New chemical tools for probing activity and inhibition of the NAD+ -dependent lysine deacylase sirtuin 2

Swyter, Sören and Schiedel, Matthias and Monaldi, Daria and Szunyogh, Sándor and Lehotzky, Attila and Ovádi, Judit (2018) New chemical tools for probing activity and inhibition of the NAD+ -dependent lysine deacylase sirtuin 2. PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY B-BIOLOGICAL SCIENCES, 373 (1748). pp. 1-13. ISSN 0962-8436

Text Swyteretal_Phylos.TransactionB_2018.pdf Restricted to Repository staff only Download (1MB) | Request a copy

Abstract

Sirtuins are NAD+ -dependent protein deacylases capable of cleaving off acetyl as well as other acyl groups from the ε-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. © 2018 The Author(s) Published by the Royal Society. All rights reserved.

Actions (login required)

Edit Item