Dysregulated metabolic networks contribute to the pathophysiology of a large number of diseases including cancer, inflammation, neurodegenerative diseases, atherosclerosis, obesity, and diabetes. These changes not only include fundamental rewiring of cell metabolism (e.g. glycolytic switch or ‘Warburg effect’ in cancer), but also impact the levels of signaling molecules and the activities of enzymes that regulate them (e.g., elevated prostaglandins due to increased cylooxygenase 2 in inflammation). We, and others, have found that metabolic pathways, and their constituent enzymes and metabolites, are often interconnected in disease states giving rise to highly integrated signaling networks that play vital roles in pathogenesis. This connectivity offers an exciting potential to perturb and control entire biochemical networks by identifying and targeting key nodal control points. Exerting such control over dysregulated biochemical pathways holds great promise for the treatment of complex diseases.Research in our group focuses on discovering and functionally characterizing dysregulated metabolic networks in disease using functional proteomic and metabolomic platforms, in order to identify enzymes that represent nodal points of control for pharmacological intervention and therapy.
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TechnologySystems biology methods have assisted in characterizing the extraordinarily complex biochemical pathways of cells and tissues. These methods include, but are not limited to, DNA microarrays and mass-spectrometry-based proteomics. Although these platforms have led to identification of the genes and proteins that make up mammalian systems, understanding the pathophysiological roles that these biomolecules and their parent networks play in physiological and pathological processes remains challenging. This is because: 1) enzymes can be regulated by post-translational events in vivo, which are poorly accounted for by standard gene and protein expression profiling; 2) a large swath of the proteome remains functionally uncharacterized and is therefore difficult to assemble into larger biochemical networks, 3) many enzymes and metabolites display difficult physicochemical properties that complicate their analysis in biological samples, and 4) many metabolic pathways that enzymes regulate in a disease-specific context are not understood. Our group addresses these challenges by applying innovative metabolomic and proteomic approaches to mapping biochemical pathways that support disease.
Activity-based protein profilingTo address some of the aforementioned challenges, our lab utilizes a chemoproteomic platform termed activity-based protein profiling, originally developed in Ben Cravatt's laboratory at The Scripps Research Institute. ABPP utilizes chemical probes to interrogate the functional state of large numbers of enzymes in native biological systems. ABPP probes consist of two key elements: 1) a reactive group for binding and covalently labeling the active sites of many members of a given enzyme class (or classes), and 2) a reporter tag for the detection, enrichment, and identification of labeled enzymes from proteomes. There are currently ABPP probes for a multitude of enzyme classes, including many that play central roles in metabolism. ABPP directly addresses many of the challenges mentioned above. First, ABPP probes allow enrichment of specific classes of proteins based on shared functional properties and therefore assist in the characterization of low abundance proteins.
Second, these probes selectively label active enzymes, but not their inactive forms, facilitating the characterization of changes in enzyme activity that occur without alterations in protein or transcript expression. Third, ABPP can also be usedc as a competitive screen to identify both reversible and irreversible enzyme inhibitors and also to confirm target inhibition in situ because inhibitors can compete with the activity-based probe. Using competitive ABPP, enzymes can be assayed in native proteomes and inhibitor potency and selectivity can be assessed across a proteome-wide scale even for uncharacterized enzymes that lack known substrates. Competitive ABPP has already led to the discovery of selective inhibitors for several enzymes, which have in turn been used to test the function of these proteins in living systems.
Metabolomics for determining enzyme function and dysregulated metabolic pathways in diseaseTo annotate the roles of metabolic pathways perturbed in disease, our lab uses an untargeted liquid chromatography-mass spectrometry (LC-MS) platform, termed discovery metabolite profiling (DMP), which allows for global and unbiased comparison of metabolites under disruption of enzymatic function or differing disease states. Using selective inhibitors developed through competitive ABPP efforts or RNA interference technology, we can specifically block the function of an enzyme of interest, and then profile the metabolites that the enzyme regulates. In this manner, we can comprehensively identify not only the substrates and products of an enzyme in specific (patho)physiological contexts, but also annotate the metabolic networks that the enzyme regulates. Collectively, our metabolomics platforms allow us to identify novel biochemical roles of already characterized enzymes or identify the metabolic roles of completely uncharacterized enzymes.
Mapping Dysregulated Metabolic Pathways in Disease
Neuroinflammatory diseasesAlthough neuroinflammation is meant as a defense mechanism against neurotoxic insult, many groups have now established that chronic and non-resolving neuroinflammation can lead to neurotoxicity and neurodegeneration. Therefore, identifying agents that can suppress neuroinflammatory processes may provide therapeutic benefit towards neurological diseases. One major direction of our lab is to identify and characterize metabolic pathways critical to neuroinflammation and neurodegeneration.
