Substrates and Cyclic Peptide Inhibitors of the Oligonucleotide‐Activated Sirtuin 7**

Abstract

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 ε‐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 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 increase in the acetylation H3K18.

Current Trends in Sirtuin Activator and Inhibitor Development

“…HEK293T cells treated with Compound 41 demonstrated increased acetylation of the Sirt7 substrate histone H3K18, consistent with on-target cellular Sirt7 inhibition [ 241 ]. Compound 41 represents a substantial improvement in Sirt7 inhibitor potency and sirtuin isoform specificity [ 237 , 241 ]. Therefore, Compound 41 demonstrates that peptide lariats are an exciting new avenue for sirtuin inhibitors, especially since their precise mode of sirtuin inhibition is unknown [ 241 ].…”

“…This screen generated two cyclic peptide inhibitors with trifluoroacetyl-lysine warheads that inhibited Sirt2 with IC 50 values of 3.2–3.7 nM and demonstrated ~9–150-fold selectivity for Sirt2 over Sirt1/3 [ 239 ]. Recently, the same cell-free translation peptide screen used to identify the first cyclic peptide inhibitors [ 239 ] was employed to develop non-mechanism-based cyclic peptide and peptide lariat sirtuin inhibitors [ 241 ]. Peptide lariat Compound 41 ( Figure 6 ) was found to be a novel Sirt7 inhibitor (IC 50 = 2.7 μM) that exhibited ~55-fold selectivity for Sirt7 over Sirt6, with minimal impacts on Sirt1/2/3/5 at 10 μM [ 241 ].…”

“…Recently, the same cell-free translation peptide screen used to identify the first cyclic peptide inhibitors [ 239 ] was employed to develop non-mechanism-based cyclic peptide and peptide lariat sirtuin inhibitors [ 241 ]. Peptide lariat Compound 41 ( Figure 6 ) was found to be a novel Sirt7 inhibitor (IC 50 = 2.7 μM) that exhibited ~55-fold selectivity for Sirt7 over Sirt6, with minimal impacts on Sirt1/2/3/5 at 10 μM [ 241 ]. HEK293T cells treated with Compound 41 demonstrated increased acetylation of the Sirt7 substrate histone H3K18, consistent with on-target cellular Sirt7 inhibition [ 241 ].…”

“…Peptide lariat Compound 41 ( Figure 6 ) was found to be a novel Sirt7 inhibitor (IC 50 = 2.7 μM) that exhibited ~55-fold selectivity for Sirt7 over Sirt6, with minimal impacts on Sirt1/2/3/5 at 10 μM [ 241 ]. HEK293T cells treated with Compound 41 demonstrated increased acetylation of the Sirt7 substrate histone H3K18, consistent with on-target cellular Sirt7 inhibition [ 241 ]. Compound 41 represents a substantial improvement in Sirt7 inhibitor potency and sirtuin isoform specificity [ 237 , 241 ].…”

Sulfur(VI) Fluoride Exchange Chemistry in Solid-Phase Synthesis of Compound Arrays: Discovery of Histone Deacetylase Inhibitors

“…We also performed Western blot-based cellular thermal shift assays (CETSA), , which we have previously used for investigating inhibitors targeting sirtuins (NAD + -dependent class III HDACs). − Because HDAC11 has very low expression levels in most cell lines, we were not able to produce a viable melting curve for this enzyme. Thus, we tested the most potent HDAC6 inhibitor of our collection ( E24 ).…”