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Benjamin F Cravatt - One of the best experts on this subject based on the ideXlab platform.
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Global Portrait of Protein Targets of Metabolites of the Neurotoxic Compound BIA 10–2474
2019Co-Authors: Zhen Huang, Armand B. Cognetta, Daisuke Ogasawara, Uthpala I. Seneviratne, Christopher Am W. Ende, Deane M. Nason, Kimberly Lapham, John Litchfield, Douglas S. Johnson, Benjamin F CravattAbstract:Clinical investigation of the fatty acid amide Hydrolase (FAAH) inhibitor BIA 10–2474 resulted in serious adverse neurological events. Structurally unrelated FAAH inhibitors tested in humans have not presented safety concerns, suggesting that BIA 10–2474 has off-target activities. A recent activity-based protein profiling (ABPP) study revealed that BIA 10–2474 and one of its major metabolites inhibit multiple members of the Serine Hydrolase class to which FAAH belongs. Here, we extend these studies by performing a proteome-wide analysis of covalent targets of BIA 10–2474 metabolites. Using alkynylated probes for click chemistry-ABPP in human cells, we show that des-methylated metabolites of BIA 10–2474 covalently modify the conserved catalytic cysteine in aldehyde dehydrogenases, including ALDH2, which has been implicated in protecting the brain from oxidative stress-related damage. These findings indicate that BIA 10–2474 and its metabolites have the potential to inhibit multiple mechanistically distinct enzyme classes involved in nervous system function
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selective irreversible inhibitors of the wnt deacylating enzyme notum developed by activity based protein profiling
ACS Medicinal Chemistry Letters, 2018Co-Authors: Radu M Suciu, Armand B. Cognetta, Zachary E Potter, Benjamin F CravattAbstract: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.
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Selective Irreversible Inhibitors of the Wnt-Deacylating Enzyme NOTUM Developed by Activity-Based Protein Profiling
2018Co-Authors: Radu M Suciu, Armand B. Cognetta, Zachary E Potter, Benjamin F CravattAbstract: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
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Remodeling Natural Products: Chemistry and Serine Hydrolase Activity of a Rocaglate-Derived β‑Lactone
2016Co-Authors: Neil J Lajkiewicz, Benjamin F Cravatt, Micah J. Niphakis, B. Cognetta, John A PorcoAbstract:*S Supporting Information ABSTRACT: Flavaglines are a class of natural products with potent insecticidal and anticancer activities. β-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 β-lactone. In addition to in vitro inhibition of the Serine Hydrolases ABHD10 and ACOT1/2, the most potent β-lactone enantiomer was also found to inhibit these enzymes, as well as the Serine peptidases CTSA and SCPEP1, in PC3 cells. Flavaglines, specifically rocaglate derivatives, are cyclopenta[b]-benzofuran natural products isolated from the genus Aglaia that have been shown to be potent anticancer agents.1 Figure 1 shows three natural products: silvestrol (1),2 methyl rocaglat
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activity based protein profiling of oncogene driven changes in metabolism reveals broad dysregulation of pafah1b2 and 1b3 in cancer
ACS Chemical Biology, 2015Co-Authors: Rebecca A Kohnz, Benjamin F Cravatt, Ku-lung Hsu, Jae Won Chang, Melinda M Mulvihill, Antonio Sorrentino, Sourav Bandyopadhyay, Andrei Goga, Daniel K. NomuraAbstract:Targeting dysregulated metabolic pathways is a promising therapeutic strategy for eradicating cancer. Understanding how frequently altered oncogenes regulate metabolic enzyme targets would be useful in identifying both broad-spectrum and targeted metabolic therapies for cancer. Here, we used activity-based protein profiling to identify Serine Hydrolase activities that were consistently upregulated by various human oncogenes. Through this profiling effort, we found oncogenic regulatory mechanisms for several cancer-relevant Serine Hydrolases and discovered that platelet activating factor acetylHydrolase 1B2 and 1B3 (PAFAH1B2 and PAFAH1B3) activities were consistently upregulated by several oncogenes, alongside previously discovered cancer-relevant Hydrolases fatty acid synthase and monoacylglycerol lipase. While we previously showed that PAFAH1B2 and 1B3 were important in breast cancer, our most recent profiling studies have revealed that these enzymes may be dysregulated broadly across many types of cance...
Daniele Piomelli - One of the best experts on this subject based on the ideXlab platform.
