Benjamin F. Cravatt, PhD - US grants

cell communication molecule; hypothermia; sleep; hormone regulation /control mechanism; hormone metabolism; amides; oleate; enzyme activity; serotonin; hydrolysis; sleep deprivation; body temperature regulation; high performance liquid chromatography; cats; laboratory rat; cerebrospinal fluid; mass spectrometry;

The objective of this proposal is to develop and utilize novel chemical probes for activity-based proteomics investigations aimed at understanding the role that hydrolytic enzymes play in tumor metastasis. With the post-genome era rapidly approaching, new strategies for the functional analysis of proteins are needed. To date, proteomics efforts have primarily been concerned with recording variations in protein level rather than activity. The ability to profile classes of proteins based on changes in their activity would greatly accelerate both the assignment of protein function and the identification of potential pharmaceutical targets. The work described in this proposal will focus on designing, synthesizing, and testing novel active site-directed probes that record dynamics in the expression and function of entire enzyme families. In this application, the PIs will use synthetic chemistry, biochemistry, and molecular and cell biology techniques towards the goals of 1) identifying proteases that support or impede tumor metastasis, 2) imaging extracellular "hot spots" of proteolytic activity associated with tumor cell migration and invasion, and 3) developing activity-based probes selective for the matrix metalloproteinase family of enzymes. Through these studies the PI hopes to clarify which, of the many, hydrolytic enzymes implicated in cancer actually participate in proteolytic events important for the progression of the disease. The enzymes will in turn represent valuable targets for pharmaceutical efforts aimed at suppressing cancer metastasis.

DESCRIPTION: The objective of this proposal is to understand the catalytic, structural, and cell biological features of fatty acid amide hydrolase (FAAH), a mammalian membrane-bound enzyme responsible for the catabolism of the fatty acid amide family of endogenous signaling lipids. Representative fatty acid amides degraded by FAAH include the endocannabinoid anandamide and the sleep-inducing lipid oleamide. Fatty acid amides have been shown to induce a remarkable array of pharmacological effects in mammals, including sleep, analgesia, hypothermia, and learning and memory defects. The impressive bioactivity of fatty acid amides suggests that FAAH might serve as an attractive target for therapeutic efforts aimed at influencing pain, sleep, and memory systems. The elucidation of FAAH's catalytic, structural, and cellular features would provide a foundation for the design of FAAH-specific chemical inhibitors to be employed as agents for both the study of fatty acid amide-based physiological processes and the potential pharmaceutical treatment of pathologies associated with these systems. In this application, the molecular and cellular properties of FAAH will be examined using a multidisciplinary approach, employing biochemistry, molecular biology, immunochemistry, and synthetic chemistry techniques towards the goals of determining: 1) the catalytic mechanism and origins of substrate selectivity for FAAH-mediated amide hydrolysis, 2) the x-ray crystal structure of a FAAH-oleyl phosphonate inhibitor complex, 3) the domains of FAAH responsible for self-association and membrane binding, and 4) the cellular and subcellular localization of FAAH in mammalian tissue. These proposed studies should provide molecular tools for the chemical and genetic regulation of FAAH in vivo, allowing for a direct evaluation of the potential costs and benefits of therapeutic strategies that target the endocannabinioid system for the treatment of pain, sleep, and/or mood disorders.

DESCRIPTION (provided by applicant): Fatty acid amides (FAAs) represent a novel family of endogenous signaling lipids implicated in a broad range of neurophysiological processes, including pain, sleep, feeding, and memory. A prototype FAA anandamide may serve as an endogenous ligand for the central cannabinoid (CB1) receptor. Although anandamide binds and activates the CB1 receptor in vitro, this compound induces only weak cannabinoid behavioral effects in vivo, possibly as a result of its rapid catabolism. Currently, our understanding of the mechanisms by which anandamide and related FAAs are produced and catabolized in vivo remains limited. The objective of this proposal is to identify and characterize enzymes that regulate the levels and activities of FAAs in vivo. One candidate enzyme responsible for terminating FAA signals in vivo is fatty acid amide hydrolase (FAAH), which hydrolyzes anandamide and related FAAs in vitro. To test the role that FAAH plays in regulating FAA function in vivo, we have created a mouse model in which this enzyme has been genetically deleted (FAAH-/- mice). FAAH-/- mice are severely impaired in their ability to degrade FAAs and exhibit an array of intense CB1 -dependent behavioral responses to anandamide, including hypomotility, analgesia, catalepsy, and hypothermia. FAAH-/- mice possess greatly increased endogenous brain levels of anandamide (and several other FAAs) and display reduced pain sensation that is reversed by the CB1 antagonist SR141716A. These data indicate that FAAH is a key regulator of FAA signaling in vivo, setting an endocannabinoid tone that modulates pain perception. In this application, we propose to extend our pharmacological and behavioral analysis of FAAH +/+,+/-, and -/- about, and -/- mice to examine the role that anandamide and related FAAs play in regulating multiple forms of acute and chronic pain sensation. We will also use these animal models to elucidate interactions between the endogenous cannabinoid and opioid systems. Finally, we plan to isolate and molecularly characterize enzymes that biosynthesis FAAs. These studies will elucidate key enzymes that regulate FAA signaling in vivo, and these proteins may represent new targets for the treatment of pain, addiction, and other neurological disorders.

The objective of Project I is to understand the molecular and cellular mechanisms of action of fatty acid amide hydrolase (FAAH), a mammalian membrane-bound enzyme responsible for the catabolism of the fatty acid amide (FAA) family of endogenous signaling lipids. FAAs represent an emerging class of chemical messengers that influence a variety of behavioral processes, including, pain sensation, anxiety, sleep, and feeding. Recent genetic and pharmacological studies have demonstrated that FAAH is a principal regulator of FAA-based signaling events in vivo, suggesting that this enzyme may represent an attractive target for the treatment of neurological disorders like pain and anxiety. Nonetheless, many questions remain regarding the function of FAAH and its suitability as a therapeutic target. For example, among the numerous hydrolases present in mammalian proteomes, how does FAAH exert such extraordinary control over the levels and activity of FAAs in vivo? Might FAAH recruit these hydrophobic substrates directly from cellular membranes, thereby giving the enzyme privileged access to these lipid messengers? Likewise, does FAAH operate alone in neurons, or might this enzyme participate in higher-order protein complexes that regulate its localization and/or activity? Our recent determination of the FAAH structure by x-ray crystallography has inspired several new hypotheses to explain this enzyme's mode of action. In this project, we aim to test these hypotheses using a multidisciplinary approach, employing synthetic chemistry, enzymology, cell biology, and proteomic techniques towards the goals of: 1) determining the roles that FAAH channels play in catalysis, 2) characterizing the molecular interactions between FAAH and cell membranes, 3) identifying FAAH-associated proteins in brain and liver, and 4) comparing the cellular stability of WT-FAAH and its natural P129T variant associated with problem drug use. We anticipate that these studies will not only enhance our understanding of the molecular and cellular mechanism of action of FAAH, but also uncover novel ways to target this enzyme with inhibitors for research and therapeutic purposes.

