by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. Selective blockade of FAAH and MAGL is critical for determining the roles of the eCBs in DSE/DSI and understanding how their action is regulated. 4-Nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184) is a recently developed, highly selective, and potent MAGL inhibitor that increases 2-AG but not AEA concentrations in mouse brain. Here, we report that JZL184 prolongs DSE in Purkinje neurons in cerebellar slices and DSI in CA1 pyramidal neurons in hippocampal slices. The effect of JZL184 on DSE/DSI is mimicked by the nonselective MAGL inhibitor methyl arachidonyl fluorophosphonate. In contrast, neither the selective FAAH inhibitor cyclohexylcarbamic acid 3'-carbomoylbiphenyl-3-yl ester (URB597) nor FAAH knockout has a significant effect on DSE/DSI. JZL184 produces greater enhancement of DSE/DSI in mouse neurons than that in rat neurons. The latter finding is consistent with biochemical studies showing that JZL184 is more potent in inhibiting mouse MAGL than rat MAGL. These results indicate that the degradation of 2-AG by MAGL is the rate-limiting step that determines the time course of DSE/DSI and that JZL184 is a useful tool for the study of 2-AG-mediated signaling.
ESTHER : Pan_2009_J.Pharmacol.Exp.Ther_331_591
PubMedSearch : Pan_2009_J.Pharmacol.Exp.Ther_331_591
PubMedID: 19666749

Title : Identification of selective inhibitors of uncharacterized enzymes by high-throughput screening with fluorescent activity-based probes - Bachovchin_2009_Nat.Biotechnol_27_387
Author(s) : Bachovchin DA , Brown SJ , Rosen H , Cravatt BF
Ref : Nat Biotechnol , 27 :387 , 2009
Abstract : High-throughput screening to discover small-molecule modulators of enzymes typically relies on highly tailored substrate assays, which are not available for poorly characterized enzymes. Here we report a general, substrate-free method for identifying inhibitors of uncharacterized enzymes. The assay measures changes in the kinetics of covalent active-site labeling with broad-spectrum, fluorescent probes in the presence of inhibitors by monitoring the fluorescence polarization signal. We show that this technology is applicable to enzymes from at least two mechanistic classes, regardless of their degree of functional annotation, and can be coupled with secondary proteomic assays that use competitive activity-based profiling to rapidly determine the specificity of screening hits. Using this method, we identify the bioactive alkaloid emetine as a selective inhibitor of the uncharacterized cancer-associated hydrolase RBBP9. Furthermore, we show that the detoxification enzyme GSTO1, also implicated in cancer, is inhibited by several electrophilic compounds found in public libraries, some of which display high selectivity for this protein.
ESTHER : Bachovchin_2009_Nat.Biotechnol_27_387
PubMedSearch : Bachovchin_2009_Nat.Biotechnol_27_387
PubMedID: 19329999
Gene_locus related to this paper: human-ABHD11

Title : Selective blockade of 2-arachidonoylglycerol hydrolysis produces cannabinoid behavioral effects - Long_2009_Nat.Chem.Biol_5_37
Author(s) : Long JZ , Li W , Booker L , Burston JJ , Kinsey SG , Schlosburg JE , Pavon FJ , Serrano AM , Selley DE , Parsons LH , Lichtman AH , Cravatt BF
Ref : Nat Chemical Biology , 5 :37 , 2009
Abstract : 2-Arachidonoylglycerol (2-AG) and anandamide are endocannabinoids that activate the cannabinoid receptors CB1 and CB2. Endocannabinoid signaling is terminated by enzymatic hydrolysis, a process that for anandamide is mediated by fatty acid amide hydrolase (FAAH), and for 2-AG is thought to involve monoacylglycerol lipase (MAGL). FAAH inhibitors produce a select subset of the behavioral effects observed with CB1 agonists, which suggests a functional segregation of endocannabinoid signaling pathways in vivo. Testing this hypothesis, however, requires specific tools to independently block anandamide and 2-AG metabolism. Here, we report a potent and selective inhibitor of MAGL called JZL184 that, upon administration to mice, raises brain 2-AG by eight-fold without altering anandamide. JZL184-treated mice exhibited a broad array of CB1-dependent behavioral effects, including analgesia, hypothermia and hypomotility. These data indicate that 2-AG endogenously modulates several behavioral processes classically associated with the pharmacology of cannabinoids and point to overlapping and unique functions for 2-AG and anandamide in vivo.
ESTHER : Long_2009_Nat.Chem.Biol_5_37
PubMedSearch : Long_2009_Nat.Chem.Biol_5_37
PubMedID: 19029917

Title : Characterization of monoacylglycerol lipase inhibition reveals differences in central and peripheral endocannabinoid metabolism - Long_2009_Chem.Biol_16_744
Author(s) : Long JZ , Nomura DK , Cravatt BF
Ref : Chemical Biology , 16 :744 , 2009
Abstract : Monoacylglycerol lipase (MAGL) is a principal degradative enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG). We recently reported a piperidine carbamate, JZL184, that inhibits MAGL with high potency and selectivity. Here, we describe a comprehensive mechanistic characterization of JZL184. We provide evidence that JZL184 irreversibly inhibits MAGL via carbamoylation of the enzyme's serine nucleophile. Functional proteomic analysis of mice treated with JZL184 revealed that this inhibitor maintains good selectivity for MAGL across a wide range of central and peripheral tissues. Interestingly, MAGL blockade produced marked, tissue-specific differences in monoglyceride metabolism, with brain showing the most dramatic elevations in 2-AG and peripheral tissues often showing greater changes in other monoglycerides. Collectively, these studies indicate that MAGL exerts tissue-dependent control over endocannabinoid and monoglyceride metabolism and designate JZL184 as a selective tool to characterize the functions of MAGL in vivo.
ESTHER : Long_2009_Chem.Biol_16_744
PubMedSearch : Long_2009_Chem.Biol_16_744
PubMedID: 19635411

