Nucleotide-binding domain and leucine-rich repeat pyrin-domain containing protein 1 (NLRP1) is an inflammasome sensor that mediates the activation of caspase-1 to induce cytokine maturation and pyroptosis(1-4). Gain-of-function mutations of NLRP1 cause severe inflammatory diseases of the skin(4-6). NLRP1 contains a function-to-find domain that auto-proteolyses into noncovalently associated subdomains(7-9), and proteasomal degradation of the repressive N-terminal fragment of NLRP1 releases its inflammatory C-terminal fragment (NLRP1 CT)(10,11). Cytosolic dipeptidyl peptidases 8 and 9 (hereafter, DPP8/DPP9) both interact with NLRP1, and small-molecule inhibitors of DPP8/DPP9 activate NLRP1 by mechanisms that are currently unclear(10,12-14). Here we report cryo-electron microscopy structures of the human NLRP1-DPP9 complex alone and with Val-boroPro (VbP), an inhibitor of DPP8/DPP9. The structures reveal a ternary complex that comprises DPP9, full-length NLRP1 and the NLRPT CT. The binding of the NLRP1 CT to DPP9 requires full-length NLRP1, which suggests that NLRP1 activation is regulated by the ratio of NLRP1 CT to full-length NLRP1. Activation of the inflammasome by ectopic expression of the NLRP1 CT is consistently rescued by co-expression of autoproteolysis-deficient full-length NLRP1. The N terminus of the NLRP1 CT inserts into the DPP9 active site, and VbP disrupts this interaction. Thus, VbP weakens the NLRP1-DPP9 interaction and accelerates degradation of the N-terminal fragment(10) to induce inflammasome activation. Overall, these data demonstrate that DPP9 quenches low levels of NLRP1 CT and thus serves as a checkpoint for activation of the NLRP1 inflammasome.
CARD8 is a germline-encoded pattern recognition receptor that detects intracellular danger signals. Like the related inflammasome sensor NLRP1, CARD8 undergoes constitutive autoprocessing within its function-to-find domain (FIIND), generating two polypeptides that stay associated and autoinhibited. Certain pathogen-and danger-associated activities, including the inhibition of the serine dipeptidases DPP8 and DPP9 (DPP8/9), induce the proteasome-mediated degradation of the N-terminal(NT) fragment,releasing the C-terminal (CT) fragment to form a caspase-1 activating inflammasome. DPP8/9 also bind directly to the CARD8 FIIND, but the role that this interaction plays in CARD8 inflammasome regulation is not yet understood. Here, we solved several cryo-EM structures of CARD8 bound to DPP9, with or without the DPP inhibitor Val-boroPro (VbP), which revealed a ternary complex composed of one DPP9, the full-length CARD8, and one CARD8-CT. Through structure-guided biochemical and cellular experiments, we demonstrated thatDPP9's structure restrains CARD8-CT after proteasomal degradation. Moreover, although DPP inhibitors do not directly displace CARD8 from DPP9in vitro,we show that they can nevertheless destabilize this complex in cells. Overall, these results demonstrate that DPP8/9 inhibitors cause CARD8 inflammasome activation via at least two distinct mechanisms, one upstream and one downstream of the proteasome.
CARD8 detects intracellular danger signals and forms a caspase-1 activating inflammasome. Like the related inflammasome sensor NLRP1, CARD8 autoprocesses into noncovalently associated N-terminal (NT) and C-terminal (CT) fragments and binds the cellular dipeptidyl peptidases DPP8 and 9 (DPP8/9). Certain danger-associated signals, including the DPP8/9 inhibitor Val-boroPro (VbP) and HIV protease, induce proteasome-mediated NT degradation and thereby liberate the inflammasome-forming CT. Here, we report cryoelectron microscopy (cryo-EM) structures of CARD8 bound to DPP9, revealing a repressive ternary complex consisting of DPP9, full-length CARD8, and CARD8-CT. Unlike NLRP1-CT, CARD8-CT does not interact with the DPP8/9 active site and is not directly displaced by VbP. However, larger DPP8/9 active-site probes can directly weaken this complex in vitro, and VbP itself nevertheless appears to disrupt this complex, perhaps indirectly, in cells. Thus, DPP8/9 inhibitors can activate the CARD8 inflammasome by promoting CARD8 NT degradation and by weakening ternary complex stability.