DESCRIPTION (provided by applicant): The main goals of this Program Project are to: 1) understand the molecular and cellular mechanisms by which the integral membrane enzyme fatty acid amide hydrolase (FAAH) degrades neuromodulatory fatty acid amides (FAAs), and 2) determine the physiological and potential pathological consequences of chemically inhibiting FAAH in vivo. FAAs represent an emerging class of lipid messengers that influence a variety of behavioral processes, including, pain sensation, anxiety, sleep, and feeding. Recent genetic and pharmacological studies have demonstrated that FAAH is a principal regulator of FAA-based signaling events in vivo, suggesting that this enzyme may represent an attractive target for the treatment of neurological disorders like pain and anxiety. Nonetheless, many questions remain regarding the mechanism of action of FAAH and its suitability as a therapeutic target. For example, among the numerous hydrolases present in mammalian proteomes, how does FAAH exert such extraordinary control over the levels and activity of FAAs? Might FAAH recruit its FAA substrates directly from cell membranes in order to expedite their inactivation? Can potent and selective reversible inhibitors of FAAH be generated that, upon pharmacological administration, reproduce the neurochemical and behavioral effects observed in FAAH-knockout mice? It is the goal of this application to address these important questions by embarking on a multidisciplinary program aimed at: 1) testing the function of unique domains in FAAH, including its provocative collection of channels and membrane-binding sites (Project I), 2) determining the crystal structures of key FAAH species, including the apo-enzyme, inhibitor/product complexes, and mutants like the natural P129T variant associated with problem drug use (Project II), and 3) evaluating the neurochemical and behavioral effects of FAAH inhibitors in vivo (Project III). The knowledge gained from these studies will also be applied towards the design of increasingly potent and selective FAAH inhibitors (Core), which should prove of great value as both research tools and potential therapeutic agents.

Proteases are suspected to play major roles in cancer, including the activation/inactivation of growth factors and the degradation of extracellular matrix components to promote cancer cell migration and invasion. Consistent with this premise, transcript and protein levels for many proteases are upregulated in cancer cell lines and primary tumors. However, whether these changes in protease expression correlate with changes in protease activity remains a critical, but largely unanswered question. Indeed, proteases are regulated by a complex series of post- translational events, meaning that their expression levels, as measured by conventional genomic and proteomic methods, may fail to accurately report on the activity of these enzymes. To address this important problem, we have introduced a chemical proteomics technology referred to as activity-based protein profiling (ABPP) that utilizes active site-directed probes to determine the functional state of large numbers of proteases directly in whole cell, tissue, and fluid samples. We have applied ABPP to identify several protease activities upregulated in human cancer cells and primary tumors. Recently, we have created an advanced antibody- based microarray platform for ABPP that enables profiling of protease activities with an unprecedented combination of sensitivity, resolution, and throughput, while requiring only minute quantities of proteome. The goal of this R33application is to extend these studies to create the first ABPP microarray for the parallel analysis of key cancer-associated protease activities in any proteomic sample. These studies will deliver valuable new reagents and methods for the functional characterization of proteases that will be made freely available to the cancer research community. We envision that the general implementation of these innovative technologies will greatly accelerate the discovery of proteases with altered activity in human cancer. These proteases may in turn represent valuable new markers and targets for the diagnosis and treatment of cancer.

[unreadable] DESCRIPTION (provided by applicant): A single nucleotide polymorphism has been correlated with human populations at increased risk for drug and alcohol abuse. Affected individuals carry a variant form of fatty acid amide hydrolase, an enzyme responsible for setting cannabinoid tone in the brain. Biochemical data on this point mutant have revealed no significant deviations from its wild-type counterpart. However, fluids samples from affected individuals have revealed that carriers of this mutation express levels of this enzyme at roughly half that of wild-type cohorts. Could this observation provide an explanation for the observed drug abuse behaviors, or does this mutation manifest its effects in unpredicted ways? The only clear way to address these questions is to create a living model system. This proposal seeks to create a mouse harboring the same mutation found in the human population studies. Once this "knock-in" mouse is engineered, a series of behavioral tests will be used to determine if the animals replicate the drug abuse behaviors correlated in humans. Further, possible mechanisms to explain these behaviors will be sought. These mechanisms include the possible alteration of endogenous cannabinoid tone and/or the selective dysregulation of individual fatty acid amide metabolites. If this mouse model is found to exhibit increased susceptibility to drug abuse behaviors, it will provide a very powerful platform for future research into the evolution of these behaviors and possible means of pharmacological intervention. The National Institute on Drug Abuse has called drug abuse and addiction "one of America's most challenging public health problems", rivaling both diabetes and cancer in terms of costs to society. Identifying genetic contributions to drug abuse behaviors can provide powerful insights into etiology and may one day offer personalized treatment options for those at risk for these health concerns. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Small-molecule metabolic and signaling pathways regulate nearly all cellular and physiological activities. Alterations in these biochemical networks likely contribute to the development of several diseases, including cancer. Nonetheless, the discovery and functional characterization of biochemical pathways that support cancer have, to date, been hindered by a lack of technologies that can globally inventory their molecular components in biological systems. To address this central problem, we have introduced a set of chemical proteomic and metabolomic technologies termed activity-based protein profiling (ABPP) and metabolite enrichment by tagging and proteolytic release (METPR), respectively. ABPP, which utilizes active site-directed chemical probes to record the functional state of enzymes directly in native proteomes, has led to the discovery of enzyme activities that are elevated in aggressive cancer cells. Several of these enzymes are completely unannotated, suggesting that they regulate novel metabolic pathways in cancer. We have shown that ABPP, when coupled with untargeted metabolite profiling, facilitates assignment of biochemical functions to unannotated enzymes that support cancer pathogenesis. METPR is an advanced metabolomics method that employs chemical probes to tag, enrich, and profile endogenous small molecules of any physicochemical class. In this application, we propose to unite our enzyme and small molecule profiling methods to create the first integrated "systems biology" platform for the global analysis of biochemical pathways altered in human cancer cells. Specifically, we will: 1) identify enzyme activities and metabolites that are elevated in aggressive cancer cells, 2) assemble these biomolecules into metabolic pathways and test their function in cancer, and 3) confirm that these pathogenesis-related biochemical pathways are dysregulated in primary human cancers. Collectively, these studies should yield fundamental insights into the molecular basis of cancer and, at the same time, achieve several of the overarching goals of this RFA, including determining: 1) how pathways are differentially regulated in disease states (i.e., aggressive versus non-aggressive cancer cells), 2) new connections between pathways (i.e., annotation of uncharacterized enzymes that regulate biochemical networks in cancer), and 3) the suitability of innovative metabolomic technologies (i.e., METPR) for basic and translational applications.