Title : Dual roles of brain serine hydrolase KIAA1363 in ether lipid metabolism and organophosphate detoxification - Nomura_2008_Toxicol.Appl.Pharmacol_228_42
Author(s) : Nomura DK , Fujioka K , Issa RS , Ward AM , Cravatt BF , Casida JE
Ref : Toxicol Appl Pharmacol , 228 :42 , 2008
Abstract : Serine hydrolase KIAA1363 is an acetyl monoalkylglycerol ether (AcMAGE) hydrolase involved in tumor cell invasiveness. It is also an organophosphate (OP) insecticide-detoxifying enzyme. The key to understanding these dual properties was the use of KIAA1363 +/+ (wildtype) and -/- (gene deficient) mice to define the role of this enzyme in brain and other tissues and its effectiveness in vivo in reducing OP toxicity. KIAA1363 was the primary AcMAGE hydrolase in brain, lung, heart and kidney and was highly sensitive to inactivation by chlorpyrifos oxon (CPO) (IC50 2 nM) [the bioactivated metabolite of the major insecticide chlorpyrifos (CPF)]. Although there was no difference in hydrolysis product monoalkylglycerol ether (MAGE) levels in +/+ and -/- mouse brains in vivo, isopropyl dodecylfluorophosphonate (30 mg/kg) and CPF (100 mg/kg) resulted in 23-51% decrease in brain MAGE levels consistent with inhibition of AcMAGE hydrolase activity. On incubating +/+ and -/- brain membranes with AcMAGE and cytidine-5'-diphosphocholine, the absence of KIAA1363 activity dramatically increased de novo formation of platelet-activating factor (PAF) and lyso-PAF, signifying that metabolically-stabilized AcMAGE can be converted to this bioactive lipid in brain. On considering detoxification, KIAA1363 -/- mice were significantly more sensitive than +/+ mice to ip-administered CPF (100 mg/kg) and parathion (10 mg/kg) with increased tremoring and mortality that correlated for CPF with greater brain acetylcholinesterase inhibition. Docking AcMAGE and CPO in a KIAA1363 active site model showed similar positioning of their acetyl and trichloropyridinyl moieties, respectively. This study establishes the relevance of KIAA1363 in ether lipid metabolism and OP detoxification.
ESTHER : Nomura_2008_Toxicol.Appl.Pharmacol_228_42
PubMedSearch : Nomura_2008_Toxicol.Appl.Pharmacol_228_42
PubMedID: 18164358
Gene_locus related to this paper: human-NCEH1

Title : Evaluation of fatty acid amides in the carrageenan-induced paw edema model - Wise_2008_Neuropharmacol_54_181
Author(s) : Wise LE , Cannavacciulo R , Cravatt BF , Martin BF , Lichtman AH
Ref : Neuropharmacology , 54 :181 , 2008
Abstract : While it has long been recognized that Delta(9)-tetrahydrocannabinol (THC), the primary psychoactive constituent of cannabis, and other cannabinoid receptor agonists possess anti-inflammatory properties, their well known CNS effects have dampened enthusiasm for therapeutic development. On the other hand, genetic deletion of fatty acid amide hydrolase (FAAH), the enzyme responsible for degradation of fatty acid amides, including endogenous cannabinoid N-arachidonoyl ethanolamine (anandamide; AEA), N-palmitoyl ethanolamine (PEA), N-oleoyl ethanolamine (OEA), and oleamide, also elicits anti-edema, but does not produce any apparent cannabinoid effects. The purpose of the present study was to investigate whether exogenous administration of FAAs would augment the anti-inflammatory phenotype of FAAH (-/-) mice in the carrageenan model. Thus, we evaluated the effects of the FAAs AEA, PEA, OEA, and oleamide in wild-type and FAAH (-/-) mice. For comparison, we evaluated the anti-edema effects of THC, dexamethasone (DEX), a synthetic glucocorticoid, diclofenac (DIC), a nonselective cyclooxygenase (COX) inhibitor, in both genotypes. A final study determined if tolerance to the anti-edema effects of PEA occurs after repeated dosing. PEA, THC, DEX, DIC elicited significant decreases in carrageenan-induced paw edema in wild-type mice. In contrast OEA produced a less reliable anti-edema effect than these other drugs, and AEA and oleamide failed to produce any significant decreases in paw edema. Moreover, none of the agents evaluated augmented the anti-edema phenotype of FAAH (-/-) mice, suggesting that maximal anti-edema effects had already been established. PEA was the most effective FAA in preventing paw edema and its effects did not undergo tolerance. While the present findings do not support a role for AEA in preventing carrageenan-induced edema, PEA administration and FAAH blockade elicited anti-edema effects of an equivalent magnitude as produced by THC, DEX, and DIC in this assay.
ESTHER : Wise_2008_Neuropharmacol_54_181
PubMedSearch : Wise_2008_Neuropharmacol_54_181
PubMedID: 17675189

