Institute of Pharmaceutical Sciences, University of Freiburg, Albertstraße 19, 79104 Freiburg im Breisgau, Germany.
Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK.
Philos Trans R Soc Lond B Biol Sci. 2018 Jun 5;373(1748). doi: 10.1098/rstb.2017.0083.
Sirtuins are NAD-dependent protein deacylases capable of cleaving off acetyl as well as other acyl groups from the ɛ-amino group of lysines in histones and other substrate proteins. They have been reported as promising drug targets, and thus modulators of their activity are needed as molecular tools to uncover their biological function and as potential therapeutics. Here, we present new assay formats that complement existing assays for sirtuin biochemistry and cellular target engagement. Firstly, we report the development of a homogeneous fluorescence-based activity assay using unlabelled acylated peptides. Upon deacylation, the free lysine residue reacts with fluorescamine to form a fluorophore. Secondly, using click chemistry with a TAMRA-azide on a propargylated sirtuin inhibitor, we prepared the first fluorescently labelled small-molecule inhibitor of Sirt2. This is used in a binding assay, which is based on fluorescence polarization. We used it successfully to map potential inhibitor-binding sites and also to show cellular Sirt2 engagement. By means of these new assays, we were able to identify and characterize novel Sirt2 inhibitors out of a focused library screen. The binding of the identified Sirt2 inhibitors was rationalized by molecular docking studies. These new chemical tools thus can enhance further sirtuin research.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.
去乙酰化酶是 NAD 依赖性的蛋白去酰基酶，能够从组蛋白和其他底物蛋白的赖氨酸 ε-氨基上切割掉乙酰基以及其他酰基。它们已被报道为很有前途的药物靶点，因此需要它们的活性调节剂作为分子工具来揭示它们的生物学功能和作为潜在的治疗方法。在这里，我们提出了新的测定格式，补充了现有的去乙酰化酶生物化学和细胞靶标结合的测定。首先，我们报告了使用未标记的酰化肽开发的均相荧光活性测定法。去酰化后，游离赖氨酸残基与荧光胺反应形成荧光团。其次，我们使用点击化学和炔丙基化的 Sirt2 抑制剂上的 TAMRA-叠氮化物，制备了第一个荧光标记的 Sirt2 小分子抑制剂。该抑制剂用于基于荧光偏振的结合测定。我们成功地使用它来绘制潜在抑制剂结合位点，并显示细胞 Sirt2 结合。通过这些新的测定方法，我们能够从聚焦文库筛选中鉴定和表征新型 Sirt2 抑制剂。通过分子对接研究来合理化鉴定的 Sirt2 抑制剂的结合。这些新的化学工具因此可以增强进一步的去乙酰化酶研究。本文是“表观遗传化学生物学前沿”讨论会议的一部分。