DESCRIPTION (provided by applicant): Of the many fascinating discoveries made by genome sequencing projects, perhaps none is more provocative than the realization that eukaryotic and prokaryotic organisms universally possess a huge number of uncharacterized enzymes. This finding belies the commonly held notion that our knowledge of cell metabolism is near completion and underscores the vast landscape of unannotated metabolic and signaling networks that operate in our cells and tissues. The functional annotation of uncharacterized enzymatic pathways, thus, represents a grand challenge for researchers in the post-genomic era. To achieve this goal, selective pharmacological tools to perturb these pathways would be of particular value. A pressing question, however, immediately arises: how can one rapidly and systematically develop potent and selective small-molecule inhibitors for uncharacterized enzymes? Here, we propose to solve this key problem using an innovative chemical proteomic technology termed activity-based protein profiling (ABPP). Specifically, we will develop a streamlined, "library-versus-library" platform for competitive ABPP that enables the parallel discovery of inhibitors for numerous uncharacterized enzymes directly in native proteomes. As an initial application, we will use this platform to develop potent and selective inhibitors for the serine hydrolase superfamily of enzymes. The serine hydrolase class represents an excellent target for inhibitor discovery by ABPP for multiple reasons: 1) it is an exceptionally large family of enzymes (~110 members in humans) that contains numerous uncharacterized members (~40-50% of the family), 2) it is enriched in enzymes that play key roles in mammalian signaling networks, especially in the nervous system, and 3) ABPP probes and inhibitor libraries for serine hydrolases are in place to enable library-versus-library screening. If successful, the studies proposed in this application will have a profound and immediate impact on both the biological and chemical research communities. The biology community will receive a bounty of new pharmacological tools in the form of serine hydrolase inhibitors that have been optimized for potency and selectivity in vitro and in vivo. The chemistry community will receive a validated methodological process for large-scale inhibitor discovery in the form of a fully developed library-versus-library competitive ABPP platform that can be applied, in principle, to any enzyme class. We believe that this application is well-suited for the EUREKA program because: 1) it tackles n difficult technical problem of extraordinary biological and biomedical significance using cutting-edge methodologies, and 2) the magnitude of impact of our proposed studies, which should provide transforming research tools and methodologies for the biological and chemistry communities, is exceptionally high. A large fraction of drugs used to treat human disease inhibit enzymes. A huge number of uncharacterized enzymes are encoded by the human genome, suggesting that many as- of-yet undiscovered drug targets may exist in our cells and tissues. The goal of this application is to develop an innovative platform to systematically discover inhibitors for uncharacterized enzymes of relevance to human health and disease.

D6.0 SELECTIVITY PROFILING CORE: A key challenge to the MLPCN strategy is the need to have sufficient capacity for selectivity screening data, essential for the optimization of selective chemical probes. The TSRI Center has instituted two generic approaches that enable these data to be developed and integrated in chemical optimization for broad ranges of biological targets. This area is one example of Center-driven scientific choices that have been of high value and impact broadly. Two such approaches, one for transcription and one for broad profiling of enzyme inhibition by catalytic family, are reduced to practice and presented.

DESCRIPTION (provided by applicant): This project aims to develop a revolutionary screening platform that will allow for the rapid isolation of hundreds of high affinity and specificity synthetic ligands for proteins in a highly parallel fashion. The central feature of the proposal is to label dozens to hundreds of mechanistically related proteins simultaneously using reagents developed for Activity-Based Protein Profiling (ABPP). This mixture of labeled proteins will then be screened en masse against a several million member combinatorial library of peptoids displayed on beads. Peptoid-displaying beads that retain labeled protein will be collected by fluorescence-activated flow sorting. Mass spectrometry-based techniques will then be employed to identify the protein captured on the bead as well as the sequence of the peptoid. These peptoids can be employed as capture agents in the creation of protein-detecting microarrays. They will also be modified for use as high potency, photo-triggered pharmacological inhibitors of their target proteins. This will involve appendage of a Ru(II)-containing complex to the peptoid. When irradiated with visible light, the ruthenium complex generates singlet oxygen, which is capable of destroying proteins in the immediate vicinity. These novel reagents will be used to probe the roles of the target proteins in biological assays. If successful, this effort will revolutionize the discovery of specific protein ligands, a goal identified by the NIH as a strategic priority. Because the ligands will be peptoids, which can be easily synthesized in large quantities even by laboratories lacking specialized organic chemistry skills, these ligands will be far more widely accessible to the research community than would be the case for most other classes of protein binding-molecules. Therefore, we believe that this project is transformative in its potential scope and impact. PUBLIC HEALTH RELEVANCE: The identification of large numbers of protein ligands has been identified by the NIH as a high priority for biomedical research. Such ligands could be employed as reagents to construct tools for the discovery of diagnostically useful disease biomarkers. The synthetic compounds that we plan to identify could also serve as drug leads for a variety of therapeutically interesting targets.