Title : Activation of the endocannabinoid system by organophosphorus nerve agents - Nomura_2008_Nat.Chem.Biol_4_373
Author(s) : Nomura DK , Blankman JL , Simon GM , Fujioka K , Issa RS , Ward AM , Cravatt BF , Casida JE
Ref : Nat Chemical Biology , 4 :373 , 2008
Abstract : Delta(9)-tetrahydrocannabinol (THC), the psychoactive ingredient of marijuana, has useful medicinal properties but also undesirable side effects. The brain receptor for THC, CB(1), is also activated by the endogenous cannabinoids anandamide and 2-arachidonylglycerol (2-AG). Augmentation of endocannabinoid signaling by blockade of their metabolism may offer a more selective pharmacological approach compared with CB(1) agonists. Consistent with this premise, inhibitors of the anandamide-degrading enzyme fatty acid amide hydrolase (FAAH) produce analgesic and anxiolytic effects without cognitive defects. In contrast, we show that dual blockade of the endocannabinoid-degrading enzymes monoacylglycerol lipase (MAGL) and FAAH by selected organophosphorus agents leads to greater than ten-fold elevations in brain levels of both 2-AG and anandamide and to robust CB(1)-dependent behavioral effects that mirror those observed with CB(1) agonists. Arachidonic acid levels are decreased by the organophosphorus agents in amounts equivalent to elevations in 2-AG, which indicates that endocannabinoid and eicosanoid signaling pathways may be coordinately regulated in the brain.
ESTHER : Nomura_2008_Nat.Chem.Biol_4_373
PubMedSearch : Nomura_2008_Nat.Chem.Biol_4_373
PubMedID: 18438404

Title : Selectivity of inhibitors of endocannabinoid biosynthesis evaluated by activity-based protein profiling - Hoover_2008_Bioorg.Med.Chem.Lett_18_5838
Author(s) : Hoover HS , Blankman JL , Niessen S , Cravatt BF
Ref : Bioorganic & Medicinal Chemistry Lett , 18 :5838 , 2008
Abstract : The endocannabinoid 2-arachidonoylglycerol (2-AG) has been implicated as a key retrograde mediator in the nervous system based on pharmacological studies using inhibitors of the 2-AG biosynthetic enzymes diacyglycerol lipase alpha and beta (DAGL-alpha/beta). Here, we show by competitive activity-based protein profiling that the DAGL-alpha/beta inhibitors, tetrahydrolipstatin (THL) and RHC80267, block several brain serine hydrolases with potencies equal to or greater than their inhibitory activity against DAGL enzymes. Interestingly, a minimal overlap in target profiles was observed for THL and RHC80267, suggesting that pharmacological effects observed with both agents may be viewed as good initial evidence for DAGL-dependent events.
ESTHER : Hoover_2008_Bioorg.Med.Chem.Lett_18_5838
PubMedSearch : Hoover_2008_Bioorg.Med.Chem.Lett_18_5838
PubMedID: 18657971

Title : Exploration of a fundamental substituent effect of alpha-ketoheterocycle enzyme inhibitors: Potent and selective inhibitors of fatty acid amide hydrolase - DeMartino_2008_Bioorg.Med.Chem.Lett_18_5842
Author(s) : DeMartino JK , Garfunkle J , Hochstatter DG , Cravatt BF , Boger DL
Ref : Bioorganic & Medicinal Chemistry Lett , 18 :5842 , 2008
Abstract : A series of C4 substituted alpha-ketooxazoles were examined as inhibitors of the serine hydrolase fatty acid amide hydrolase in efforts that further define and generalize a fundamental substituent effect on enzyme inhibitory potency. Thus, a plot of the Hammett sigma(m) versus -logK(i) provided a linear correlation (R(2)=0.90) with a slope of 3.37 (rho=3.37), that is of a magnitude that indicates that of the electron-withdrawing character of the substituent dominates its effects (a one unit change in sigma(m) provides a >1000-fold change in K(i)).
ESTHER : DeMartino_2008_Bioorg.Med.Chem.Lett_18_5842
PubMedSearch : DeMartino_2008_Bioorg.Med.Chem.Lett_18_5842
PubMedID: 18639454

Title : Anandamide biosynthesis catalyzed by the phosphodiesterase GDE1 and detection of glycerophospho-N-acyl ethanolamine precursors in mouse brain - Simon_2008_J.Biol.Chem_283_9341
Author(s) : Simon GM , Cravatt BF
Ref : Journal of Biological Chemistry , 283 :9341 , 2008
Abstract : Anandamide (AEA) is an endogenous ligand of cannabinoid receptors and a well characterized mediator of many physiological processes including inflammation, pain, and appetite. The biosynthetic pathway(s) for anandamide and its N-acyl ethanolamine (NAE) congeners remain enigmatic. Previously, we proposed an enzymatic route for producing NAEs that involves the double-O-deacylation of N-acyl phosphatidylethanolamines (NAPEs) by alpha/beta-hydrolase 4 (ABDH4 or Abh4) to form glycerophospho (GP)-NAEs, followed by conversion of these intermediates to NAEs by an unidentified phosphodiesterase. Here, we report the detection and measurement of GP-NAEs, including the anandamide precursor glycerophospho-N-arachidonoylethanolamine (GP-NArE), as endogenous constituents of mouse brain tissue. Inhibition of the phosphodiesterase-mediated degradation of GP-NAEs ex vivo resulted in a striking accumulation of these lipids in brain extracts, suggesting a rapid endogenous flux through this pathway. Furthermore, we identify the glycerophosphodiesterase GDE1, also known as MIR16, as a broadly expressed membrane enzyme with robust GP-NAE phosphodiesterase activity. Together, these data provide evidence for a multistep pathway for the production of anandamide in the nervous system by the sequential actions of Abh4 and GDE1.
ESTHER : Simon_2008_J.Biol.Chem_283_9341
PubMedSearch : Simon_2008_J.Biol.Chem_283_9341
PubMedID: 18227059

Title : A functional proteomic strategy to discover inhibitors for uncharacterized hydrolases -
Author(s) : Li W , Blankman JL , Cravatt BF
Ref : Journal of the American Chemical Society , 129 :9594 , 2007
PubMedID: 17629278