Several cytosolic pattern-recognition receptors (PRRs) form multiprotein complexes called canonical inflammasomes in response to intracellular danger signals. Canonical inflammasomes recruit and activate caspase-1 (CASP1), which in turn cleaves and activates inflammatory cytokines and gasdermin D (GSDMD), inducing pyroptotic cell death. Inhibitors of the dipeptidyl peptidases DPP8 and DPP9 (DPP8/9) activate both the human NLRP1 and CARD8 inflammasomes. NLRP1 and CARD8 have different N-terminal regions but have similar C-terminal regions that undergo autoproteolysis to generate two non-covalently associated fragments. Here, we show that DPP8/9 inhibition activates a proteasomal degradation pathway that targets disordered and misfolded proteins for destruction. CARD8's N terminus contains a disordered region of -160 amino acids that is recognized and destroyed by this degradation pathway, thereby freeing its C-terminal fragment to activate CASP1 and induce pyroptosis. Thus, CARD8 serves as an alarm to signal the activation of a degradation pathway for disordered and misfolded proteins.
NLRP1 is a cytosolic inflammasome sensor that mediates activation of caspase-1, which in turn induces cytokine maturation and pyroptotic cell death1-6. Gain-of-function NLPR1 mutations cause skin inflammatory diseases including carcinoma, keratosis, and papillomatosis7-14. NLRP1 contains a unique function-to-find domain (FIIND) that autoproteolyzes into noncovalently associated subdomains15-18. Proteasomal degradation of the autoinhibitory N-terminal fragment (NT) activates NLRP1 by releasing the inflammatory C-terminal fragment (CT)19,20. Cytosolic dipeptidyl peptidases 8 and 9 (DPP8/9) interact with NLRP1, and small-molecule DPP8/9 inhibitors activate NLRP1 by poorly characterized mechanisms11,19,21. Here, we report cryo-EM structures of the human NLRP1-DPP9 complex, alone and in complex with the DPP8/9 inhibitor Val-boroPro (VbP). Surprisingly, the NLRP1-DPP9 complex is a ternary complex comprised of DPP9, one intact FIIND of a non-degraded full-length NLRP1 (NLRP1-FL) and one NLRP1-CT freed by NT degradation. The N-terminus of the NLRP1-CT unfolds and inserts into the DPP9 active site but is not cleaved by DPP9, and this binding is disrupted by VbP. Structure-based mutagenesis reveals that the binding of NLRP1-CT to DPP9 requires NLRP1-FL and vice versa, and inflammasome activation by ectopic NLRP1-CT expression is rescued by co-expressing autoproteolysis-deficient NLRP1-FL. Collectively, these data indicate that DPP9 functions as a 'bomb-diffuser' to prevent NLRP1-CTs from inducing inflammation during homeostatic protein turnover.
Canonical inflammasomes are innate immune signaling platforms that are formed in response to intracellular pathogen-associated signals and trigger caspase-1-dependent pyroptosis. Inflammasome formation and signaling is thought to mainly occur in myeloid cells, and in particular monocytes and macrophages. Here we show that small molecule inhibitors of dipeptidyl peptidases 8 and 9 (DPP8/9), which activate the related CARD8 and NLRP1 inflammasomes, also activate pyroptosis in human and rodent resting lymphocytes. We found that both CD4(+) and CD8(+) T cells were particularly sensitive to these inhibitors, although the sensitivity of T cells, like macrophages, varied considerably between species. In human T cells, we show that CARD8 mediates DPP8/9 inhibitor-induced pyroptosis. Intriguingly, although activated human T cells express the key proteins known to be required for CARD8-mediated pyroptosis, these cells were completely resistant to DPP8/9 inhibitors. Overall, these data show that resting lymphoid cells can activate at least one inflammasome, revealing additional cell types and states poised to undergo rapid pyroptotic cell death in response to danger-associated signals.