Using the functional proteomic and metabolomic platforms described above, we have identified one such nodal enzyme called monoacylglycerol lipase (MAGL), which controls multiple lipid signaling pathways to modulate a variety of pathophysiological processes including pain, inflammation, and neurodegeneration. MAGL, as its name suggests, had been previously shown by others to hydrolyze monoacylglycerol lipids as the ultimate step of fat lipolysis in vitro. One of these monoacylglycerols, 2-arachidonoylglycerol (2-AG), is an endogenous signaling lipid for the cannabinoid receptor, and is also broken down by MAGL. The cannabinoid system is most popularly known through tetrahydrocannabinol, the active component of marijuana, which binds cannabinoid receptors to elicit its associated high, but also provides beneficial effects such as pain relief and anti-inflammation.
In the brain, we found that MAGL serves as a nodal control point to regulate both the cannabinoid signaling lipid 2-AG, as well as its breakdown product arachidonic acid--a substrate for the synthesis of a diverse network of inflammatory eicosanoid signaling lipids. Eicosanoids are suppressed by commonly used anti-inflammatory agents such as ibuprofen and aspirin. One aim in our lab is to determine whether blocking MAGL can bidirectionally enhance cannabinoid signaling to elicit pain relieving effects, while also suppressing eicosanoid signaling to provide protection against neuroinflammation and neurodegeneration.
Cancer MetabolismCancer cells have fundamentally altered cellular metabolism that provide a biochemical foundation for tumors to progress in their etiology. These alterations include aerobic glycolysis, glutamine-dependent anaplerosis, and de novo lipid biosynthesis, which serve as metabolic platforms for supporting tumorigenicity. Although these factors are important in the transformation of cells from a non-cancerous to a cancerous state, much less is understood about the metabolic pathways that confer malignancy during tumor progression. Since most cancer deaths are related to cancer malignancy and metastasis, understanding metabolic pathways that contribute to these pathogenic features of cancer is critical to both diagnosis and treatment. Another major direction of our lab is to identify and characterize dysregulated metabolic networks in cancer.
Using ABPP to profile serine hydrolase activities across a panel of non-aggressive versus aggressive cancer cells from a variety of cancer types (melanoma, breast, ovarian, and prostate cancers), we previously identified MAGL as one such “cancer pathogenicity” enzyme that is highly expressed in malignant cancers and is both necessary and sufficient to drive the aggressiveness of multiple types of human cancer cells. Using functional metabolomic platforms, we discovered that MAGL has a novel pathophysiological role in cancer of controlling a free fatty acid network that is enriched in protumorigenic signaling lipids. A focus of our lab is to further annotate the biochemical, molecular, and potential clinical role of MAGL in cancer.
In addition, using both functional proteomic and genomic platforms, we have identified a multitude of additional metabolic enzymes that are also highly expressed across multiple types of aggressive cancer cells. Another focus of our lab is to disrupt these other dysregulated metabolic pathways in cancer with RNA interference technology and/or small-molecule inhibitors to understand the metabolic role of these enzymes and determine whether we can impair tumor cell pathogenicity in the hopes of offering novel ways to treat the most aggressive forms of cancer.
Although our inflammatory response is meant as a defense mechanism against various noxious stimuli such as infection or tissue injury, chronic or uncontrolled inflammation can lead to a wide range of pathologies including sepsis, cancer, arthritis, pain, neurodegenerative disease, obesity, diabetes, and atherosclerosis. Although there are effective anti-inflammatory therapies including non-steroidal anti-inflammatory drugs (NSAIDs), glucocorticoids and anti-cytokine therapies, many of these agents have significant untoward side effects that discourage chronic treatment. Therefore, identifying novel biochemical pathways that promote chronic inflammation is crucial not only for understanding the molecular basis of inflammatory disease states, but also for devising new ways for their treatment. Another direction of our research group is to map metabolic pathways that are critical to the inflammatory process by identifying anti-inflammatory small molecules, their cognate protein targets, and the metabolic/signaling pathways that they regulate.
Piro JR, Benjamin DI, Duerr JM, Pi Y, Gonzales C, Wood KM, Schwartz JW, Nomura DK(#), Samad TA(#). (2012) A dysregulated endocannabinoid-eicosanoid network supports pathogenesis in a mouse model of Alzheimer's disease. Cell Reports 1, 617-623. (#co-corresponding author)
Nomura DK(#), Morrison BE, Blankman JL, Long JZ, Kinsey SG, Marcondes MC, Ward AM, Hahn YK, Lichtman AH, Conti B, Cravatt BF(#). (2011) Endocannabinoid hydrolysis generates brain eicosanoids that promote neuroinflammation. Science 334, 809-813. (#co-corresponding author)
Nomura DK(#), Lombardi DP, Chang JW, Niessen S, Ward AM, Long JZ, Hoover HH, Cravatt BF(#). (2011) Monoacylglycerol lipase exerts bidirectional control over endocannabinoid and fatty acid pathways to support prostate cancer pathogenesis. Chem. Biol. 18, 846-856. (#co-corresponding author)
Chang JW, Nomura DK, Cravatt BF. (2011) A potent and selective inhibitor of KIAA1363/AADACL1 that impairs prostate cancer pathogenesis. Chem Biol. 18, 476-484.