Title : A comprehensive profile of brain enzymes that hydrolyze the endocannabinoid 2-arachidonoylglycerol - Blankman_2007_Chem.Biol_14_1347
Author(s) : Blankman JL , Simon GM , Cravatt BF
Ref : Chemical Biology , 14 :1347 , 2007
Abstract : Endogenous ligands for cannabinoid receptors ("endocannabinoids") include the lipid transmitters anandamide and 2-arachidonoylglycerol (2-AG). Endocannabinoids modulate a diverse set of physiological processes and are tightly regulated by enzymatic biosynthesis and degradation. Termination of anandamide signaling by fatty acid amide hydrolase (FAAH) is well characterized, but less is known about the inactivation of 2-AG, which can be hydrolyzed by multiple enzymes in vitro, including FAAH and monoacylglycerol lipase (MAGL). Here, we have taken a functional proteomic approach to comprehensively map 2-AG hydrolases in the mouse brain. Our data reveal that approximately 85% of brain 2-AG hydrolase activity can be ascribed to MAGL, and that the remaining 15% is mostly catalyzed by two uncharacterized enzymes, ABHD6 and ABHD12. Interestingly, MAGL, ABHD6, and ABHD12 display distinct subcellular distributions, suggesting that they may control different pools of 2-AG in the nervous system.
ESTHER : Blankman_2007_Chem.Biol_14_1347
PubMedSearch : Blankman_2007_Chem.Biol_14_1347
PubMedID: 18096503
Gene_locus related to this paper: human-ABHD6 , human-ABHD12 , human-MGLL

Title : Evaluation of fatty acid amide hydrolase inhibition in murine models of emotionality - Naidu_2007_Psychopharmacology.(Berl)_192_61
Author(s) : Naidu PS , Varvel SA , Ahn K , Cravatt BF , Martin BR , Lichtman AH
Ref : Psychopharmacology (Berl) , 192 :61 , 2007
Abstract : RATIONALE: Manipulations of the endocannabinoid/fatty acid amide hydrolase (FAAH) signaling systems result in conflicting and paradoxical effects in rodent models of emotional reactivity. OBJECTIVES: In the present study, we tested the hypothesis that the inhibition of FAAH would elicit significant effects in murine models used to screen anxiolytic and antidepressant drugs. MATERIALS AND
METHODS: FAAH (-/-) mice and wild-type mice treated with FAAH inhibitors (URB597 and OL-135) were evaluated in standard behavioral screening models for antidepressant (i.e., tail suspension and forced-swim tests) and anxiolytic (i.e., elevated plus maze) agents. The doses of URB597 and OL-135 selected were based on their ability to augment the pharmacological effects (i.e., analgesia, catalepsy, and hypothermia) of exogenously administered anandamide.
RESULTS: FAAH (-/-) mice, anandamide-injected FAAH (-/-) mice, or wild-type mice injected with FAAH inhibitors or anandamide failed to exhibit significant effects in standard tests of emotional reactivity, although the antidepressant desipramine and the anxiolytic agent midazolam were active in the appropriate assays. FAAH- (-/-) and URB597-treated mice finally displayed significant effects in the tail suspension test when substantial methodological changes were made (i.e., altered ambient light and increased sample sizes).
CONCLUSIONS: Although FAAH suppression can elicit significant effects under some instances in which consequential procedural modifications are made, the present results indicate that the pharmacological inhibition or genetic deletion of FAAH is ineffective in standard mouse models of emotional reactivity. It remains to be established whether the effects of FAAH inhibition in these modified tasks are predictive of their efficacy in treating emotional disorders.
ESTHER : Naidu_2007_Psychopharmacology.(Berl)_192_61
PubMedSearch : Naidu_2007_Psychopharmacology.(Berl)_192_61
PubMedID: 17279376

Title : Assessment of anandamide's pharmacological effects in mice deficient of both fatty acid amide hydrolase and cannabinoid CB1 receptors - Wise_2007_Eur.J.Pharmacol_557_44
Author(s) : Wise LE , Shelton CC , Cravatt BF , Martin BR , Lichtman AH
Ref : European Journal of Pharmacology , 557 :44 , 2007
Abstract : In the present study, we investigated whether anandamide produces its behavioral effects through a cannabinoid CB(1) receptor mechanism of action. The behavioral effects of anandamide were evaluated in mice that lacked both fatty acid amide hydrolase (FAAH) and cannabinoid CB(1) receptors (DKO) as compared to FAAH (-/-), cannabinoid CB(1) (-/-), and wild type mice. Anandamide produced analgesia, catalepsy, and hypothermia in FAAH (-/-) mice, but failed to elicit any of these effects in the other three genotypes. In contrast, anandamide decreased locomotor behavior regardless of genotype, suggesting the involvement of multiple mechanisms of action, including its products of degradation. These findings indicate that the cannabinoid CB(1) receptor is the predominant target mediating anandamide's behavioral effects.
ESTHER : Wise_2007_Eur.J.Pharmacol_557_44
PubMedSearch : Wise_2007_Eur.J.Pharmacol_557_44
PubMedID: 17217945

