Discovery of metabolic pathways using phenotypic screening and chemical proteomics

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PROJECT SUMMARY Obesity is a major public health problem that is characterized by a vast expansion of adipose tissue. Under conditions of chronic excess energy intake, adipocytes expand to store surplus nutrients. In obesity, chronically enlarged adipocytes can no longer respond to insulin, properly secrete insulin-sensitizing hormones, or retain excess lipids, which then deposit in tissues such as liver and muscle where they hinder insulin action. Obesity- linked adipocyte dysfunction drives the pathogenesis of metabolic disorders such as type 2 diabetes (T2D) and nonalcoholic fatty liver disease (NALFD). Agents that can revert obesity-driven adipocyte defects and restore normal lipid partitioning amongst tissues are useful insulin sensitizers in humans. The Saez and Cravatt labs previously developed an innovative strategy that integrates phenotypic screening with chemical proteomics to streamline the identification of protein targets of bioactive small molecules and rapidly uncover new metabolic targets on a proteome-wide scale. By screening unique libraries of small molecules for desirable phenotypes in adipocytes (e.g., increased lipid retention), they identified in tandem physiologically relevant proteins and chemical tools to perturb the function of these proteins to expedite their functional characterization and therapeutic validation in T2D. In past work, they applied this strategy to study adipocyte physiology, and identified and therapeutically validated new targets for T2D (e.g., Ces3, PGRMC2). In this project, I will advance this ligand-protein target discovery strategy by screening for desirable effects in adipocytes (e.g., increased lipid retention) a library of chemical probes that are stereochemically defined. The introduction of stereochemistry allows for a set of chemical probes to have similar physiochemical properties, yet differ in the ability to engage proteins, thus facilitating the identification of the relevant protein targets of bioactive probes. My goal is to apply this integrated phenotypic screening and chemical proteomics strategy using this new library of stereoselective probes to discover metabolic pathways that can be pharmacologically modulated to revert obesity-induced adipocyte defects. It is my expectation that some of the novel pathways I discover may provide the basis for the development of new therapeutic strategies for obesity-linked conditions. Extensive preliminary data included in the proposal establishes the feasibility of this approach. I have already identified several probes that stereoselectively promote adipocyte differentiation and lipid storage. In one instance, I have also identified the proteins that interact with the proadipogenic compound. Thus, I am poised to identify novel metabolic pathways that can be targeted to treat obesity-driven maladies.