Hsu KL

References (27)

Title : Endocannabinoid biosynthetic enzymes regulate pain response via LKB1-AMPK signaling - Chen_2023_Proc.Natl.Acad.Sci.U.S.A_120_e2304900120
Author(s) : Chen M , Shin M , Ware TB , Donvito G , Muchhala KH , Mischel R , Mustafa MA , Serbulea V , Upchurch CM , Leitinger N , Akbarali HI , Lichtman AH , Hsu KL
Ref : Proc Natl Acad Sci U S A , 120 :e2304900120 , 2023
Abstract : Diacylglycerol lipase-beta (DAGLbeta) serves as a principal 2-arachidonoylglycerol (2-AG) biosynthetic enzyme regulating endocannabinoid and eicosanoid metabolism in immune cells including macrophages and dendritic cells. Genetic or pharmacological inactivation of DAGLbeta ameliorates inflammation and hyper-nociception in preclinical models of pathogenic pain. These beneficial effects have been assigned principally to reductions in downstream proinflammatory lipid signaling, leaving alternative mechanisms of regulation largely underexplored. Here, we apply quantitative chemical- and phospho-proteomics to find that disruption of DAGLbeta in primary macrophages leads to LKB1-AMPK signaling activation, resulting in reprogramming of the phosphoproteome and bioenergetics. Notably, AMPK inhibition reversed the antinociceptive effects of DAGLbeta blockade, thereby directly supporting DAGLbeta-AMPK crosstalk in vivo. Our findings uncover signaling between endocannabinoid biosynthetic enzymes and ancient energy-sensing kinases to mediate cell biological and pain responses.
ESTHER : Chen_2023_Proc.Natl.Acad.Sci.U.S.A_120_e2304900120
PubMedSearch : Chen_2023_Proc.Natl.Acad.Sci.U.S.A_120_e2304900120
PubMedID: 38109529

Title : Diacylglycerol Lipase-beta Is Required for TNF-alpha Response but Not CD8(+) T Cell Priming Capacity of Dendritic Cells - Shin_2019_Cell.Chem.Biol_26_1036
Author(s) : Shin M , Buckner A , Prince J , Bullock TNJ , Hsu KL
Ref : Cell Chemical Biology , 26 :1036 , 2019
Abstract : Diacylglycerol lipase-beta (DAGLbeta) hydrolyzes arachidonic acid (AA)-esterified diacylglycerols to produce 2-arachidonoylglycerol (2-AG) and downstream prostanoids that mediate inflammatory responses of macrophages. Here, we utilized DAGL-tailored activity-based protein profiling and genetic disruption models to discover that DAGLbeta regulates inflammatory lipid and protein signaling pathways in primary dendritic cells (DCs). DCs serve as an important link between innate and adaptive immune pathways by relaying innate signals and antigen to drive T cell clonal expansion and prime antigen-specific immunity. We discovered that disruption of DAGLbeta in DCs lowers cellular 2-AG and AA that is accompanied by reductions in lipopolysaccharide (LPS) stimulated tumor necrosis factor alpha secretion. Cell-based vaccination studies revealed that DC maturation ex vivo and immunogenicity in vivo was surprisingly unaffected by DAGLbeta inactivation. Collectively, we identify DAGLbeta pathways as a means for attenuating DC inflammatory signaling while sparing critical adaptive immune functions and further expand the utility of targeting lipid pathways for immunomodulation.
ESTHER : Shin_2019_Cell.Chem.Biol_26_1036
PubMedSearch : Shin_2019_Cell.Chem.Biol_26_1036
PubMedID: 31105063

Title : Lipid-metabolizing serine hydrolases in the mammalian central nervous system: endocannabinoids and beyond - Shin_2019_Biochim.Biophys.Acta.Mol.Cell.Biol.Lipids_1864_907
Author(s) : Shin M , Ware TB , Lee HC , Hsu KL
Ref : Biochimica & Biophysica Acta Molecular & Cellular Biology Lipids , 1864 :907 , 2019
Abstract : The metabolic serine hydrolases hydrolyze ester, amide, or thioester bonds found in broad small molecule substrates using a conserved activated serine nucleophile. The mammalian central nervous system (CNS) express a diverse repertoire of serine hydrolases that act as (phospho)lipases or lipid amidases to regulate lipid metabolism and signaling vital for normal neurocognitive function and CNS integrity. Advances in genomic DNA sequencing have provided evidence for the role of these lipid-metabolizing serine hydrolases in neurologic, psychiatric, and neurodegenerative disorders. This review briefly summarizes recent progress in understanding the biochemical and (patho)physiological roles of these lipid-metabolizing serine hydrolases in the mammalian CNS with a focus on serine hydrolases involved in the endocannabinoid system. The development and application of specific inhibitors for an individual serine hydrolase, if available, are also described. This article is part of a Special Issue entitled Novel functions of phospholipase A2 Guest Editors: Makoto Murakami and Gerard Lambeau.
ESTHER : Shin_2019_Biochim.Biophys.Acta.Mol.Cell.Biol.Lipids_1864_907
PubMedSearch : Shin_2019_Biochim.Biophys.Acta.Mol.Cell.Biol.Lipids_1864_907
PubMedID: 30905349