Title : The putative endocannabinoid transport blocker LY2183240 is a potent inhibitor of FAAH and several other brain serine hydrolases - Alexander_2006_J.Am.Chem.Soc_128_9699
Author(s) : Alexander JP , Cravatt BF
Ref : Journal of the American Chemical Society , 128 :9699 , 2006
Abstract : How lipid transmitters move within and between cells to communicate signals remains an important and largely unanswered question. Integral membrane transporters, soluble lipid-binding proteins, and metabolic enzymes have all been proposed to collaboratively regulate lipid signaling dynamics in vivo. Assignment of the relative contributions made by each of these classes of proteins requires selective pharmacological agents to perturb their individual functions. Recently, LY2183240, a heterocyclic urea inhibitor of the putative endocannabinoid (EC) transporter, was shown to disrupt the cellular uptake of the lipid EC anandamide and promote analgesia in vivo. Here, we show that LY2183240 is a potent, covalent inhibitor of the EC-degrading enzyme fatty acid amide hydrolase (FAAH). LY2183240 inactivates FAAH by carbamylation of the enzyme's serine nucleophile. More global screens using activity-based proteomic probes identified several additional serine hydrolases that are also inhibited by LY2183240. These results indicate that the blockade of anandamide uptake observed with LY2183240 may be due primarily to the inactivation of FAAH, providing further evidence that this enzyme serves as a metabolic driving force that promotes the diffusion of anandamide into cells. More generally, the proteome-wide target promiscuity of LY2183240 designates the heterocyclic urea as a chemotype with potentially excessive protein reactivity for drug design.
ESTHER : Alexander_2006_J.Am.Chem.Soc_128_9699
PubMedSearch : Alexander_2006_J.Am.Chem.Soc_128_9699
PubMedID: 16866524

Title : Serine hydrolase KIAA1363: toxicological and structural features with emphasis on organophosphate interactions - Nomura_2006_Chem.Res.Toxicol_19_1142
Author(s) : Nomura DK , Durkin KA , Chiang KP , Quistad GB , Cravatt BF , Casida JE
Ref : Chemical Research in Toxicology , 19 :1142 , 2006
Abstract : Serine hydrolase KIAA1363 is highly expressed in invasive cancer cells and is the major protein in mouse brain diethylphosphorylated by and hydrolyzing low levels of chlorpyrifos oxon (CPO) (the activated metabolite of a major insecticide). It is also the primary CPO-hydrolyzing enzyme in spinal cord, kidney, heart, lung, testis, and muscle but not liver, a pattern of tissue expression confirmed by fluorophosphonate-rhodamine labeling. KIAA1363 gene deletion using homologous recombination reduces CPO binding, hydrolysis, and metabolism 3-29-fold on incubation with brain membranes and homogenates determined with 1 nM [(3)H-ethyl]CPO and the inhibitory potency for residual CPO with butyrylcholinesterase as a biomarker. Studies with knockout mice further show that KIAA1363 partially protects brain AChE and monoacylglycerol lipase from CPO-induced in vivo inhibition. Surprisingly, mouse brain KIAA1363 and AChE are similar in in vitro sensitivity to seven methyl, ethyl, and propyl but not higher alkyl OP insecticides and analogues, prompting structural comparisons of the active sites of KIAA1363 and AChE relative to OP potency and selectivity. Homology modeling based largely on the Archaeoglobus fulgidus esterase crystal structure indicates that KIAA1363 has a catalytic triad of S191, D348, and H378, a GDSAG motif, and an oxyanion hole of H113, G114, G115, and G116. Excellent selectivity for KIAA1363 is achieved on OP structure optimization with long alkyl chain substituents suggesting that KIAA1363 has larger acyl and leaving group pockets than those of AChE. KIAA1363 reactivates faster than AChE presumably due to differences in the uncoupling of the catalytic triad His upon phosphorylation. The structural modeling of KIAA1363 helps us understand OP structure-activity relationships and the toxicological relevance of this detoxifying enzyme.
ESTHER : Nomura_2006_Chem.Res.Toxicol_19_1142
PubMedSearch : Nomura_2006_Chem.Res.Toxicol_19_1142
PubMedID: 16978018
Gene_locus related to this paper: human-NCEH1 , mouse-Q8BLF1

Title : An enzyme that regulates ether lipid signaling pathways in cancer annotated by multidimensional profiling - Chiang_2006_Chem.Biol_13_1041
Author(s) : Chiang KP , Niessen S , Saghatelian A , Cravatt BF
Ref : Chemical Biology , 13 :1041 , 2006
Abstract : Hundreds, if not thousands, of uncharacterized enzymes currently populate the human proteome. Assembly of these proteins into the metabolic and signaling pathways that govern cell physiology and pathology constitutes a grand experimental challenge. Here, we address this problem by using a multidimensional profiling strategy that combines activity-based proteomics and metabolomics. This approach determined that KIAA1363, an uncharacterized enzyme highly elevated in aggressive cancer cells, serves as a central node in an ether lipid signaling network that bridges platelet-activating factor and lysophosphatidic acid. Biochemical studies confirmed that KIAA1363 regulates this pathway by hydrolyzing the metabolic intermediate 2-acetyl monoalkylglycerol. Inactivation of KIAA1363 disrupted ether lipid metabolism in cancer cells and impaired cell migration and tumor growth in vivo. The integrated molecular profiling method described herein should facilitate the functional annotation of metabolic enzymes in any living system.
ESTHER : Chiang_2006_Chem.Biol_13_1041
PubMedSearch : Chiang_2006_Chem.Biol_13_1041
PubMedID: 17052608
Gene_locus related to this paper: human-NCEH1 , mouse-Q8BLF1