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deficiency of lipoprotein lipase in neurons modifies the regulation of energy balance and leads to obesity
Cell Metabolism, 2011Co-Authors: Hong Wang, Daniele Piomelli, Giuseppe Astarita, Matthew D Taussig, Kalyani G Bharadwaj, Nicholas V Dipatrizio, Klausarmin Nave, Ira J Goldberg, Robert H EckelAbstract:Free fatty acids (FFAs) suppress appetite when injected into the hypothalamus. To examine whether lipoprotein lipase (LPL), a Serine Hydrolase that releases FFAs from circulating triglyceride (TG)-rich lipoproteins, might contribute to FFA-mediated signaling in the brain, we created neuron-specific LPL-deficient mice. Homozygous mutant (NEXLPL-/-) mice were hyperphagic and became obese by 16 weeks of age. These traits were accompanied by elevations in the hypothalamic orexigenic neuropeptides, AgRP and NPY, and were followed by reductions in metabolic rate. The uptake of TG-rich lipoprotein fatty acids was reduced in the hypothalamus of 3-month-old NEXLPL-/- mice. Moreover, deficiencies in essential fatty acids in the hypothalamus were evident by 3 months, with major deficiencies of long-chain n-3 fatty acids by 12 months. These results indicate that TG-rich lipoproteins are sensed in the brain by an LPL-dependent mechanism and provide lipid signals for the central regulation of body weight and energy balance.
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discovery of potent and reversible monoacylglycerol lipase inhibitors
Chemistry & Biology, 2009Co-Authors: Alvin R King, Alessio Lodola, Daniele Piomelli, Kwang-mook Jung, Emmanuel Y Dotsey, Azar Ghomian, Yan Qiu, Marco MorAbstract:Monoacylglycerol lipase (MGL) is a Serine Hydrolase involved in the biological deactivation of the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG). Previous efforts to design MGL inhibitors have focused on chemical scaffolds that irreversibly block the activity of this enzyme. Here, we describe two naturally occurring terpenoids, pristimerin and euphol, which inhibit MGL activity with high potency (median effective concentration, IC(50) = 93 nM and 315 nM, respectively) through a reversible mechanism. Mutational and modeling studies suggest that the two agents occupy a common hydrophobic pocket located within the putative lid domain of MGL, and each reversibly interacts with one of two adjacent cysteine residues (Cys(201) and Cys(208)) flanking such pocket. This previously unrecognized regulatory region might offer a molecular target for potent and reversible inhibitors of MGL.
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a critical cysteine residue in monoacylglycerol lipase is targeted by a new class of isothiazolinone based enzyme inhibitors
British Journal of Pharmacology, 2009Co-Authors: Alvin R King, Alessio Lodola, Caterina Carmi, Jin Fu, Daniele PiomelliAbstract:Background and purpose: Monoacylglycerol lipase (MGL) is a presynaptic Serine Hydrolase that inactivates the endocannabinoid neurotransmitter, 2-arachidonoyl-sn-glycerol. Recent studies suggest that cysteine residues proximal to the enzyme active site are important for MGL function. In the present study, we characterize the role of cysteines in MGL function and identify a series of cysteine-reactive agents that inhibit MGL activity with nanomolar potencies by interacting with cysteine residue 208.
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design synthesis and structure activity relationships of alkylcarbamic acid aryl esters a new class of fatty acid amide Hydrolase inhibitors
Journal of Medicinal Chemistry, 2003Co-Authors: Giorgio Tarzia, Andrea Duranti, Andrea Tontini, Giovanni Piersanti, Silvia Rivara, P V Plazzi, Chris Park, Satish Kathuria, Daniele PiomelliAbstract:Fatty acid amide Hydrolase (FAAH), an intracellular Serine Hydrolase enzyme, participates in the deactivation of fatty acid ethanolamides such as the endogenous cannabinoid anandamide, the intestinal satiety factor oleoylethanolamide, and the peripheral analgesic and anti-inflammatory factor palmitoylethanolamide. In the present study, we report on the design, synthesis, and structure−activity relationships (SAR) of a novel class of potent, selective, and systemically active inhibitors of FAAH activity, which we have recently shown to exert potent anxiolytic-like effects in rats. These compounds are characterized by a carbamic template substituted with alkyl or aryl groups at their O- and N-termini. Most compounds inhibit FAAH, but not several other Serine Hydrolases, with potencies that depend on the size and shape of the substituents. Initial SAR investigations suggested that the requirements for optimal potency are a lipophilic N-alkyl substituent (such as n-butyl or cyclohexyl) and a bent O-aryl subst...
