A Novel Continuous Assay for the Deacylase Sirtuin 5 and Other Deacetylases

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• 作者：Claudia Roessler ; Christian T眉ting ; Marat Meleshin ; Clemens Steegborn ; Mike Schutkowski
• 刊名：Journal of Medicinal Chemistry
• 出版年：2015
• 出版时间：September 24, 2015
• 年：2015
• 卷：58
• 期：18
• 页码：7217-7223
• 全文大小：341K
• ISSN：1520-4804

文摘

Sirtuins are NAD⁺ dependent lysine deacylases involved in many regulatory processes like control of metabolic pathways, DNA repair, and stress response. Modulators of sirtuin activity are needed as tools for uncovering the biological function of these enzymes and as potential therapeutics. Systematic discovery of such modulators is hampered by the lack of efficient and simple continuous activity assays running at low sirtuin concentrations in microtiter plates. Here we describe an improved continuous sirtuin 5 assay based on the coupling of the sirtuin reaction to a proteolytic cleavage using internally fluorescence-quenched substrates. Systematic optimization of a carbamoyl phosphate synthetase 1 derived, glutarylated peptide yielded a Sirt5 substrate with k_cat/K_M value of 337鈥?00 M^鈥? s^鈥?, which represents the best sirtuin substrate described so far. These extraordinary substrate properties allowed reliable determination of K_i values for different inhibitors in the presence of only 10 nM sirtuin in microtiter plate format. Assay conditions could be transferred effectively to other lysine deacetylases, like sirtuin 2 and sirtuin 3, which now enables more efficient development of sirtuin targeting drugs.