Title : Endocannabinoid biosynthesis proceeding through glycerophospho-N-acyl ethanolamine and a role for alpha\/beta-hydrolase 4 in this pathway - Simon_2006_J.Biol.Chem_281_26465
Author(s) : Simon GM , Cravatt BF
Ref : Journal of Biological Chemistry , 281 :26465 , 2006
Abstract : N-Acyl ethanolamines (NAEs) are a large class of signaling lipids implicated in diverse physiological processes, including nociception, cognition, anxiety, appetite, and inflammation. It has been proposed that NAEs are biosynthesized from their corresponding N-acyl phosphatidylethanolamines (NAPEs) in a single enzymatic step catalyzed by a phospholipase D (NAPE-PLD). The recent generation of NAPE-PLD(-/-) mice has revealed that these animals possess lower brain levels of saturated NAEs but essentially unchanged concentrations of polyunsaturated NAEs, including the endogenous cannabinoid anandamide. These findings suggest the existence of additional enzymatic routes for the production of NAEs in vivo. Here, we report evidence for an alternative pathway for NAE biosynthesis that proceeds through the serine hydrolase-catalyzed double-deacylation of NAPE to generate glycerophospho-NAE, followed by the phosphodiesterase-mediated cleavage of this intermediate to liberate NAE. Furthermore, we describe the functional proteomic isolation and identification of a heretofore uncharacterized enzyme alpha/beta-hydrolase 4 (Abh4) as a lysophospholipase/phospholipase B that selectively hydrolyzes NAPEs and lysoNAPEs. Abh4 accepts lysoNAPEs bearing both saturated and polyunsaturated N-acyl chains as substrates and displays a distribution that closely mirrors lysoNAPE-lipase activity in mouse tissues. These results support the existence of an NAPE-PLD-independent route for NAE biosynthesis and suggest that Abh4 plays a role in this metabolic pathway by acting as a (lyso)NAPE-selective lipase.
ESTHER : Simon_2006_J.Biol.Chem_281_26465
PubMedSearch : Simon_2006_J.Biol.Chem_281_26465
PubMedID: 16818490
Gene_locus related to this paper: human-ABHD4

Title : Proteomic profiling of metalloprotease activities with cocktails of active-site probes - Sieber_2006_Nat.Chem.Biol_2_274
Author(s) : Sieber SA , Niessen S , Hoover HS , Cravatt BF
Ref : Nat Chemical Biology , 2 :274 , 2006
Abstract : Metalloproteases are a large, diverse class of enzymes involved in many physiological and disease processes. Metalloproteases are regulated by post-translational mechanisms that diminish the effectiveness of conventional genomic and proteomic methods for their functional characterization. Chemical probes directed at active sites offer a potential way to measure metalloprotease activities in biological systems; however, large variations in structure limit the scope of any single small-molecule probe aimed at profiling this enzyme class. Here, we address this problem by creating a library of metalloprotease-directed probes that show complementary target selectivity. These probes were applied as a 'cocktail' to proteomes and their labeling profiles were analyzed collectively using an advanced liquid chromatography-mass spectrometry platform. More than 20 metalloproteases were identified, including members from nearly all of the major branches of this enzyme class. These findings suggest that chemical proteomic methods can serve as a universal strategy to profile the activity of the metalloprotease superfamily in complex biological systems.
ESTHER : Sieber_2006_Nat.Chem.Biol_2_274
PubMedSearch : Sieber_2006_Nat.Chem.Biol_2_274
PubMedID: 16565715

Title : A brain detoxifying enzyme for organophosphorus nerve poisons - Nomura_2005_Proc.Natl.Acad.Sci.U.S.A_102_6195
Author(s) : Nomura DK , Leung D , Chiang KP , Quistad GB , Cravatt BF , Casida JE
Ref : Proc Natl Acad Sci U S A , 102 :6195 , 2005
Abstract : Organophosphorus (OP) insecticides and chemical warfare agents act primarily by inhibiting acetylcholinesterase. There are many secondary targets for OP toxicants as observed for example with the major insecticide chlorpyrifos and its bioactivated metabolite chlorpyrifos oxon (CPO). Therefore, it was surprising that the predominant mouse brain protein labeled in vitro by [(3)H-ethyl]CPO (1 nM) (designated CPO-binding protein or CPO-BP) is not one of these known OP toxicant targets. CPO-BP is a 50-kDa membrane-bound serine hydrolase measured by derivatization with [(3)H]CPO and SDS/PAGE or filtration binding assay. It appears to undergo rapid diethylphosphorylation by [(3)H]CPO followed by either dephosphorylation and reactivation or aging on loss of an ethyl group. CPO and several other OP toxicants potently inhibit CPO-BP in vivo (i.p., 2 h) (50% inhibition at 2-25 mg/kg) and in vitro (50% inhibition at 8-68 nM). Using three chemical labeling reagents, i.e., [(3)H]CPO and the activity-based proteomic probes fluorophosphonate-biotin and fluorophosphonate-rhodamine, mouse brain CPO-BP is identified as serine hydrolase KIAA1363 of unknown function. Brains from KIAA1363(-/-) mice show greatly reduced levels of CPO labeling and hydrolytic metabolism compared to brains from wild-type mice. KIAA1363 therefore is the principal enzyme for metabolizing low levels of CPO in brain and may play a more general role in detoxification of OP nerve poisons.
ESTHER : Nomura_2005_Proc.Natl.Acad.Sci.U.S.A_102_6195
PubMedSearch : Nomura_2005_Proc.Natl.Acad.Sci.U.S.A_102_6195
PubMedID: 15840715
Gene_locus related to this paper: human-NCEH1 , mouse-Q8BLF1

