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

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 a...