Brent R. Martin - One of the best experts on this subject based on the ideXlab platform.
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affinity based selectivity profiling of an in class selective competitive inhibitor of acyl protein thioesterase 2
ACS Medicinal Chemistry Letters, 2017Co-Authors: Sang Joon Won, Joseph D Eschweiler, Jaimeen D Majmudar, Fei San Chong, Sin Ye Hwang, Brandon T Ruotolo, Brent R. MartinAbstract:Activity-based protein profiling (ABPP) has revolutionized the discovery and optimization of active-site ligands across distinct enzyme families, providing a robust platform for in-class selectivity profiling. Nonetheless, this approach is less straightforward for profiling reversible inhibitors and does not access proteins outside the ABPP probe’s target profile. While the active-site competitive acyl protein thioesterase 2 inhibitor ML349 (Ki = 120 nM) is highly selective within the Serine Hydrolase enzyme family, it could still interact with other cellular targets. Here we present a chemoproteomic workflow to enrich and profile candidate ML349-binding proteins. In human cell lysates, biotinylated-ML349 enriches a recurring set of proteins, including metabolite kinases and flavin-dependent oxidoreductases that are potentially enhanced by avidity-driven multimeric interactions. Confirmatory assays by native mass spectrometry and fluorescence polarization quickly rank-ordered these weak off-targets, provi...
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acyl protein thioesterase inhibitors as probes of dynamic s palmitoylation
MedChemComm, 2014Co-Authors: Dahvid Davda, Brent R. MartinAbstract:Protein palmitoylation describes the hydrophobic post-translational modification of cysteine residues in certain proteins, and is required for the spatial organization and composition of cellular membrane environments. Certain palmitoylated proteins are processed by acyl protein thioesterase (APT) enzymes, which catalyze thioester hydrolysis of palmitoylated cysteine residues. Inhibiting APT enzymes disrupts Ras trafficking and attenuates oncogenic growth signaling, highlighting these enzymes as potential therapeutic targets. As members of the Serine Hydrolase enzyme family, APT enzymes can be assayed by fluorophosphonate activity-based protein profiling (ABPP) methods, allowing rapid profiling of inhibitor selectivity and potency. In this review, we discuss recent progress in the development of potent and selective inhibitors to APT enzymes, including both reversible and irreversible inhibitor chemotypes. These examples highlight how ABPP methods integrate with medicinal chemistry for the discovery and optimization of inhibitors in complex proteomes.
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click generated triazole ureas as ultrapotent in vivo active Serine Hydrolase inhibitors
Nature Chemical Biology, 2011Co-Authors: Alexander Adibekian, Daniel A Bachovchin, Sherry Niessen, Brent R. Martin, Chu Wang, Ku-lung Hsu, Heather HooverAbstract:Serine Hydrolases are a diverse enzyme class representing ∼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 (α,β-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.
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click generated triazole ureas as ultrapotent in vivo active Serine Hydrolase inhibitors
Nature Chemical Biology, 2011Co-Authors: Alexander Adibekian, Daniel A Bachovchin, Sherry Niessen, Brent R. Martin, Chu Wang, Heather Hoover, Benjamin F CravattAbstract:Investigations of Serine Hydrolases have been frustrated by a lack of selective chemical inhibitors. Profiling of synthetically accessible 1,2,3-triazole ureas in cells and mice now identifies several effective compounds, application of which yields new insights into N-acetylation by APEH.
Mario Van Der Stelt - One of the best experts on this subject based on the ideXlab platform.
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structure kinetics relationships and molecular dynamics show crucial role for heterocycle leaving group in irreversible diacylglycerol lipase inhibitors
Journal of Medicinal Chemistry, 2019Co-Authors: Antonius P A Janssen, Jacob M A Van Hengst, Olivier J M Bequignon, Gerard J P Van Westen, Hui Deng, Mario Van Der SteltAbstract:Drug discovery programs of covalent irreversible, mechanism-based enzyme inhibitors often focus on optimization of potency as determined by IC50-values in biochemical assays. These assays do not allow the characterization of the binding activity (Ki) and reactivity (kinact) as individual kinetic parameters of the covalent inhibitors. Here, we report the development of a kinetic substrate assay to study the influence of the acidity (pKa) of heterocyclic leaving group of triazole urea derivatives as diacylglycerol lipase (DAGL)-α inhibitors. Surprisingly, we found that the reactivity of the inhibitors did not correlate with the pKa of the leaving group, whereas the position of the nitrogen atoms in the heterocyclic core determined to a large extent the binding activity of the inhibitor. This finding was confirmed and clarified by molecular dynamics simulations on the covalently bound Michaelis–Menten complex. A deeper understanding of the binding properties of covalent Serine Hydrolase inhibitors is expecte...