Title : Reversible inhibitors of fatty acid amide hydrolase that promote analgesia: evidence for an unprecedented combination of potency and selectivity - Lichtman_2004_J.Pharmacol.Exp.Ther_311_441
Author(s) : Lichtman AH , Leung D , Shelton CC , Saghatelian A , Hardouin C , Boger DL , Cravatt BF
Ref : Journal of Pharmacology & Experimental Therapeutics , 311 :441 , 2004
Abstract : Fatty acid amide hydrolase (FAAH) is the primary catabolic regulator of several bioactive lipid amides in vivo, including the endogenous cannabinoid anandamide and the sleep-inducing substance oleamide. Inhibitors of FAAH are considered a potential therapeutic approach for the treatment of several nervous system disorders, including pain, anxiety, and insomnia. However, for FAAH inhibitors to achieve clinical utility, they must not only display efficacy in vivo but also selectivity for this enzyme relative to the numerous other serine hydrolases present in mammalian proteomes. Here, we report a general strategy for evaluating the pharmacological activity and target specificity of FAAH inhibitors and its implementation to develop the first class of selective reversible inhibitors of this enzyme that are highly efficacious in vivo. Using a series of functional proteomics, analytical chemistry, and behavioral pharmacology assays, we have identified a class of alpha-keto-heterocycles that show unprecedented selectivity for FAAH relative to other mammalian hydrolases, and, when administered to rodents, raise central nervous system levels of anandamide and promote cannabinoid receptor 1-dependent analgesia in several assays of pain sensation. These studies provide further evidence that FAAH may represent an attractive therapeutic target and describe a general route by which inhibitors of this enzyme can be optimized to achieve exceptional potency, selectivity, and efficacy in vivo.
ESTHER : Lichtman_2004_J.Pharmacol.Exp.Ther_311_441
PubMedSearch : Lichtman_2004_J.Pharmacol.Exp.Ther_311_441
PubMedID: 15229230

Title : Discovering potent and selective reversible inhibitors of enzymes in complex proteomes - Leung_2003_Nat.Biotechnol_21_687
Author(s) : Leung D , Hardouin C , Boger DL , Cravatt BF
Ref : Nat Biotechnol , 21 :687 , 2003
Abstract : To realize the promise of genomics-based therapeutics, new methods are needed to accelerate the discovery of small molecules that selectively modulate protein activity. Toward this end, advances in combinatorial synthesis have provided unprecedented access to large compound libraries of considerable structural complexity and diversity, shifting the bottleneck in drug discovery to the development of efficient screens for protein targets. Screening for reversible enzyme inhibitors typically requires extensive target-specific work, including protein expression and purification, as well as the development of specific substrate assays. Here we report a proteomic method for the discovery of reversible enzyme inhibitors that avoids these steps. We show that competitive profiling of a library of candidate serine hydrolase inhibitors in complex proteomes with activity-based chemical probes identifies nanomolar reversible inhibitors of several enzymes simultaneously, including the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH), triacylglycerol hydrolase (TGH) and an uncharacterized membrane-associated hydrolase that lacks known substrates. The strategy tests inhibitors against numerous enzymes in parallel, assigning both potency and selectivity factors to each agent. In this way, promiscuous inhibitors were readily rejected in favor of equally potent compounds with 500-fold or greater selectivity for their targets.
ESTHER : Leung_2003_Nat.Biotechnol_21_687
PubMedSearch : Leung_2003_Nat.Biotechnol_21_687
PubMedID: 12740587

Title : Enzyme activity profiles of the secreted and membrane proteome that depict cancer cell invasiveness - Jessani_2002_Proc.Natl.Acad.Sci.U.S.A_99_10335
Author(s) : Jessani N , Liu Y , Humphrey M , Cravatt BF
Ref : Proc Natl Acad Sci U S A , 99 :10335 , 2002
Abstract : By primarily measuring changes in transcript and protein abundance, conventional genomics and proteomics methods may fail to detect significant posttranslational events that regulate protein activity and, ultimately, cell behavior. To address these limitations, activity-based proteomic technologies that measure dynamics in protein function on a global scale would be of particular value. Here, we describe the application of a chemical proteomics strategy to quantitatively compare enzyme activities across a panel of human breast and melanoma cancer cell lines. A global analysis of the activity, subcellular distribution, and glycosylation state for the serine hydrolase superfamily resulted in the identification of a cluster of proteases, lipases, and esterases that distinguished cancer lines based on tissue of origin. Strikingly, nearly all of these enzyme activities were down-regulated in the most invasive cancer lines examined, which instead up-regulated a distinct set of secreted and membrane-associated enzyme activities. These invasiveness-associated enzymes included urokinase, a secreted serine protease with a recognized role in tumor progression, and a membrane-associated hydrolase KIAA1363, for which no previous link to cancer had been made. Collectively, these results suggest that invasive cancer cells share discrete proteomic signatures that are more reflective of their biological phenotype than cellular heritage, highlighting that a common set of enzymes may support the progression of tumors from a variety of origins and thus represent attractive targets for the diagnosis and treatment of cancer.
ESTHER : Jessani_2002_Proc.Natl.Acad.Sci.U.S.A_99_10335
PubMedSearch : Jessani_2002_Proc.Natl.Acad.Sci.U.S.A_99_10335
PubMedID: 12149457
Gene_locus related to this paper: human-NCEH1 , mouse-Q8BLF1

Title : Supersensitivity to anandamide and enhanced endogenous cannabinoid signaling in mice lacking fatty acid amide hydrolase - Cravatt_2001_Proc.Natl.Acad.Sci.U.S.A_98_9371
Author(s) : Cravatt BF , Demarest K , Patricelli MP , Bracey MH , Giang DK , Martin BR , Lichtman AH
Ref : Proc Natl Acad Sci U S A , 98 :9371 , 2001
Abstract : The medicinal properties of marijuana have been recognized for centuries, but clinical and societal acceptance of this drug of abuse as a potential therapeutic agent remains fiercely debated. An attractive alternative to marijuana-based therapeutics would be to target the molecular pathways that mediate the effects of this drug. To date, these neural signaling pathways have been shown to comprise a cannabinoid receptor (CB(1)) that binds the active constituent of marijuana, tetrahydrocannabinol (THC), and a postulated endogenous CB(1) ligand anandamide. Although anandamide binds and activates the CB(1) receptor in vitro, this compound induces only weak and transient cannabinoid behavioral effects in vivo, possibly a result of its rapid catabolism. Here we show that mice lacking the enzyme fatty acid amide hydrolase (FAAH(-/-)) are severely impaired in their ability to degrade anandamide and when treated with this compound, exhibit an array of intense CB(1)-dependent behavioral responses, including hypomotility, analgesia, catalepsy, and hypothermia. FAAH(-/-)-mice possess 15-fold augmented endogenous brain levels of anandamide and display reduced pain sensation that is reversed by the CB(1) antagonist SR141716A. Collectively, these results indicate that FAAH is a key regulator of anandamide signaling in vivo, setting an endogenous cannabinoid tone that modulates pain perception. FAAH may therefore represent an attractive pharmaceutical target for the treatment of pain and neuropsychiatric disorders.
ESTHER : Cravatt_2001_Proc.Natl.Acad.Sci.U.S.A_98_9371
PubMedSearch : Cravatt_2001_Proc.Natl.Acad.Sci.U.S.A_98_9371
PubMedID: 11470906

