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

Abstract

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 dual inhibition of Sirt2 results in strongly reduced levels of the oncogene 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.

Comparison of Cellular Target Engagement Methods for the Tubulin Deacetylases Sirt2 and HDAC6: NanoBRET, CETSA, Tubulin Acetylation, and PROTACs

“…Whereas Nano-BRET-based target engagement assays for Zn 2+ -dependent HDACs, including HDAC6, have been available for several years, 12 the first method for a member of the NAD + -dependent HDACs, Sirt2, has only very recently been reported. 6 Application of the NanoBRET-based Sirt2 target engagement assay enabled the development of small molecule inhibitors with low nanomolar Sirt2 affinities in cells. Moreover, this method was used to prove cellular target engagement for several literature-known Sirt2 inhibitors and provided addi- tional evidence of the low on-target specificity of the broadly used Sirt2 probe Sirtinol.…”

“…Moreover, this method was used to prove cellular target engagement for several literature-known Sirt2 inhibitors and provided addi- tional evidence of the low on-target specificity of the broadly used Sirt2 probe Sirtinol. 6 Cellular target engagement studies are of fundamental importance in order to confirm relevant drug targets and to evaluate the cellular on-target activity of biologically active compounds. For the tubulin deacetylases Sirt2 and HDAC6, which are both relevant drug targets, substantial progress in establishing methods to study cellular target engagement has been made in recent years.…”

“…Furthermore, the overall effect of selective Sirt2 inhibition on tubulin acetylation is not highly pronounced and often challenging to demonstrate via Western blot, due to its low dynamic range of detection. This might be one reason, why several recently published studies preferred immunofluorescence microscopy over Western blotting to prove cellular inhibition of Sirt2-mediated α-tubulin deacetylation. , Additionally, antibody-based methods for the detection of α-tubulin acetylation are time and labor intensive because of their heterogeneous assay protocols, thereby limiting the throughput of these methods. Due to the aforementioned drawbacks of using α-tubulin acetylation as a readout for cellular inhibition of Sirt2- or HDAC6-mediated deacetylation, several alternative methods have been recently established to demonstrate cellular target engagement for Sirt2 and HDAC6.…”

“…Moreover, the assay readout can be performed with a plate reader in a highly accurate and high-throughput manner. Whereas NanoBRET-based target engagement assays for Zn 2+ -dependent HDACs, including HDAC6, have been available for several years, the first method for a member of the NAD + -dependent HDACs, Sirt2, has only very recently been reported . Application of the NanoBRET-based Sirt2 target engagement assay enabled the development of small molecule inhibitors with low nanomolar Sirt2 affinities in cells.…”

“…Application of the NanoBRET-based Sirt2 target engagement assay enabled the development of small molecule inhibitors with low nanomolar Sirt2 affinities in cells. Moreover, this method was used to prove cellular target engagement for several literature-known Sirt2 inhibitors and provided additional evidence of the low on-target specificity of the broadly used Sirt2 probe Sirtinol …”