Inflammasomes are multiprotein complexes that activate inflammatory cytokines and induce pyroptosis in response to intracellular danger-associated signals. NLRP1 and CARD8 are related germline-encoded pattern recognition receptors that form inflammasomes, but their activation mechanisms and biological purposes have not yet been fully established. Both NLRP1 and CARD8 undergo post-translational autoproteolysis to generate two non-covalently associated polypeptide chains. NLRP1 and CARD8 activators induce the proteasome-mediated destruction of the N-terminal fragment, liberating the C-terminal fragment to form an inflammasome. Here, we review the danger-associated stimuli that have been reported to activate NLRP1 and/or CARD8, including anthrax lethal toxin, Toxoplasma gondii, Shigella flexneri and the small molecule DPP8/9 inhibitor Val-boroPro, focusing on recent mechanistic insights and highlighting unresolved questions. In addition, we discuss the recently identified disease-associated mutations in NLRP1 and CARD8, the potential role that DPP9's protein structure plays in inflammasome regulation, and the emerging link between NLRP1 and metabolism. Finally, we summarize all of this latest research and consider the possible biological purposes of these enigmatic inflammasomes.
Intracellular pathogens and danger signals trigger the formation of inflammasomes, which activate inflammatory caspases and induce pyroptosis. The anthrax lethal factor metalloprotease and small-molecule DPP8/9 inhibitors both activate the NLRP1B inflammasome, but the molecular mechanism of NLRP1B activation is unknown. In this study, we used genome-wide CRISPR-Cas9 knockout screens to identify genes required for NLRP1B-mediated pyroptosis. We discovered that lethal factor induces cell death via the N-end rule proteasomal degradation pathway. Lethal factor directly cleaves NLRP1B, inducing the N-end rule-mediated degradation of the NLRP1B N terminus and freeing the NLRP1B C terminus to activate caspase-1. DPP8/9 inhibitors also induce proteasomal degradation of the NLRP1B N terminus but not via the N-end rule pathway. Thus, N-terminal degradation is the common activation mechanism of this innate immune sensor.
Intracellular pathogenic structures or activities stimulate the formation of inflammasomes, which recruit and activate caspase-1 and trigger an inflammatory form of cell death called pyroptosis. The well-characterized mammalian inflammasome sensor proteins all detect one specific type of signal, for example double-stranded DNA or bacterial flagellin. Remarkably, NLRP1 was the first protein discovered to form an inflammasome, but the pathogenic signal that NLRP1 detects has not yet been identified. NLRP1 is highly polymorphic, even among inbred rodent strains, and it has been suggested that these diverse NLRP1 alleles may have evolved to detect entirely different stimuli. Intriguingly, inhibitors of the serine proteases DPP8 and DPP9 (DPP8/9) were recently shown to activate human NLRP1, its homolog CARD8, and several mouse NLRP1 alleles. Here, we show now that DPP8/9 inhibitors activate all functional rodent NLRP1 alleles, indicating that DPP8/9 inhibition induces a signal detected by all NLRP1 proteins. Moreover, we discovered that the NLRP1 allele sensitivities to DPP8/9 inhibitor-induced and Toxoplasma gondii-induced pyroptosis are strikingly similar, suggesting that DPP8/9 inhibition phenocopies a key activity of T. gondii. Overall, this work indicates that the highly polymorphic NLRP1 inflammasome indeed senses a specific signal like the other mammalian inflammasomes.
The dipeptidyl peptidases (DPPs) regulate hormones, cytokines, and neuropeptides by cleaving dipeptides after proline from their amino termini. Due to technical challenges, many DPP substrates remain unknown. Here, we introduce a simple method, termed CHOPS (chemical enrichment of protease substrates), for the discovery of protease substrates. CHOPS exploits a 2-pyridinecarboxaldehyde (2PCA)-biotin probe, which selectively biotinylates protein N-termini except those with proline in the second position. CHOPS can, in theory, discover substrates for any protease, but is particularly well suited to discover canonical DPP substrates, as cleaved but not intact DPP substrates can be identified by gel electrophoresis or mass spectrometry. Using CHOPS, we show that DPP8 and DPP9, enzymes that control the Nlrp1 inflammasome through an unknown mechanism, do not directly cleave Nlrp1. We further show that DPP9 robustly cleaves short peptides but not full-length proteins. More generally, this work delineates a practical technology for identifying protease substrates, which we anticipate will complement available "N-terminomic" approaches.
