New chemical tools for probing activity and inhibition of the NAD + -dependent lysine deacylase sirtuin 2
| Autor: | Sándor Szunyogh, Judit Ovádi, Wolfgang Sippl, Sören Swyter, Daria Monaldi, Tobias Rumpf, Attila Lehotzky, Manfred Jung, Matthias Schiedel |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
biology 010405 organic chemistry Chemistry Ligand binding assay Lysine Chemical biology SIRT2 Fluorescamine 01 natural sciences General Biochemistry Genetics and Molecular Biology 0104 chemical sciences 03 medical and health sciences chemistry.chemical_compound 030104 developmental biology Biochemistry Sirtuin biology.protein Click chemistry NAD+ kinase General Agricultural and Biological Sciences |
| Zdroj: | Philosophical Transactions of the Royal Society B: Biological Sciences. 373:20170083 |
| ISSN: | 1471-2970 0962-8436 |
| DOI: | 10.1098/rstb.2017.0083 |
| Popis: | 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'. |
| Databáze: | OpenAIRE |
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