DESCRIPTION (provided by applicant): The brain is arguably the most complex organ in higher mammals in terms of diversity of resident cell types, anatomical organization, and intricate modes of intercellular communication and protein regulation. Each of these factors presents a tremendous technical challenge for researchers interested in applying large-scale biological approaches to the study of brain physiology and pathology. Proteomics, for instance, is most adept at mapping protein expression and post-translational events in biological samples enriched in a single cell type (e.g., cultured cells or relatively homogenous organs, such as liver). The brain, however, offers a much more complicated scenario. Indeed, brain disorders such as drug dependence and withdrawal may be due to changes in the molecular composition and function of only a small number of neurons or neuronal circuits dispersed among a large background of unaffected cells. Neuronal plasticity is furthermore known to rely heavily on post-translational events that regulate protein structure and activity, often within specific subcellular compartments (e.g., the synapse). Detecting and quantifying such 'rare'biochemical events is beyond the capacity of contemporary proteomics methods. As a result, the tremendous opportunity afforded by proteomics to discover new proteins and pathways that contribute to higher-order brain function has, to date, gone unrealized. This proposal addresses this Grand Opportunity (GO) through creation of the first proteomics platform for quantitative analysis of protein expression and post-translational modification in the mammalian brain. This platform, termed qPEMM (for quantitative Protein Expression and Modification in Mammals) will be applied to create a brain atlas of proteomic changes that occur in nicotine-dependent rodents. Tobacco usage remains one the most prevalent forms of substance abuse and exerts a tremendous cost on world health and economy. While it is known that nicotine is the major component in tobacco smoke responsible for addiction, we currently have only a limited understanding of the biochemical alterations caused by nicotine in the nervous system. This project will yield the first large-scale anatomical inventory of nicotine- induced changes in protein expression and post-translational modification in the mammalian brain. These changes will identify key protein pathways that are dysregulated by nicotine, which should in turn serve as fertile ground for future studies aimed at deciphering the neurochemical basis for nicotine dependence. In this way, these proteomic studies will serve as a powerful hypothesis-generating engine that can be leveraged many times over to spawn new basic and translational research programs aimed at understanding and eventually treating nicotine addiction. More generally, this project will deliver a fully integrated and portable quantitative proteomic platform that can be adopted by biological researchers around the world to accelerate the growth and pace of scientific discovery. PUBLIC HEALTH RELEVANCE: Tobacco usage remains one of the most prevalent forms of substance abuse and exerts a tremendous cost on world health and economy. Here, we propose to complete the first large-scale anatomical inventory of nicotine-induced changes in protein expression and post-translational modification in the mammalian brain. These studies should yield new hypotheses to explain the mechanistic basis for nicotine addiction and, through doing so, reveal novel diagnostic and therapeutic strategies to treat this debilitating disease.

DESCRIPTION (provided by applicant): Enzymes are central components of nearly all metabolic and signaling pathways in cells and tissues. The dysregulation of enzymes and their endogenous inhibitory proteins contributes to the development of several diseases, including cancer. Nonetheless, efforts to elucidate the function of specific enzymes in cancer have, to date, suffered from a lack of techniques that can assess the activity of these proteins in complex biological systems. To address this problem, we have introduced a chemical proteomic strategy termed activity-based protein profiling (ABPP) that utilizes active site-directed probes to measure changes in enzyme activity directly in native proteomes. To date, we have generated activity-based probes that target numerous enzyme classes, including proteases, lipases, histone deacetylases, and cytochrome P450s. In the previous funding period, we applied ABPP to identify several enzyme activities dysregulated in aggressive human cancer cells, including the serine proteases uPA and tPA, and the previously uncharacterized transmembrane hydrolase KIAA1363. We have shown that these enzymes play important roles in supporting the malignant properties of cancer cells, although the biochemical mechanisms for these effects remain to be fully elucidated. We have also introduced advanced "tag-free" versions of ABPP that exploit the versatility of click chemistry and resolving power of mass spectrometry to enable high-content profiling of small molecule-protein interactions in living systems. Finally, we have developed a complementary proteomic platform for globally mapping the endogenous substrates of proteases termed PROTOMAP (PROtein TOpography and Migration Analysis Platform). In this competitive renewal application, we will apply our suite of ABPP and PROTOMAP technologies to test three major hypotheses of high significance to the fields of cancer and chemical biology: 1) KIAA1363 promotes cancer aggressiveness through regulation of an ether lipid signaling network, 2) dysregulated proteases contribute to cancer pathogenicity by activating and/or inactivating key signaling pathways, and 3) tag-free ABPP will offer a general and quantitative technology to map small molecule-protein interactions in living systems. We anticipate that these studies will define key enzymatic pathways that support cancer malignancy and contain new biomarkers and therapeutic targets, as well as produce methodological advances that greatly expand the scope and utility of the ABPP and PROTOMAP technologies. PUBLIC HEALTH RELEVANCE: A large fraction of human enzymes remain uncharacterized in terms of their function in health and disease. We have developed advanced chemical technologies to functionally characterize enzymes directly in native biological systems. The goal of this application is to further develop and apply these technologies to identify enzymes that play important roles in cancer, which may serve as valuable new biomarkers and therapeutic targets.

2-arachidonylglycerol; Active Sites; Agonist; Amides; anandamide; Anti-inflammatory; Anti-Inflammatory Agents; base; Behavior; Binding (Molecular Function); Binding Proteins; Biochemical; Biochemical Pathway; Biological; Brain; Carrier Proteins; Catalysis; Cell Cycle Kinetics; chemical genetics; Chemicals; Chemistry; Degradation Pathway; depression; Disease; Drug Delivery Systems; Drug usage; Endocannabinoids; Environment; Enzymatic Biochemistry; enzyme structure; Enzymes; Ethanolamines; factor A; fatty acid amide hydrolase; Fatty Acids; Funding; Genetic; Goals; Human; Hydrolase; Hydrolysis; in vivo; Individual; inhibitor/antagonist; insight; Kinetics; Knowledge; Link; Lipid A; Lipid Bilayers; Lipids; Maps; Mediating; Membrane; Membrane Lipids; Metabolic; Metabolic Diseases; Metabolism; metabolomics; Methods; Modeling; Molecular Biology; Monoacylglycerol Lipases; mouse model; Mus; mutant; Mutation; Nervous system structure; Neurons; Obesity; Pain; Pathway interactions; Physiological; Physiology; Play; Process; Property; protein transport; Proteins; Proteomics; Recruitment Activity; Role; Signal Transduction; Site; Structure; System; Taurine; Testing; tool; Travel; TRP channel; uptake

