High-throughput assays for the development of SIRT5-specific inhibitors

描述（由申请人提供）：Sirtuins是一类被称为烟酰胺腺嘌呤二核苷酸（NAD）依赖性去乙酰化酶的酶。人类有7种sirtuins， SIRT1-7，已被证明可以调节多种生物过程，包括衰老、转录和代谢。然而，在7种人类sirtuins中，只有SIRT1、2和3具有强大的去乙酰化酶活性，而SIRT4-7具有非常低或没有去乙酰化酶活性。我们的实验室最近证明SIRT5具有非常弱的去乙酰酶活性，可以非常有效地催化赖氨酸残基水解丙二酰基和琥珀酰基。此外，从牛肝脏线粒体中鉴定出几种丙二酰化和琥珀酰化蛋白，表明赖氨酸丙二酰化和琥珀酰化是以前未被识别的蛋白质翻译后修饰。这一发现提出了许多有趣的问题。例如，蛋白质丙二酰化和琥珀酰化是否会发生在许多蛋白质上，还是只局限于少数蛋白质？蛋白质丙二酰化/琥珀酰化和sirt5催化的去丙二酰化/去琥珀酰化的生理功能是什么？丙二酰化/琥珀酰化如何调节蛋白质功能？细胞渗透性sirt5特异性抑制剂将非常有助于解决这些问题。这些抑制剂可以在细胞或模式生物中积累丙二酰化和琥珀酰化的蛋白质，以促进这些蛋白质的鉴定，帮助研究丙二酰化/琥珀酰化如何影响蛋白质的功能，并为蛋白质丙二酰化/琥珀酰化的生物学功能提供见解。在本提案中，我们详细介绍了开发一种高通量测定方法的计划，该方法可用于筛选特异性抑制SIRT5的化合物。该试验将利用荧光琥珀酰肽，其本身不荧光，但在SIRT5水解丙二酰/琥珀酰后变为荧光。用这种方法鉴定出的抑制剂随后将接受市售的去乙酰化酶筛选，以消除那些也能抑制其他sirt5的抑制剂，从而获得sirt5特异性抑制剂。

DESCRIPTION (provided by applicant): 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.