Sirtuins are a class of enzymes known as nicotinamide adenine dinucleotide (NAD)- dependent deacetylases. Humans have seven sirtuins, SIRT1-7, that have been shown to regulate a variety of biological processes, including aging, transcription, and metabolism. However, among the seven human sirtuins, only SIRT1, 2, and 3 have robust deacetylase activity, while SIRT4-7 have very little or no deacetylase activity. Our laboratory has recently demonstrated that SIRT5, which has very weak deacetylase activity, can catalyze the hydrolysis of malonyl and succinyl groups from lysine residues very efficiently. Furthermore, several malonylated and succinylated proteins were identified from bovine liver mitochondria, demonstrating that lysine malonylation and succinylation are previously unrecognized protein posttranslational modifications. This discovery raised many interesting questions. For example, do protein malonylation and succinylation occur to many proteins or are they only limited to a few proteins? What is the physiological function of protein malonylation/succinylation and SIRT5-catalyzed demalonylation/desuccinylation? How do malonylation/succinylation regulate protein function? Cell- permeable SIRT5-specific inhibitors will be extremely helpful for addressing these questions. Such inhibitors can be used in cells or model organisms to accumulate malonylated and succinylated proteins to facilitate the identification of these proteins, to help study how malonylation/succinylation affects the functions of the proteins, and to provide insights into the biological function of protein malonylation/succinylation. In this proposal, we detail a plan to develop a high-throughput assay that can be used to screen for compounds that can specifically inhibit SIRT5. This assay will utilize a fluorogenic succinyl peptide, which by itself is not fluorescent but becomes fluorescent upon the hydrolysis of malonyl/succinyl by SIRT5. Inhibitors identified this way will then be subjected to a commercially available deacetylase screen to eliminate those that can also inhibit other sirtuins to give SIRT5-specific inhibitors. PUBLIC HEALTH RELEVANCE: Sirtuins, a class of enzymes known as NAD-dependent deacetylase, have been shown to regulate many important biological processes. Our laboratory recently discovered that SIRT5, one of the seven human sirtuins that has weak deacetylase activity, can efficiently remove two novel protein posttranslational modifications, lysine malonylation and succinylation. SIRT5-specific inhibitors are urgently needed for studying the physiological function of these novel protein posttranslational modifications. This project aims to develop a high-throughput screening assay to develop cell permeable SIRT5-specific inhibitors.