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design and synthesis of quenched activity based probes for diacylglycerol lipase and α β Hydrolase domain containing protein 6
Chemistry-an Asian Journal, 2018Co-Authors: E J Van Rooden, Antonius P A Janssen, M Kohsiek, Roy M Kreekel, A C M Van Esbroeck, A M C H Van Den Nieuwendijk, R J B H N Van Den Berg, Hermen S Overkleeft, Mario Van Der SteltAbstract:Diacylglycerol lipases (DAGL) are responsible for the biosynthesis of the endocannabinoid 2‐arachidonoylglycerol. The fluorescent activity‐based probes DH379 and HT‐01 have been previously shown to label DAGLs and to cross‐react with the Serine Hydrolase ABHD6. Here, we report the synthesis and characterization of two new quenched activity‐based probes 1 and 2, the design of which was based on the structures of DH379 and HT‐01, respectively. Probe 1 contains a BODIPY‐FL and a 2,4‐dinitroaniline moiety as a fluorophore–quencher pair, whereas probe 2 employs a Cy5‐fluorophore and a cAB40‐quencher. The fluorescence of both probes was quenched with relative quantum yields of 0.34 and 0.0081, respectively. The probes showed target inhibition as characterized in activity‐based protein profiling assays using human cell‐ and mouse brain lysates, but were unfortunately not active in living cells, presumably due to limited cell permeability.
Sherry Niessen - One of the best experts on this subject based on the ideXlab platform.
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highly selective inhibitors of monoacylglycerol lipase bearing a reactive group that is bioisosteric with endocannabinoid substrates
Chemistry & Biology, 2012Co-Authors: Jae Won Chang, Sherry Niessen, Chu Wang, Micah J. Niphakis, Armand B. Cognetta, Matthew W. Buczynski, Loren H. Parsons, Kenneth M Lum, Megan L Matthews, Benjamin F CravattAbstract:Summary 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.
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competitive activity based protein profiling identifies aza β lactams as a versatile chemotype for Serine Hydrolase inhibition
Journal of the American Chemical Society, 2012Co-Authors: A Zuhl, Justin T Mohr, Daniel A Bachovchin, Sherry Niessen, Jacob M Berlin, Maximilian Dochnahl, Maria P Lopezalberca, Gregory C Fu, Benjamin F CravattAbstract: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-β-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 α,β-Hydrolase domain-containing 10 (ABHD10). ABPP-guided medicinal chemistry yielded a compound ABL303 that potently (IC50 ≈ 30 nM) and selectively inactivated ABHD10 in vitro and in living cel...
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click generated triazole ureas as ultrapotent in vivo active Serine Hydrolase inhibitors
Nature Chemical Biology, 2011Co-Authors: Alexander Adibekian, Daniel A Bachovchin, Sherry Niessen, Brent R. Martin, Chu Wang, Ku-lung Hsu, Heather HooverAbstract:Serine Hydrolases are a diverse enzyme class representing ∼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 (α,β-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.
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click generated triazole ureas as ultrapotent in vivo active Serine Hydrolase inhibitors
Nature Chemical Biology, 2011Co-Authors: Alexander Adibekian, Daniel A Bachovchin, Sherry Niessen, Brent R. Martin, Chu Wang, Heather Hoover, Benjamin F CravattAbstract:Investigations of Serine Hydrolases have been frustrated by a lack of selective chemical inhibitors. Profiling of synthetically accessible 1,2,3-triazole ureas in cells and mice now identifies several effective compounds, application of which yields new insights into N-acetylation by APEH.