Inflammasomes are multiprotein complexes formed in response to pathogens. NLRP1 and CARD8 are related proteins that form inflammasomes, but the pathogen-associated signal(s) and the molecular mechanisms controlling their activation have not been established. Inhibitors of the serine dipeptidyl peptidases DPP8 and DPP9 (DPP8/9) activate both NLRP1 and CARD8. Interestingly, DPP9 binds directly to NLRP1 and CARD8, and this interaction may contribute to the inhibition of NLRP1. Here, we use activity-based probes, reconstituted inflammasome assays, and mass spectrometry-based proteomics to further investigate the DPP9-CARD8 interaction. We show that the DPP9-CARD8 interaction, unlike the DPP9-NLRP1 interaction, is not disrupted by DPP9 inhibitors or CARD8 mutations that block autoproteolysis. Moreover, wild-type, but not catalytically inactive mutant, DPP9 rescues CARD8-mediated cell death in DPP9 knockout cells. Together, this work reveals that DPP9's catalytic activity and not its binding to CARD8 restrains the CARD8 inflammasome and thus suggests the binding interaction likely serves some other biological purpose.
Small-molecule inhibitors of the serine dipeptidases DPP8 and DPP9 (DPP8/9) induce a lytic form of cell death called pyroptosis in mouse and human monocytes and macrophages(1,2). In mouse myeloid cells, Dpp8/9 inhibition activates the inflammasome sensor Nlrp1b, which in turn activates pro-caspase-1 to mediate cell death(3), but the mechanism of DPP8/9 inhibitor-induced pyroptosis in human myeloid cells is not yet known. Here we show that the CARD-containing protein CARD8 mediates DPP8/9 inhibitor-induced pro-caspase-1-dependent pyroptosis in human myeloid cells. We further show that DPP8/9 inhibitors induce pyroptosis in the majority of human acute myeloid leukemia (AML) cell lines and primary AML samples, but not in cells from many other lineages, and that these inhibitors inhibit human AML progression in mouse models. Overall, this work identifies an activator of CARD8 in human cells and indicates that its activation by small-molecule DPP8/9 inhibitors represents a new potential therapeutic strategy for AML.
Val-boroPro (PT-100, Talabostat) induces powerful anti-tumor immune responses in syngeneic cancer models, but its mechanism of action has not yet been established. Val-boroPro is a non-selective inhibitor of post-proline-cleaving serine proteases, and the inhibition of the highly related cytosolic serine proteases Dpp8 and Dpp9 (Dpp8/9) by Val-boroPro was recently demonstrated to trigger an immunostimulatory form of programmed cell death known as pyroptosis selectively in monocytes and macrophages. Here we show that Dpp8/9 inhibition activates the inflammasome sensor protein Nlrp1b, which in turn activates pro-caspase-1 to mediate pyroptosis. This work reveals a previously unrecognized mechanism for activating an innate immune pattern recognition receptor and suggests that Dpp8/9 serve as an intracellular checkpoint to restrain Nlrp1b and the innate immune system.
Val-boroPro (Talabostat, PT-100), a nonselective inhibitor of post-proline cleaving serine proteases, stimulates mammalian immune systems through an unknown mechanism of action. Despite this lack of mechanistic understanding, Val-boroPro has attracted substantial interest as a potential anticancer agent, reaching phase 3 trials in humans. Here we show that Val-boroPro stimulates the immune system by triggering a proinflammatory form of cell death in monocytes and macrophages known as pyroptosis. We demonstrate that the inhibition of two serine proteases, DPP8 and DPP9, activates the pro-protein form of caspase-1 independent of the inflammasome adaptor ASC. Activated pro-caspase-1 does not efficiently process itself or IL-1beta but does cleave and activate gasdermin D to induce pyroptosis. Mice lacking caspase-1 do not show immune stimulation after treatment with Val-boroPro. Our data identify what is to our knowledge the first small molecule that induces pyroptosis and reveals a new checkpoint that controls the activation of the innate immune system.