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 : Deregulation of the endocannabinoid system and therapeutic potential of ABHD6 blockade in the cuprizone model of demyelination - Manterola_2018_Biochem.Pharmacol_157_189
Author(s) : Manterola A , Bernal-Chico A , Cipriani R , Canedo-Antelo M , Moreno-Garcia A , Martin-Fontecha M , Perez-Cerda F , Sanchez-Gomez MV , Ortega-Gutierrez S , Brown JM , Hsu KL , Cravatt B , Matute C , Mato S
Ref : Biochemical Pharmacology , 157 :189 , 2018
Abstract : Multiple sclerosis (MS) is a chronic demyelinating disease of unknown etiology in which tissue pathology suggests both immune-dependent attacks to oligodendroglia and primary oligodendrocyte demise. The endocannabinoid system has been crucially involved in the control of autoimmune demyelination and cannabinoid-based therapies exhibit therapeutic potential, but also limitations, in MS patients. In this context, growing evidence suggests that targeting the hydrolysis of the main endocannabinoid 2-arachidonoylglycerol (2-AG) may offer a more favorable benefit-to-risk balance in MS than existing cannabinoid medicines. Here we evaluated the modulation of endocannabinoid signaling and the therapeutic potential of targeting the 2-AG hydrolytic enzyme alpha/beta-hydrolase domain-containing 6 (ABHD6) in the cuprizone model of non-immune dependent demyelination. The concentrations of N-arachidonoylethanolamine (anandamide, AEA) and its congener N-palmitoylethanolamine (PEA) were reduced following 6weeks of cuprizone feeding. Deregulation of AEA and PEA levels was not due to differences in the expression of the hydrolytic and biosynthetic enzymes fatty acid amide hydrolase and N-acylphosphatidylethanolamine-phospholipase D, respectively. Conversely, we measured elevated transcript levels of 2-AG hydrolytic enzymes monoacylglycerol lipase, ABHD6 and ABHD12 without changes in bulk 2-AG concentration. Upregulated CB1 and CB2 receptors expression, ascribed in part to microglia, was also detected in the brain of cuprizone-treated mice. Administration of an ABHD6 inhibitor partially attenuated myelin damage, astrogliosis and microglia/macrophage reactivity associated to cuprizone feeding. However, ABHD6 blockade was ineffective at engaging protective or differentiation promoting effects in oligodendrocyte cultures. These results show specific alterations of the endocannabinoid system and modest beneficial effects resulting from ABHD6 inactivation in a relevant model of primary demyelination.
ESTHER : Manterola_2018_Biochem.Pharmacol_157_189
PubMedSearch : Manterola_2018_Biochem.Pharmacol_157_189
PubMedID: 30075103
Gene_locus related to this paper: human-ABHD6

Title : Liposomal Delivery of Diacylglycerol Lipase-Beta Inhibitors to Macrophages Dramatically Enhances Selectivity and Efficacy in Vivo - Shin_2018_Mol.Pharm_15_721
Author(s) : Shin M , Snyder HW , Donvito G , Schurman LD , Fox TE , Lichtman AH , Kester M , Hsu KL
Ref : Mol Pharm , 15 :721 , 2018
Abstract : Diacylglycerol lipase-beta (DAGLbeta) hydrolyzes arachidonic acid (AA)-containing diacylglycerols to produce bioactive lipids including endocannabinoids and AA-derived eicosanoids involved in regulation of inflammatory signaling. Previously, we demonstrated that DAGLbeta inactivation using the triazole urea inhibitor KT109 blocked macrophage inflammatory signaling and reversed allodynic responses of mice in inflammatory and neuropathic pain models. Here, we tested whether we could exploit the phagocytic capacity of macrophages to localize delivery of DAGLbeta inhibitors to these cells in vivo using liposome encapsulated KT109. We used DAGLbeta-tailored activity-based probes and chemical proteomic methods to measure potency and selectivity of liposomal KT109 in macrophages and tissues from treated mice. Surprisingly, delivery of approximately 5 mug of liposomal KT109 was sufficient to achieve approximately 80% inactivation of DAGLbeta in macrophages with no apparent activity in other tissues in vivo. Our macrophage-targeted delivery resulted in a >100-fold enhancement in antinociceptive potency compared with free compound in a mouse inflammatory pain model. Our studies describe a novel anti-inflammatory strategy that is achieved by targeted in vivo delivery of DAGLbeta inhibitors to macrophages.
ESTHER : Shin_2018_Mol.Pharm_15_721
PubMedSearch : Shin_2018_Mol.Pharm_15_721
PubMedID: 28901776

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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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