Continuous Sirtuin/HDAC (histone deacetylase) activity assay using thioamides as PET (Photoinduced Electron Transfer)-based fluorescence quencher

Histone deacylase 11 and human sirtuins are able to remove fatty acid-derived acyl moieties from the epsilon-amino group of lysine residues. Specific substrates are needed for investigating the biological functions of these enzymes. Additionally, appropriate screening systems are required for identification of modulators of enzymatic activities of HDAC11 and sirtuins. We designed and synthesized a set of activity probes by incorporation of a thioamide quencher unit into the fatty acid-derived acyl chain and a fluorophore in the peptide sequence. Systematic variation of both fluorophore and quencher position resulted super-substrates with catalytic constants of up to 15,000,000 M(-1)s(-1) for human sirtuin 2 (Sirt2) enabling measurements using enzyme concentrations down to 100 pM in microtiter plate-based screening formats. It could be demonstrated that the stalled intermediate formed by the reaction of Sirt2-bound thiomyristoylated peptide and NAD(+) has IC50 values below 200 pM.

Automated summary

The research found that Histone deacylase 11 and human sirtuins can remove fatty acid-derived acyl moieties from lysine residues, requiring specific substrates and screening systems. By designing and synthesizing activity probes, highly catalytic super-substrates were created, enabling enzyme measurements down to 100 pM in microtiter plate-based screening formats. The IC50 values of stalled intermediates formed by Sirt2-bound peptides and NAD(+) were demonstrated to be below 200 pM.