Antibodies; Biochemical; Biological; Breeding; Budgets; Cells; Chemicals; Communication; design; Documentation; Endocannabinoids; enzyme structure; Enzymes; Evaluation; Genetic; Hydrolase; in vivo; Individual; inhibitor/antagonist; Manuscripts; Metabolic; mouse model; Preparation; Proteins; Structure

DESCRIPTION (provided by applicant): The endocannabinoids (ECs) A/-arachidonoyl ethanolamine (anandamide, AEA) and 2-arachidonoylglycerol (2-AG) are lipid transmitters that activate the central and peripheral cannabinoid receptors (CB1 and CB2, respectively) to regulate a broad range of physiological processes, including pain sensation, inflammation, cognition, emotional state, and feeding. The magnitude and duration of EC signaling are tightly regulated by hydrolytic enzymes. However, our understanding of the distinct biochemical pathways that terminate AEA and 2-AG signaling in vivo remains incomplete. Key outstanding questions include: 1) what are the mechanisms and three-dimensional structures of EC hydrolases? 2) what are the neurochemical and physiological consequences of perturbing the function of EC hydrolases in vivo? 3) do multiple hydrolases coordinately control 2-AG metabolism in vivo? and 4) do AEA- and 2-AG-dependent EC pathways regulate distinct mammalian behaviors? In this Program Project, we have assembled a multidisciplinary research team that aims to address these questions by developing and implementing cutting-edge chemical, enzymological, genetic, proteomic, structural, and behavioral pharmacology methods. Specifically, we plan to: 1) characterize the biochemical and cellular mechanisms for terminating EC signaling the nervous system (Project 1), 2) determine the crystal structures of key EC hydrolases, including apo-enzymes, inhibitor complexes, and active-site mutants (Project 2), and 3) evaluate the neurochemical and behavioral effects of disrupting EC hydrolases in vivo (Projects 3 and 4). The knowledge gained from these studies will be further applied towards the design of increasingly potent and selective inhibitors of EC hydrolases (Core), which should prove of great value as both research tools and potential therapeutic agents for the treatment of a range of human diseases, including chronic pain, depression, anxiety, and metabolic disorders. PUBLIC HEALTH RELEVANCE: The endogenous cannabinoid system regulates a broad range of neurophysiological processes. Elucidation of the enzymes that regulate endogenous cannabinoids and their mechanisms of action may lead to the identification of new therapeutic targets for the treatment of human disorders such as chronic pain, depression, and anxiety.

DESCRIPTION (provided by applicant): Opioid addiction from the chronic use of prescription analgesics and illicit agents represents a major unmet public health crisis. Current pharmacotherapies for opioid dependence, such as opioid maintenance therapy (e.g., methadone and buprenorphine), reduce the need for illicit substances and alleviate opioid withdrawal, but possess a similar side effect profile as other opioids and can also trigger severe opioid withdrawal responses. Consequently, a strong need remains for the development of new treatment strategies that would relieve patients of opioid dependence without transferring this dependency to another drug. D9-tetrahydrocannabinol (THC), the primary psychoactive constituent in Cannabis sativa, has long been known to reduce naloxone- precipitated withdrawal symptoms in opioid-dependent animals. However, THC and other CB1 receptor agonists display several cannabimimetic side effects, including marijuana-like subjective activity, that limit their general therapeutic potential. Alternatively, increasing brain levels of the endogenous cannabinoids anandamide and 2-arachidonylglycerol (2-AG) though the blockade of their respective catabolic enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) represents a promising therapeutic approach that lacks many of the undesirable side effects of direct-acting CB1 receptor agonists. We have recently developed JZL184, the first highly potent, selective, and orally active MAGL inhibitor. This compound causes significant elevations in brain 2-AG levels and robustly reduces the magnitude of naloxone-precipitated somatic withdrawal responses in morphine-dependent mice, but elicits far fewer cannabimimetic effects compared with THC. While FAAH inhibitors produce significant effects in preclinical models of pain and anxiety, our preliminary data show that these compounds lack efficacy in reducing opioid withdrawal effects. Thus, the overall objective of this proposal is to test whether optimized MAGL inhibitors will reduce the constellation of withdrawal symptoms in opioid-dependent mice and rhesus monkeys undergoing abstinence. In the proposed experiments, we will characterize the metabolism, pharmacokinetics, target selectivity, safety profile, and effectiveness of JZL184 and structurally related analogues in ameliorating withdrawal symptoms in established rodent and nonhuman primate models of opioid dependence. The following three major hypotheses will be tested: 1) MAGL inhibitors will reduce somatic and affective withdrawal signs in opioid- dependent rodents; 2) MAGL inhibitors will reduce opioid withdrawal symptoms and withdrawal-related increases in heroin self-administration in opioid-dependent rhesus monkeys; and 3) inhibition of MAGL will produce minimal side effects compared to direct opioid and cannabinoid receptor agonists. The ultimate goal of this application is to identify a potent, selective, orally active, safe, and efficacious MAGL inhibitor that prevents opioid withdrawal in mouse and monkey preclinical models and is ready for IND-enabling toxicology en route to clinical development as a novel therapeutic for treating opioid abuse. PUBLIC HEALTH RELEVANCE: Opioid addicts undergoing abstinence suffer from a constellation of physiological and behavioral withdrawal signs that cause great suffering and high-potential for relapse to drug usage; new targeted therapies are desperately needed to assist addicts in alleviating their dependence on opioid drugs while minimizing the potential for relapse. Here, we propose an innovative new way to treat opioid withdrawal with selective inhibitors of the endocannabinoid-degrading enzyme monoacylglycerol lipase (MAGL) and detail a plan to test this premise by developing best-in-class MAGL inhibitors with good translational potential for evaluation in animal models of opioid dependence and withdrawal. If successful, this research program could deliver next- generation therapies for opioid addiction that show excellent efficacy with limited potential for side effects and drug relapse.