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selectivity of inhibitors of endocannabinoid biosynthesis evaluated by activity based protein profiling
Bioorganic & Medicinal Chemistry Letters, 2008Co-Authors: Heather Hoover, Jacqueline L Blankman, Sherry Niessen, Benjamin F CravattAbstract:The endogenous cannabinoid (endocannabinoid) system consists of a set of G-protein coupled receptors (CB1 and CB2), natural lipid ligands [N-arachidonoyl ethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG)], and enzymatic pathways for ligand biosynthesis and degradation.1 The CB1 receptor is widely distributed throughout the mammalian nervous system, raising provocative questions about how endocannabinoid signaling is regulated in specific brain regions and neural circuits. Unlike more classical neurotransmitter systems, such as the monoamine or glutamatergic systems, where receptor diversification serves as a key mechanism to vary signaling outputs, the endocannabinoid system appears to achieve this goal, at least in part, by producing multiple ligands. Indeed, the biosynthetic and degradative pathways for anandamide and 2-AG are mediated by distinct sets of enzymes and accumulating evidence suggests that these pathways are differentially regulated in the nervous system.1 Two key enzymes implicated in the biosynthesis of 2-AG are diacylglycerol lipase (DAGL)-α and β.2 DAGL-α and β are both integral membrane proteins with four predicted transmembrane domains followed by a catalytic domain that conforms to the general sequence requirements for a Serine Hydrolase (including the presence of the canonical GXSXG active site motif). Multiple lines of evidence suggest that DAGL-α/β play a role in regulating 2-AG biosynthesis in neurons. For instance, overexpression of DAGL-α in the mouse neuroblastoma cell line Neuro-2a results in a significant increase in basal 2-AG levels.3 Conversely, RNA interference-mediated knockdown of DAGL-α in Neuro-2a cells reduced basal levels of 2-AG and blocked the production of this endocannabinoid stimulated by agonists of group 1 metabotropic glutamate receptors. 3 DAGL-α/β are inhibited by two small-molecule agents, RHC80267 and tetrahydrolipstatin (THL)2,4 (Figure 1), which have been used to block many of the CB1-dependent forms of neuronal plasticity observed in in vitro preparations.5 These findings have invoked 2-AG as the principle endocannabinoid involved in retrograde signaling in the nervous system. However, this conclusion is predicated on the assumption that RHC80267 and THL selectively target DAGL-α/β in the nervous system, a hypothesis that, to date, has gone largely untested. These agents likely inhibit DAGL-α/β by covalent reaction with the enzymes' Serine nucleophile (forming carbamoylated and esterified products, respectively), which suggests that the compounds might target additional Serine Hydrolases in the nervous system via a similar mechanism. This is certainly the case in peripheral tissues, where, for example, THL has found clinical utility as an anti-obesity agent due to blockade of pancreatic lipases in the intestine.6 Figure 1 Structures of inhibitors of the 2-AG biosynthetic enzymes DAGL-α/β. To more globally assess the selectivity of RHC80267 and THL, we analyzed these inhibitors by competitive activity-based protein profiling (ABPP).7 ABPP is a chemical proteomic method that utilizes active site-directed small-molecule probes to assess the functional state of numerous enzymes in parallel directly in native biological systems. In competitive ABPP, inhibitors are evaluated for their ability to impair probe labeling of target enzymes.8 Because ABPP probes typically label many members from a given enzyme class, competitive profiling experiments provide an excellent assessment of both the potency and selectivity of inhibitors. Inhibitors of DAGL-α/β are most commonly used in nervous system preparations;3,5 we therefore elected to profile these agents against a mouse brain proteome using fluorophosphonate (FP) ABPP probes, which broadly target enzymes from the Serine Hydrolase.9 For initial comparison, we also analyzed two lipid-based FP inhibitors, O-3841 and MAFP (Figure 1), which have also been shown to inhibit DAGL-α/β.4 The mouse brain membrane proteome was treated with each inhibitor across a broad concentration range (0.01- 100 μM) for 30 min, after which reactions were incubated with a rhodamine-tagged FP probe (FP-Rh) for 60 min, separated by SDS-PAGE, and analyzed by in-gel fluorescence scanning. O-3841 and MAFP were found to inhibit probe labeling of numerous Serine Hydrolase activities, including fatty acid amide Hydrolase (FAAH), KIAA1363, monoacylglycerol lipase (MAGL), ABHD6, and ABHD12 (Figure 2A). Most of these enzymes with the exception of KIAA1363 were more potently inhibited by MAFP than O-3841. In contrast, RHC80267 and THL showed more selective patterns of enzyme inhibition, blocking the labeling of three and two Hydrolase activities, respectively (Figure 2B). None