Nomura DK(#), Casida JE(#). (2011) Activity-based protein profiling of organophosphorus and thiocarbamate pesticides reveals multiple secondary targets in the mammalian nervous system. J Agric Food Chem. 59, 2808-2815. (*co-corresponding author)
Bachovchin DA, Mohr JT, Speers AE, Wang C, Berlin JM, Spicer TP, Fernandez-Vega V, Chase P, Hodder PS, Schűrer, Nomura DK, Rosen H, Fu GC, Cravatt BF. (2011) Academic cross-fertilization by public screening yields a remarkable class of protein phosphatase methylesterase-1 inhibitors. Proc Natl Acad Sci USA,108, 6811-6816.
Kopp F, Komatsu T, Nomura DK, Trauger SA, Thomas JR, Simon GM, Cravatt BF. (2010) The glycerophospho-metabolome and its influence on amino acid homeostasis by brain metabolomics of GDE1(-/-) mice. Chem Biol. 17, 831-840.
Schlosburg JE, Blankman JL, Long JZ, Nomura DK, Nguyen PT, Ramesh D, Kinsey SG, Booker L, Burston JK, Wise LE, Ghosh S, Selley DE, Sim-Selley LJ, Liu Q, Cravatt BF, Lichtman AH. (2010) Sustained inactivation of monoacylglycerol lipase produces functional antagonism of the brain endocannabinoid system. Nat Neurosci.13, 1113-1119.
Nomura DK, Dix MM, Cravatt BF. (2010) Chemoproteomic Approaches for Biochemical Pathway Discovery in Cancer. Nat Rev Cancer. 10, 630-638.
Nomura DK, Long JZ, Niessen S, Hoover HS, Ng S-W, Cravatt BF. (2010) Monoacylglycerol lipase regulates a fatty acid network that promotes cancer pathogenesis. Cell. 140, 49-61.
Long JZ, Nomura DK, Vann RE, Walentiny DM, Booker L, Jin X, Burston JJ, Sim-Selley LJ, Lichtman AH, Wiley JL, Cravatt BF. (2009) Dual blockade of FAAH and MAGL identifies behavioral processes regulated by endocannabinoid crosstalk in vivo. Proc Natl Acad Sci USA. 106, 20270-20275.
Long JZ, Nomura DK, Cravatt BF. (2009) Mechanistic characterization of selective monoacylglycerol lipase inhibition reveals differences in central and peripheral endocannabinoid metabolism. Chem Biol. 16, 744-753.
Ruby M, Nomura DK, Hudak CS, Mangravite LM, Chiu S, Casida JE, Krauss RM. (2008) Overactive endocannabinoid signaling impairs apolipoprotein E-mediated clearance of triglyceride-rich lipoproteins. Proc Natl Acad Sci USA. 105, 14561-14566.
Nomura DK , Ward AM, Hudak CS, Burston JJ, Issa RS, Fisher KJ, Abood ME, Wiley JL, Lichtman A, Casida JE. (2008) Monoacylglycerol lipase regulates 2-arachidonoylglycerol action and arachidonic acid levels. Bioorg Med Chem Lett. 18, 5875-5878.
Nomura DK, Blankman JL, Simon GM, Fujioka K, Issa RS, Ward AM, Cravatt BF, Casida JE. (2008) Activation of the endocannabinoid system by organophosphorus nerve agents. Nat Chem Biol. 4, 373-378.
Nomura DK, Fujioka K, Issa RS, Ward AM, Cravatt BF, Casida JE. (2008) Dual Roles of Brain Serine Hydrolase KIAA1363 in Ether Lipid Metabolism and Organophosphate Detoxification. Toxicol Appl Pharmacol. 228, 42-482.
Nomura DK, Leung D, Chiang KP, Quistad GB, Cravatt BF, Casida JE. (2005) A Brain Detoxifying Enzyme for Organophosphorus Nerve Poisons. Proc Natl Acad Sci USA. 102, 6195-6200.
Cravatt BF, Long JZ, Li W, Nomura DK. (2010) Methods and Compositions Related to Targeting Monoacylglycerol Lipase. United States Provisional Patent. International application no. PCT/US2009/006045