DESCRIPTION (provided by applicant): Enzymes perform many of the most vital functions in our cells and tissues and are the targets of numerous transformative medicines. Considering the importance of enzymes in biology and medicine, it is both provocative and humbling to realize that the human proteome contains a huge number of uncharacterized enzymes. Assigning biochemical, cellular, and physiological functions to these enzymes represents a grand challenge for researchers in the post-genomic era. To achieve this goal, selective pharmacological tools to perturb enzymes are needed. A pressing question, however, immediately arises: how can one rapidly and systematically discover potent and selective inhibitors for uncharacterized enzymes? Over the past decade, our lab has pioneered the development and application of an innovative chemoproteomic solution to this problem termed activity-based protein profiling (ABPP). The objective of this proposal is to use our suite of competitive ABPP platforms to develop potent, selective, and in vivo-active inhibitors for a substantial fraction of mammalian serine hydrolases (SHs), which are a large and diverse enzyme class that represent ~1% of all human proteins. SHs play critical roles in virtually all physiological and pathological processes, and are targeted by several approved drugs to treat diseases such as diabetes, obesity, and Alzheimer's disease. Despite their biological and biomedical importance, the vast majority of mammalian SHs lack selective, in vivo- active inhibitors and consequently remains poorly characterized with regards to their physiologic substrates and functions. We have created an efficient competitive ABPP platform for SH inhibitor discovery and optimization that is fully operational in our laboratory and has already yielded selective and in vivo-active inhibitors for more than 10 SHs, as well as lead inhibitors fo many additional (20+) enzymes. In most cases, these compounds represent the first pharmacological probes for studying their SH targets in living systems and are therefore in widespread use by the biology research community. In this application, we propose to use a multidisciplinary research program involving chemical synthesis, enzymology, proteomics, metabolomics, and cell and animal pharmacology to: 1) optimize the potency, selectivity, and in vivo-activity of lead SH inhibitors by competitive- ABPP guided medicinal chemistry (Aim 1), 2) screen for lead inhibitors of additional SHs by HTS-compatible ABPP (Aim 2), and 3) use optimized inhibitors in combination with metabolomics and proteomics to determine the physiologic substrates and pathways regulated by SHs in vivo (Aim 3). We have also enlisted a diverse set of biology collaborators who are interested in using our optimized inhibitors to probe the functions of SHs in (patho)physiological processes that include cancer, diabetes, and nervous system disorders. The ultimate goal of this application is to deliver potent, selective, and in vivo-active inhibitors for a substantial fraction of mammalian SHs. These inhibitors will serve as valuable research tools to probe the biological functions of SHs, as well as leads for drug development programs aimed at targeting SHs to treat human disease.

The Core's mission is the scientific and administrative support of all four Projects in the Program, providing a standardized source of reagents, resources, and protocols. A Chemistry Component will design, synthesize, and test enzyme inhibitors for potency and selectivity in vitro and in vivo before passing them on to Projects 2 and 3/4 for co-crystallization studies and pharmacological analysis, respectively. A Biochemistry Component will provide pure proteins to Program Projects 1 and 2 for functional and structural studies, respectively. A Mouse Component will manage the breeding, crossing, and distribution of genetic mouse models. These three scientific arms of the Core will combine to provide the best and most consistent reagents for use in the research aims of the Program Project as a whole. Finally, a small Administrative Component will ensure fiscal responsibility, seamless communication, and proper documentation.

DESCRIPTION (provided by applicant): Obesity-linked insulin resistance and type 2 diabetes are intimately linked to adipocyte dysfunction, increased adipocyte lipolysis, and lipid accretion in tissues other than adipose. In obesity, the hypertrophied adipocyte is not able to properiy store excess fatty acids, the rate of lipolysis is increased, and these lipids deposit in other tissues where they hamper insulin action. Inhibiting obesity-linked adipocyte lipolysis can improve insulin sensitivity. All enzymes involved in adipocyte lipolysis belong to the serine hydrolase family. Despite their importance in fat cell physiology, the majority of serine hydrolases have not been studied. Serine hydrolases (SHs) are a key enzyme family involved in metabolism and adipocyte function, v\/here they contribute to lipolysis, lipogenesis, and lipid uptake. Yet, more than 50% ofthe 120+ human serine hydrolases, including some that have been genetically linked to human disease, remain unannotated, have no known function or physiological substrates, and most lack inhibitors to aid in their characterization and therapeuti validation. Because individual SHs already constitute targets for drugs that treat metabolic disease, it is reasonable to hypothesize that important additional drug targets will be found among the numerous SHs that remain uncharacterized. Discerning which of these unannotated SHs are relevant in adipocyte function and which may serve as therapeutic targets for obesity-diabetes is a very complex problem. The critically important research challenge that this project addresses is the identification and therapeutic validation of pooriy annotated metabolic serine hydrolases that play key roles in adipocyte function. Our multidisciplinary team will achieve this goal by combining cutting-edge chemoproteomic and metabolomics methods with deep biological expertise in obesity and type 2 diabetes. Specifically, we intend to globally identify and assess the therapeutic potential of unannotated SHs active in adipocytes and whose activity is modulated in physiologic conditions and in obesity-diabetes. Some of these enzymes may be new targets for metabolic disease. In the process, we will create first-in-class chemical probes and genetic models to study adipocyte SHs that will be distributed to the larger research community.