of these enzymes correspond to DAGL-α/β, large enzymes (> 70 kDa) that appear to be poorly labeled by FP-probes.10 Figure 2 Competitive ABPP of DAGL-α/β inhibitors with a mouse brain membrane proteome. A, Profiles for the fluorophosphonate inhibitors O-3841 and MAFP. B, Profiles for RHC80267 and THL. To identify the targets of RHC80267 and THL, mouse brain proteome was treated with each inhibitor (50 and 5 μM, respectively) or DMSO (control) for 30 min and then incubated with a biotinylated FP-probe (FP-biotin, 5 μM) for 120 min. Probe-labeled proteins were enriched and characterized by a combination of avidin affinity and liquid chromatography-mass spectrometry steps, following previously described procedures.11 All samples were analyzed in triplicate. This approach, termed ABPP-MudPIT, identified 35 brain Serine Hydrolases at sufficiently high spectral counts (> 15 average counts in control proteomes) for comparative quantitation between inhibitor- and DMSO-treated proteomes (Figure 3A). Figure 3 Identification of brain Serine Hydrolase targets of RHC80267 and THL by ABPP-MudPIT. A, Average spectral count values for brain Serine Hydrolases in control (DMSO-treated), RHC80267-treated (50 μM), and THL-treated (5 μM) mouse brain proteomes. ... Candidate targets of RHC80267 and THL were defined as Serine Hydrolases that showed greater than 60% reductions in average spectral counts in inhibitor-treated versus DMSO-treated proteomes. This criterion identified six and three putative targets of RHC80267 and THL, respectively (Figure 3B). These Hydrolases included enzymes implicated in endocannabinoid metabolism, such as FAAH12 and ABHD12,13 enzymes that regulate other signaling molecules, such as acetylcholine [acetylcholinesterase (AChE)14], platelet-activating factor (PLA2g715) and lysophosphatidic acid (KIAA136316), as well as uncharacterized enzymes (BAT5). Comparison of these results to the gel-based profiles shown in Figure 2 led to the tentative assignment of the 60 kDa Hydrolase sensitive to both RHC80267 and THL as BAT5. Additional Hydrolases detected by LC-MS analysis (e.g., PLA2g7, AChE) were not visible by gel analysis, underscoring the enhanced sensitivity of the former analytical method. Neither DAGL-α nor β were detected by ABPP-MudPIT, indicating that these Hydrolase are either very low abundance in mouse brain and/or weakly labeled by FP probes.10 We next set out to confirm a subset of the Serine Hydrolase targets of RHC80267 and THL. HEK293T cells were transfected with cDNAs encoding representative targets of RHC80267 (KIAA1363, FAAH, BAT5, PLA2g7) and THL (ABHD12, BAT5, PLA2g7), and transfected proteomes were then treated with RHC80267 and THL (0.01-100 μM), followed by FP-Rh, which permitted calculation of IC50 values for inhibition of each enzyme (Figure 4). ABHD12, BAT5, and PLA2g7 were inhibited by THL with high potencies (IC50 values < 100 nM) that rivaled the reported potency for THL inhibition of DAGL-α/β (IC50 value of 60 nM4). Targets of RHC80267 were inhibited with much lower potency (IC50 values between 10-70 μM), as has also been observed with DAGL-α/β, for which treatment with 30-100 μM of RHC80267 is typically used for inhibition.2,5 Figure 4 Recombinant expression and characterization of Serine Hydrolase targets of RHC80267 and THL. Data represent average values ± standard errors for three independent experiments. Asterisk denotes lower band of double that corresponds to ABHD12 in ... Our recombinant expression studies further confirmed the selectivity that RHC80267 and THL show for individual brain Hydrolases. For instance, both FAAH and KIAA1363 were inhibited by RHC80267, but not THL, while ABHD12 was selectively blocked by THL. Only two Hydrolases, BAT5 and PLA2g7 were inhibited by both RHC80267 and THL. These results indicate that, while RHC80267 and THL each inactivate several brain Serine Hydrolases, the overlap in their target profiles beyond DAGL-α/β is quite small (Figure 5). We finally confirmed that none of the newly identified targets of RHC80267 and THL exhibited significant hydrolytic activity using 1-stearoyl-2-arachidonoyl glycerol as a substrate, indicating that these enzymes are not themselves DAGLs (Figure 6). Collectively, these data lead us to conclude that pharmacological effects observed with both RHC80267 and THL may be viewed as good initial evidence for DAGL-α/β-dependent events, especially in systems where endocannabinoid pathways have been implicated to function. On the other hand, pharmacological effects observed selectively with THL or RHC80267 may reflect the inactivation of distinct enzymatic pathways and should therefore be interpreted with caution. Figure 5 Venn diagram showing unique and overlapping Serine Hydrolase targets of RHC80267 and THL. Figure 6 DAGL activity for recombinant Hydrolases as measured using the substrate 1-stearoyl-2-arachidonoyl glycerol. Product formation (stearic acid, 2-AG) was monitored by LC-MS following general procedures described previously.13