Title : Profiling serine hydrolase activities in complex proteomes - Kidd_2001_Biochemistry_40_4005
Author(s) : Kidd D , Liu Y , Cravatt BF
Ref : Biochemistry , 40 :4005 , 2001
Abstract : Serine hydrolases represent one of the largest and most diverse families of enzymes in higher eukaryotes, comprising numerous proteases, lipases, esterases, and amidases. The activities of many serine hydrolases are tightly regulated by posttranslational mechanisms, limiting the suitability of standard genomics and proteomics methods for the functional characterization of these enzymes. To facilitate the global analysis of serine hydrolase activities in complex proteomes, a biotinylated fluorophosphonate (FP-biotin) was recently synthesized and shown to serve as an activity-based probe for several members of this enzyme family. However, the extent to which FP-biotin reacts with the complete repertoire of active serine hydrolases present in a given proteome remains largely unexplored. Herein, we describe the synthesis and utility of a variant of FP-biotin in which the agent's hydrophobic alkyl chain linker was replaced by a more hydrophilic poly(ethylene glycol) moiety (FP-peg-biotin). When incubated with both soluble and membrane proteomes for extended reaction times, FP-biotin and FP-peg-biotin generated similar "maximal coverage" serine hydrolase activity profiles. However, kinetic analyses revealed that several serine hydrolases reacted at different rates with each FP agent. These rate differences were exploited in studies that used the biotinylated FPs to examine the target selectivity of reversible serine hydrolase inhibitors directly in complex proteomes. Finally, a general method for the avidin-based affinity isolation of FP-biotinylated proteins was developed, permitting the rapid and simultaneous identification of multiple serine peptidases, lipases, and esterases. Collectively, these studies demonstrate that chemical probes such as the biotinylated FPs can greatly accelerate both the functional characterization and molecular identification of active enzymes in complex proteomes.
ESTHER : Kidd_2001_Biochemistry_40_4005
PubMedSearch : Kidd_2001_Biochemistry_40_4005
PubMedID: 11300781

Title : Exceptionally potent inhibitors of fatty acid amide hydrolase: the enzyme responsible for degradation of endogenous oleamide and anandamide - Boger_2000_Proc.Natl.Acad.Sci.U.S.A_97_5044
Author(s) : Boger DL , Sato H , Lerner AE , Hedrick MP , Fecik RA , Miyauchi H , Wilkie GD , Austin BJ , Patricelli MP , Cravatt BF
Ref : Proc Natl Acad Sci U S A , 97 :5044 , 2000
Abstract : The development of exceptionally potent inhibitors of fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of oleamide (an endogenous sleep-inducing lipid), and anandamide (an endogenous ligand for cannabinoid receptors) is detailed. The inhibitors may serve as useful tools to clarify the role of endogenous oleamide and anandamide and may prove to be useful therapeutic agents for the treatment of sleep disorders or pain. The combination of several features-an optimal C12-C8 chain length, pi-unsaturation introduction at the corresponding arachidonoyl Delta(8,9)/Delta(11,12) and oleoyl Delta(9,10) location, and an alpha-keto N4 oxazolopyridine with incorporation of a second weakly basic nitrogen provided FAAH inhibitors with K(i)s that drop below 200 pM and are 10(2)-10(3) times more potent than the corresponding trifluoromethyl ketones.
ESTHER : Boger_2000_Proc.Natl.Acad.Sci.U.S.A_97_5044
PubMedSearch : Boger_2000_Proc.Natl.Acad.Sci.U.S.A_97_5044
PubMedID: 10805767

Title : Activity-based protein profiling: the serine hydrolases - Liu_1999_Proc.Natl.Acad.Sci.U.S.A_96_14694
Author(s) : Liu Y , Patricelli MP , Cravatt BF
Ref : Proc Natl Acad Sci U S A , 96 :14694 , 1999
Abstract : With the postgenome era rapidly approaching, new strategies for the functional analysis of proteins are needed. To date, proteomics efforts have primarily been confined to recording variations in protein level rather than activity. The ability to profile classes of proteins on the basis of changes in their activity would greatly accelerate both the assignment of protein function and the identification of potential pharmaceutical targets. Here, we describe the chemical synthesis and utility of an active-site directed probe for visualizing dynamics in the expression and function of an entire enzyme family, the serine hydrolases. By reacting this probe, a biotinylated fluorophosphonate referred to as FP-biotin, with crude tissue extracts, we quickly and with high sensitivity detect numerous serine hydrolases, many of which display tissue-restricted patterns of expression. Additionally, we show that FP-biotin labels these proteins in an activity-dependent manner that can be followed kinetically, offering a powerful means to monitor dynamics simultaneously in both protein function and expression.
ESTHER : Liu_1999_Proc.Natl.Acad.Sci.U.S.A_96_14694
PubMedSearch : Liu_1999_Proc.Natl.Acad.Sci.U.S.A_96_14694
PubMedID: 10611275