Development of a NanoBRET assay to validate inhibitors of Sirt2-mediated lysine deacetylation and defatty-acylation that block prostate cancer cell migration

Sirtuin2 (Sirt2) with its NAD + -dependent deacetylase and defatty-acylase activities plays a central role in the regulation of specific cellular functions. Dysregulation of Sirt2 activity has been associated with the pathogenesis of many diseases, thus making Sirt2 a promising target for pharmaceutical intervention. Herein, we present new high affinity Sirt2 selective Sirtuin-Rearranging Ligands (SirReals) that inhibit both Sirt2-dependent deacetylation and defatty-acylation in vitro and in cells. We show that simultaneous inhibition of both Sirt2 activities results in strongly reduced levels of the oncoprotein c-Myc and an inhibition of cancer cell migration. Furthermore, we describe the development of a NanoBRET-based assay for Sirt2, thereby providing a method to study cellular target engagement for Sirt2 in a straightforward and accurately quantifiable manner. Applying this assay, we could confirm cellular Sirt2 binding of our new Sirt2 inhibitors and correlate their anticancer effects with their cellular target engagement. Sirt2 inhibitors that show simultaneous inhibition of Sirt2 deacetylase and defatty-acylase activity block prostate cancer cell migration and their target engagement is shown by a newly developed NanoBRET assay.