Title: Pyroptosis and Apoptosis Pathways Engage in Bidirectional Crosstalk in Monocytes and Macrophages Taabazuing CY, Okondo MC, Bachovchin DA Ref: Cell Chemical Biology, 24:507, 2017 : PubMed
Pyroptosis is a lytic form of programmed cell death mediated by the inflammatory caspase-1, -4, and -5. We recently discovered that small-molecule inhibitors of the serine peptidases DPP8 and DPP9 (DPP8/9) induce pro-caspase-1-dependent pyroptosis in monocytes and macrophages. Notably, DPP8/9 inhibitors, unlike microbial agents, absolutely require caspase-1 to induce cell death. Therefore, DPP8/9 inhibitors are useful probes to study caspase-1 in cells. Here, we show that, in the absence of the pyroptosis-mediating substrate gasdermin D (GSDMD), caspase-1 activates caspase-3 and -7 and induces apoptosis, demonstrating that GSDMD is the only caspase-1 substrate that induces pyroptosis. Conversely, we found that, during apoptosis, caspase-3/-7 specifically block pyroptosis by cleaving GSDMD at a distinct site from the inflammatory caspases that inactivates the protein. Overall, this work reveals bidirectional crosstalk between apoptosis and pyroptosis in monocytes and macrophages, further illuminating the complex interplay between cell death pathways in the innate immune system.
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.
The selectivity of an enzyme inhibitor is a key determinant of its usefulness as a tool compound or its safety as a drug. Yet selectivity is never assessed comprehensively in the early stages of the drug discovery process, and only rarely in the later stages, because technical limitations prohibit doing otherwise. Here, we report EnPlex, an efficient, high-throughput method for simultaneously assessing inhibitor potency and specificity, and pilot its application to 96 serine hydrolases. EnPlex analysis of widely used serine hydrolase inhibitors revealed numerous previously unrecognized off-target interactions, some of which may help to explain previously confounding adverse effects. In addition, EnPlex screening of a hydrolase-directed library of boronic acid- and nitrile-containing compounds provided structure-activity relationships in both potency and selectivity dimensions from which lead candidates could be more effectively prioritized. Follow-up of a series of dipeptidyl peptidase 4 inhibitors showed that EnPlex indeed predicted efficacy and safety in animal models. These results demonstrate the feasibility and value of high-throughput, superfamily-wide selectivity profiling and suggest that such profiling can be incorporated into the earliest stages of drug discovery.
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
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.
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.
Title: The pharmacological landscape and therapeutic potential of serine hydrolases Bachovchin DA, Cravatt BF Ref: Nat Rev Drug Discov, 11:52, 2012 : PubMed
Serine hydrolases perform crucial roles in many biological processes, and several of these enzymes are targets of approved drugs for indications such as type 2 diabetes, Alzheimer's disease and infectious diseases. Despite this, most of the human serine hydrolases (of which there are more than 200) remain poorly characterized with respect to their physiological substrates and functions, and the vast majority lack selective, in vivo-active inhibitors. Here, we review the current state of pharmacology for mammalian serine hydrolases, including marketed drugs, compounds that are under clinical investigation and selective inhibitors emerging from academic probe development efforts. We also highlight recent methodological advances that have accelerated the rate of inhibitor discovery and optimization for serine hydrolases, which we anticipate will aid in their biological characterization and, in some cases, therapeutic validation.
The serine hydrolases constitute a large class of enzymes that play important roles in physiology. There is great interest in the development of potent and selective pharmacological inhibitors of these proteins. Traditional active-site inhibitors often have limited selectivity within this superfamily and are tedious and expensive to discover. Using the serine hydrolase RBBP9 as a model target, we designed a rapid and relatively inexpensive route to highly selective peptoid-based inhibitors that can be activated by visible light. This technology provides rapid access to photo-activated tool compounds capable of selectively blocking the function of particular serine hydrolases.
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.
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.
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.
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.
The retinoblastoma (RB) tumor suppressor protein controls cell cycle progression by regulating the activity of the transcription factor E2F, which activates genes essential for DNA replication. Thus, factors that bind and regulate RB activity are considered valuable targets for preventing tumorigenesis. The enzyme RB binding protein 9 (RBBP9) is widely expressed in different tissues and upregulated in certain tumors. As a result, the identification of compounds that selectively inhibit RBBP9 activity would serve as potentially valuable probes for the study of apoptosis, cell cycle, and tumorigenesis. We previously reported a modestly potent, RBBP9 reversible inhibitor, ML081 (CID-6603320). However, ML081 exhibits high cytotoxicity. We, therefore, have now identified a newer probe, ML114 (CID-5934766), which is 10-fold more potent than ML081, exhibits no cytotoxicity, and is from an entirely different structural and mechanistic class of compounds that covalently inhibit RBBP9. This new probe will be useful for in vitro assays in which it is desirable to specifically block RBBP9 activity for primary research purposes.
