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)依赖的脱乙酰酶。人类有七个sirtuin，SIRT1-7，已被证明调节各种生物过程，包括衰老、转录和新陈代谢。然而，在7个人类sirtuin中，只有SIRT1、2和3具有较强的脱乙酰酶活性，而SIRT4-7具有很少的或没有脱乙酰酶活性。我们实验室最近证明，SIRT5具有很弱的脱乙酰酶活性，可以非常有效地催化赖氨酸残基中的丙二酸基和琥珀酸基的水解。此外，从牛肝线粒体中鉴定出几种丙二酸化和琥珀酸化的蛋白质，这表明赖氨酸丙二酸化和琥珀酸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，蛋白质丙二酸化和琥珀酸化是否发生在许多蛋白质中，还是仅限于少数几种蛋白质？蛋白质丙二酸化/琥珀酸化和SIRT5催化的去丙二酸化/去琥珀酸化的生理功能是什么？丙二酸化/琥珀酸化如何调节蛋白质功能？细胞渗透性的SIRT5特异性抑制剂将对解决这些问题非常有帮助。这些抑制剂可用于在细胞或模式生物中积累丙二酸化和琥珀酸化的蛋白质，以便于鉴定这些蛋白质，帮助研究丙二酸化/琥珀酸化如何影响蛋白质的功能，并对蛋白质丙二酸化/琥珀酸化的生物学功能提供见解。在这份提案中，我们详细介绍了一项开发高通量试验的计划，该试验可用于筛选能够特异性抑制SIRT5的化合物。这项检测将利用一种产生荧光的琥珀酸肽，这种肽本身不是荧光的，但在SIRT5对丙二酰基/琥珀酸基进行水解时会变成荧光。然后，用这种方法鉴定的抑制剂将经过商业上可用的脱乙酰酶筛选，以消除那些也可以抑制其他sirtuins的抑制剂，从而获得SIRT5特异性抑制剂。 与公共卫生相关：sirtuins是一类被称为NAD依赖脱乙酰酶的酶，已被证明调节许多重要的生物过程。我们实验室最近发现，七种具有弱脱乙酰酶活性的人类sirtuin之一的SIRT5可以有效地去除两种新的蛋白质翻译后修饰：赖氨酸丙二酰化和琥珀酸化。为了研究这些新的蛋白质翻译后修饰的生理功能，迫切需要SIRT5特异性的抑制剂。该项目旨在开发一种高通量的筛选试验，以开发细胞渗透性的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.