DESCRIPTION (provided by applicant): Opioid addiction from the chronic use of prescription analgesics and illicit agents represents a major unmet public health crisis. Current pharmacotherapies for opioid dependence, such as opioid maintenance therapy (e.g., methadone and buprenorphine), reduce the need for illicit substances and alleviate opioid withdrawal, but possess a similar side effect profile as other opioids and can also trigger severe opioid withdrawal responses. Consequently, a strong need remains for the development of new treatment strategies that would relieve patients of opioid dependence without transferring this dependency to another drug. D9-tetrahydrocannabinol (THC), the primary psychoactive constituent in Cannabis sativa, has long been known to reduce naloxone- precipitated withdrawal symptoms in opioid-dependent animals. However, THC and other CB1 receptor agonists display several cannabimimetic side effects, including marijuana-like subjective activity, that limit their general therapeutic potential. Alternatively, increasing brain levels of the endogenous cannabinoids anandamide and 2-arachidonylglycerol (2-AG) though the blockade of their respective catabolic enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) represents a promising therapeutic approach that lacks many of the undesirable side effects of direct-acting CB1 receptor agonists. We have recently developed JZL184, the first highly potent, selective, and orally active MAGL inhibitor. This compound causes significant elevations in brain 2-AG levels and robustly reduces the magnitude of naloxone-precipitated somatic withdrawal responses in morphine-dependent mice, but elicits far fewer cannabimimetic effects compared with THC. While FAAH inhibitors produce significant effects in preclinical models of pain and anxiety, our preliminary data show that these compounds lack efficacy in reducing opioid withdrawal effects. Thus, the overall objective of this proposal is to test whether optimized MAGL inhibitors will reduce the constellation of withdrawal symptoms in opioid-dependent mice and rhesus monkeys undergoing abstinence. In the proposed experiments, we will characterize the metabolism, pharmacokinetics, target selectivity, safety profile, and effectiveness of JZL184 and structurally related analogues in ameliorating withdrawal symptoms in established rodent and nonhuman primate models of opioid dependence. The following three major hypotheses will be tested: 1) MAGL inhibitors will reduce somatic and affective withdrawal signs in opioid- dependent rodents; 2) MAGL inhibitors will reduce opioid withdrawal symptoms and withdrawal-related increases in heroin self-administration in opioid-dependent rhesus monkeys; and 3) inhibition of MAGL will produce minimal side effects compared to direct opioid and cannabinoid receptor agonists. The ultimate goal of this application is to identify a potent, selective, orally active, safe, and efficacious MAGL inhibitor that prevents opioid withdrawal in mouse and monkey preclinical models and is ready for IND-enabling toxicology en route to clinical development as a novel therapeutic for treating opioid abuse.

DESCRIPTION (provided by applicant): Opioid addiction from the chronic use of prescription analgesics and illicit agents represents a major unmet public health crisis. Current pharmacotherapies for opioid dependence, such as opioid maintenance therapy (e.g., methadone and buprenorphine), reduce the need for illicit substances and alleviate opioid withdrawal, but possess a similar side effect profile as other opioids and can also trigger severe opioid withdrawal responses. Consequently, a strong need remains for the development of new treatment strategies that would relieve patients of opioid dependence without transferring this dependency to another drug. D9-tetrahydrocannabinol (THC), the primary psychoactive constituent in Cannabis sativa, has long been known to reduce naloxone- precipitated withdrawal symptoms in opioid-dependent animals. However, THC and other CB1 receptor agonists display several cannabimimetic side effects, including marijuana-like subjective activity, that limit their general therapeutic potential. Alternatively, increasing brain levels of the endogenous cannabinoids anandamide and 2-arachidonylglycerol (2-AG) though the blockade of their respective catabolic enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) represents a promising therapeutic approach that lacks many of the undesirable side effects of direct-acting CB1 receptor agonists. We have recently developed JZL184, the first highly potent, selective, and orally active MAGL inhibitor. This compound causes significant elevations in brain 2-AG levels and robustly reduces the magnitude of naloxone-precipitated somatic withdrawal responses in morphine-dependent mice, but elicits far fewer cannabimimetic effects compared with THC. While FAAH inhibitors produce significant effects in preclinical models of pain and anxiety, our preliminary data show that these compounds lack efficacy in reducing opioid withdrawal effects. Thus, the overall objective of this proposal is to test whether optimized MAGL inhibitors will reduce the constellation of withdrawal symptoms in opioid-dependent mice and rhesus monkeys undergoing abstinence. In the proposed experiments, we will characterize the metabolism, pharmacokinetics, target selectivity, safety profile, and effectiveness of JZL184 and structurally related analogues in ameliorating withdrawal symptoms in established rodent and nonhuman primate models of opioid dependence. The following three major hypotheses will be tested: 1) MAGL inhibitors will reduce somatic and affective withdrawal signs in opioid- dependent rodents; 2) MAGL inhibitors will reduce opioid withdrawal symptoms and withdrawal-related increases in heroin self-administration in opioid-dependent rhesus monkeys; and 3) inhibition of MAGL will produce minimal side effects compared to direct opioid and cannabinoid receptor agonists. The ultimate goal of this application is to identify a potent, selective, orally active, safe, and efficacious MAGL inhibitor that prevents opioid withdrawal in mouse and monkey preclinical models and is ready for IND-enabling toxicology en route to clinical development as a novel therapeutic for treating opioid abuse.

? DESCRIPTION (provided by applicant): A striking number of hereditary nervous system diseases are caused by deleterious mutations in poorly characterized enzymes from the serine hydrolase class. Over the past decade, our group has developed an innovative set of chemical proteomic and metabolomic platforms for assigning functions to uncharacterized serine hydrolases and have a special interest in applying these platforms to serine hydrolases that are causally linked by human genetics to nervous system diseases. In this grant application, we focus on the role that serine hydrolases play in the neurological disorder PHARC (polyneuropathy, hearing loss, ataxia, retinosis pigmentosa, and cataract). Specifically, we have determined that the serine hydrolase ABHD12, mutations of which cause PHARC, is a major brain lyso-phosphatidylserine (lyso-PS) lipase. ABHD12-/- mice exhibit elevated brain lyso-PS content and auditory and motor deficits coupled with heightened neuroinflammation, implicating deregulated lyso-PS signaling as a contributory factor to PHARC. Blocking the upstream enzyme(s) that produces lyso-PS could thus provide a therapeutic strategy to treat PHARC and possibly other (neuro) inflammatory diseases. We recently determined that the heretofore uncharacterized serine hydrolase ABHD16A is a major PS lipase responsible for generating lyso-PS in mammalian systems, including mouse brain and PHARC subject-derived lymphoblasts. The goal of this application is to perform a high-throughput screen of TSRI's 640,000+ compound library to identify structurally novel classes of ABHD16A inhibitors that can be progressed to selective and in vivo-active chemical probes. We have established a diverse and innovative set of biochemical, chemoproteomic, and lipidomic assays to rigorously assess the potency, selectivity, and cellular activity of ABHD16A inhibitors. We expect that the Specific Aims of this application will deliver multiple structurally distinct ABHD16A inhibitors that show suitable levels of potency (in vitro IC50 < 1 µM), selectivity (a limited number of off-targets (lss than five) across the serine hydrolase class), and cellular activity (in situ IC50 < 10 µM) for prioritization as leads for medicinal chemistry optimization into in vivo-active chemical probes. Our collaborative research team has a strong track-record of performing high-throughput screens on serine hydrolases and developing leads from these screens into selective and in vivo-active inhibitors. We are therefore well-equipped to progress the prioritized inhibitors discovered in this application toward optimized chemical probes for testing the function of ABHD16A and its lyso-PS products in PHARC and other (neuro) inflammatory disorders.

