Hodder PS

References (6)

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 : 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 : 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 : 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 : 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