Recent findings have identified protein phosphatase methylesterase-1 (PME-1) as a protector of sustained ERK pathway activity in malignant gliomas. PME-1 is a protein methylesterase that functions in the regulation of protein phosphatase 2A (PP2A) by reversible methylation. Biochemical elucidation of PME-1 would thus greatly benefit from the development of potent and selective chemical inhibitors. The probe compound ML136 (CID-44607965), containing a sulfonyl acrylonitrile core, represents the first potent, selective inhibitor of PME-1. Moreover, the probe does not appear to exhibit cytotoxicity. Thus, ML136 should serve as a useful tool for in vitro and in situ research assays in which it is desirable to specifically block PME-1 activity.
Serine hydrolases (SHs) are one of the largest and most diverse enzyme classes in mammals. They play fundamental roles in virtually all physiological processes and are targeted by drugs to treat diseases such as diabetes, obesity, and neurodegenerative disorders. Despite this, we lack biological understanding for most of the 110+ predicted mammalian metabolic SHs, in large part because of a dearth of assays to assess their biochemical activities and a lack of selective inhibitors to probe their function in living systems. We show here that the vast majority (> 80%) of mammalian metabolic SHs can be labeled in proteomes by a single, active site-directed fluorophosphonate probe. We exploit this universal activity-based assay in a library-versus-library format to screen 70+ SHs against 140+ structurally diverse carbamates. Lead inhibitors were discovered for approximately 40% of the screened enzymes, including many poorly characterized SHs. Global profiles identified carbamate inhibitors that discriminate among highly sequence-related SHs and, conversely, enzymes that share inhibitor sensitivity profiles despite lacking sequence homology. These findings indicate that sequence relatedness is not a strong predictor of shared pharmacology within the SH superfamily. Finally, we show that lead carbamate inhibitors can be optimized into pharmacological probes that inactivate individual SHs with high specificity in vivo.
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.
The retinoblastoma (RB) tumor suppressor protein controls cell cycle progression by regulating the activity of the transcription factor E2F, which in turn activates genes essential for DNA replication. Thus, factors that bind and regulate RB activity provide for valuable targets for preventing tumorigenesis. The enzyme, RB binding protein 9 (RBBP9), is widely expressed in a number of different tissues and is upregulated in certain tumors. As a result, the identification of compounds that selectively inhibit RBBP9 activity would serve as potentially valuable probes for the study of apoptosis, cell cycle, and tumorigenesis. The probe ML081 (CID- 6603320; emetine hydrochloride) represents the first non-covalent, selective RBBP9 inhibitor, and will be useful for exploring the enzymatic functions of RBBP9 in biological systems. Moreover, the tight structure-activity relationship of the emetine-RBBP9 interaction suggests that only minor modifications to the emetine structure will improve its activity. As a result, future studies will involve semi-synthetic addition of small moieties to the emetine and cephaeline scaffolds.
Title: Identification of selective inhibitors of uncharacterized enzymes by high-throughput screening with fluorescent activity-based probes Bachovchin DA, Brown SJ, Rosen H, Cravatt BF Ref: Nat Biotechnol, 27:387, 2009 : PubMed
High-throughput screening to discover small-molecule modulators of enzymes typically relies on highly tailored substrate assays, which are not available for poorly characterized enzymes. Here we report a general, substrate-free method for identifying inhibitors of uncharacterized enzymes. The assay measures changes in the kinetics of covalent active-site labeling with broad-spectrum, fluorescent probes in the presence of inhibitors by monitoring the fluorescence polarization signal. We show that this technology is applicable to enzymes from at least two mechanistic classes, regardless of their degree of functional annotation, and can be coupled with secondary proteomic assays that use competitive activity-based profiling to rapidly determine the specificity of screening hits. Using this method, we identify the bioactive alkaloid emetine as a selective inhibitor of the uncharacterized cancer-associated hydrolase RBBP9. Furthermore, we show that the detoxification enzyme GSTO1, also implicated in cancer, is inhibited by several electrophilic compounds found in public libraries, some of which display high selectivity for this protein.
