Substrates and Cyclic Peptide Inhibitors of the Oligonucleotide-Activated Sirtuin 7

The sirtuins are NAD+-dependent lysine deacylases, comprising seven isoforms (SIRT1-7) in humans, which are involved in the regulation of a plethora of biological processes, including gene expression and metabolism. The sirtuins share a common hydrolytic mechanism but display preferences for different epsilon-N-acyllysine substrates. SIRT7 deacetylates targets in nuclei and nucleoli but remains one of the lesser studied of the seven isoforms, in part due to a lack of chemical tools to specifically probe SIRT7 activity. Here we expressed SIRT7 and, using small-angle X-ray scattering, reveal SIRT7 to be a monomeric enzyme with a low degree of globular flexibility in solution. We developed a fluorogenic assay for investigation of the substrate preferences of SIRT7 and to evaluate compounds that modulate its activity. We report several mechanism-based SIRT7 inhibitors as well as de novo cyclic peptide inhibitors selected from mRNA-display library screening that exhibit selectivity for SIRT7 over other sirtuin isoforms, stabilize SIRT7 in cells, and cause an increase in the acetylation of H3 K18. Broad screening of the substrate specificity of SIRT7 revealed a strong preference for the cleavage of long-chain acyllysine modifications. The activity was substantially increased in the presence of oligonucleotides or nucleosome particles. Finally, random nonstandard peptide integrated discovery (RaPID) delivered a cyclopeptide with potency in the sub-micromolar range and selectivity for SIRT7, which binds and stabilizes SIRT7 in cells.**+image

Automated summary

Sirtuins are a group of proteins that play important roles in regulating gene expression and metabolism in humans. Among the seven isoforms, SIRT7 has been less studied, but this study reveals its specific functions in the nucleus and nucleolus, as well as compounds that modulate its activity.