Abstract There is a need to develop diabetes drugs with a mechanism of action distinct from that of established agents. Defects in adipocyte function drive the onset of systemic insulin resistance and obesity-linked diabetes. The ability of hypertrophied adipocytes to dispose of glucose and lipids and secrete insulin-sensitizing adipokines is severely compromised and this contributes to hyperglycemia, hyperlipidemia, insulin resistance, inflammation, and lipid deposition in tissues such as liver where it dampens insulin action. Agents that can revert these defects and restore natural lipid partitioning amongst tissues are useful insulin sensitizers in humans. The critical challenge that this project addresses is the identification and therapeutic validation of new molecular pathways that can be pharmacologically modulated to revert adipocyte defects and restore insulin signaling in liver and other tissues. Our multidisciplinary team will achieve this goal by combining cutting-edge phenotypic screening and chemoproteomic technologies, with deep expertise in adipose tissue and liver biology and lipid metabolism. We have developed innovative strategies that integrate phenotypic screening with chemoproteomics to streamline the identification of protein targets of bioactive small molecules, which we initially applied to enzymes of the serine hydrolase family, but have more recently extended for proteome-wide investigations. We propose to complement our initial serine hydrolase-focused approach with our new powerful platforms for integrated phenotypic screening and chemical biology to enable the rapid, systematic, and proteome-wide discovery of metabolism targets. By screening unique libraries of small-molecules for desirable phenotypes in adipocytes and hepatocytes in an unbiased manner, we will identify in tandem physiologically relevant proteins and chemical tools to perturb the function of these proteins to expedite their functional annotation and therapeutic validation in diabetes and NASH. In the process, we will create first-in-class chemical probes and genetic models to study key metabolic pathways that will be distributed to the larger research community. Our cutting-edge chemical biology platforms radically expand the portion of the proteome that can be targeted with small molecules. Application of these tools to the central problem that drives diabetes endows this project with unparalleled potential to discover new targets to treat diabetes and associated conditions.

Cancer research and treatment have greatly benefited from advances in DNA sequencing methods, which have accelerated the discovery of genes that, when mutated, promote tumorigenesis. The protein products of some of these oncogenes have served as direct targets for groundbreaking new medicines. Many oncogenes, however, code for proteins that lack chemical probes and are even considered undruggable. In these cases, our understanding of the molecular basis of cancer has not yet translated into effective new therapies. A similar gap can be found in cancer-related immunology (or immuno-oncology), where human genetics is discovering proteins that play fundamental roles in innate and adaptive immunity; yet, again, most of these proteins lack chemical probes. A critical challenge has thus emerged in cancer research ? how can the massive gains in understanding of cancer and immunology bequeathed by modern human genetics be translated into new therapies for cancer? The goal of this research program is to leverage and extend our lab?s innovative activity- based protein profiling (ABPP) technology to radically expand the druggable content of the human proteome and develop high-quality chemical probes for genetically-defined protein targets in cancer and immuno- oncology. We have recently introduced advanced ABPP platforms that evaluate small-molecule interactions across thousands of proteins in parallel directly in native biological systems. By combining proteome-wide druggability maps of human cancer and immune cells furnished by ABPP with human genetic information, we have identified several high-priority cancer targets poised for chemical probe development. Optimized chemical probes will be used by our lab and a set of expert biology collaborators to characterize the functional relevance of protein targets in cancer and cancer-related immunology. We will also describe plans for continued technology innovation to further enhance chemical probe and target discovery by ABPP, including the following objectives ? i) identify newly druggable E3 ligase systems capable of supporting targeted protein degradation in cancer cells; ii) discover chemical probes that selectively engage modified states of protein targets in cancer cells; and iii) generate advanced chemical libraries for ABPP to further increase the druggable fraction of the human cancer proteome. In summary, our research program should deliver high-quality chemical probes for, and pharmacological validation of, biologically compelling human cancer targets, providing critical knowledge to direct the future development of transformative cancer therapeutics. More generally, we envision that our research program will inspire chemical and cancer biologists to embrace the potential druggability of any human protein, as well as provide an experimental roadmap to realize this goal, for the benefit of both basic and translational research.

PROJECT SUMMARY ? RP5 Small molecules that enhance autophagy have the potential to serve as therapeutics against a phylogenetically diverse and broad range of pathogens. Nonetheless, most of the proteins that participate in, or regulate, autophagy lack chemical probes and some may even be viewed as undruggable. To accelerate the discovery of chemical probes, and ultimately drugs that promote anti-infective autophagy, new platforms are needed that can broadly and efficiently evaluate small molecule interactions for autophagy-related proteins from diverse structural and functional classes. Our laboratory has recently introduced the first chemical proteomic platforms to globally assess the druggability of proteins directly in native biological systems. These quantitative mass spectrometry-activity-based protein profiling (MS-ABPP) platforms can be applied to any cell type or state and have identified small-molecule hit ligands for many hundreds of proteins across a wide range of classes, including those previously considered undruggable (e.g., adaptors, transcription factors). Mining our current MS-ABPP data sets of the human proteome, which contain 30,000+ sites on 10,000+ proteins, has uncovered evidence of druggability for several proteins involved in degradative autophagy and/or ATG gene- dependent immunity. In RP5, we will use our MS-ABPP platforms in close collaboration with RP1-4 to discover and optimize small-molecule probes that enhance autophagy-mediated defense against infectious diseases. We will pursue the following Specific Aims: 1) the chemical proteomic identification of protein targets of autophagy-stimulating small molecules; 2) the chemical proteomic discovery of hit ligands for prioritized autophagy proteins; 3) the chemical proteomic discovery of novel, druggable proteins that regulate autophagy; and 4) the optimization of chemical probes for prioritized autophagy proteins. RP5 should furnish potent and selective chemical probes for several prioritized autophagy proteins, and these probes will be provided to RP1- 4 for biological studies. The chemical probes developed in RP5 should also serve as much needed tools for the greater autophagy and infectious disease communities, as well as valuable starting points for the development of broad-spectrum anti-infective therapies.