Discovery of SIRT3 Inhibitors Using SAMDI Mass Spectrometry

Abstract

Lysine acetylation plays a critical role in cellular regulation and is implicated in human disease. Sirtuin deacetylases remove acetyl groups from modified lysine residues, and sirtuin 3 (SIRT3) has been identified as a target for cancer therapeutics. Robust and high-throughput screening methods for these targets will be important to the development of therapeutics. This article describes the use of self-assembled monolayer desorption/ionization mass spectrometry, or SAMDI-MS-a label-free drug discovery tool-to characterize SIRT3 activity and discover inhibitors. SAMDI-MS was used to analyze a peptide array having 361 distinct acetylated peptides to identify an active SIRT3 substrate (GYK Ac RGC). This peptide was used in a screen of 100,000 small molecules to identify inhibitors of SIRT3. A total of 306 SIRT3 inhibitors were identified, with one compound, SDX-437, having an IC 50 of 700 nM with >100-fold selectivity for SIRT3 over SIRT1.

The unraveling of substrate specificity of histone deacetylase 6 domains using acetylome peptide microarrays and peptide libraries

“…The acetylome peptide microarray was developed to characterize substrate specificities of all human sirtuins and to allow the hypothesis-free assignment of novel sirtuin substrates (27). In addition, acetylome peptide microarrays were used to characterize modulators of sirtuin activity (23). This approach was then adapted to define subsite specificities of individual catalytic domains of human HDAC6 and to identify potential novel in vivo HDAC6 substrates.…”

2-Methoxyestradiol Affects Mitochondrial Biogenesis Pathway and Succinate Dehydrogenase Complex Flavoprotein Subunit A in Osteosarcoma Cancer Cells

“…Figure 6 shows the 3D and 2D ligand receptor interactions demonstrated by molecular docking. Interestingly, the crucial interaction regions reported in the literature for known inhibitors (small molecules or peptides) were observed for 2-ME at both the canonical inhibitor and allosteric sites (42)(43)(44)(45)(46) Figure 6B-E.…”

The Evolution of MALDI-TOF Mass Spectrometry toward Ultra-High-Throughput Screening: 1536-Well Format and Beyond

“…The technology offers data comparable to that of well-characterized SPE-MS methods, but with sample throughput and assay volumes more suited for full-diversity screening. While groups have reported high-throughput screens in MS-based systems previously, these often require a high-volume 384-well format, and throughput is achieved through either multiplexing 7 or compound pooling, 30 both of which require alterations to standard screening logistics and complicated data analysis. The work described here allows data input directly into standard 1536-well analysis workflows.…”