Abstract

Mammalian Sirtuin 6 (Sirt6) is an NAD+-dependent protein deacylase regulating metabolism and chromatin homeostasis. Sirt6 activation protects against metabolic and aging-related diseases, and Sirt6 inhibition is considered a cancer therapy. Available Sirt6 modulators show insufficient potency and specificity, and even partially contradictory Sirt6 effects were reported for the plant flavone quercetin. To understand Sirt6 modulation by quercetin-based compounds, we analysed their binding and activity effects on Sirt6 and other Sirtuin isoforms and solved crystal structures of compound complexes with Sirt6 and Sirt2. We find that quercetin activates Sirt6 via the isoform-specific binding site for pyrrolo[1,2-a]quinoxalines. Its inhibitory effect on other isoforms is based on an alternative binding site at the active site entrance. Based on these insights, we identified isoquercetin as a ligand that can discriminate both sites and thus activates Sirt6 with increased specificity. Furthermore, we find that quercetin derivatives that inhibit rather than activate Sirt6 exploit the same general Sirt6 binding site as the activators, identifying it as a versatile allosteric site for Sirt6 modulation. Our results thus provide a structural basis for Sirtuin effects of quercetin-related compounds and helpful insights for Sirt6-targeted drug development.

Highlights

  • Mammalian Sirtuin 6 (Sirt6) is an NAD+-dependent protein deacylase regulating metabolism and chromatin homeostasis

  • Contradictory effects on Sirt[6] activity were previously reported for the plant flavone quercetin[15,21,22,26], and we find that the compound interferes with two popular Sirtuin activity assays, which is a common problem for optical assays[32]

  • Reported inhibitory effects at lower quercetin www.nature.com/scientificreports concentrations could not be observed and likely are assay artifacts caused by the optical effects of the compound and/or the use of Sirt[6] tagged with GST, which is known to bind quercetin tightly[33]

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Summary

Introduction

Mammalian Sirtuin 6 (Sirt6) is an NAD+-dependent protein deacylase regulating metabolism and chromatin homeostasis. Sirt[6] efficiently deacetylates nucleosomal histones in vitro, and nucleosomes as well as proteins involved in glucose metabolism and DNA repair in cells[7,12] It features the conserved Sirtuin catalytic core of ~275 amino acids, with a Rossman-fold and a Zn2+-binding domain[10]. Free fatty acids and fatty acid ethanolamides at high micromolar concentrations were found to stimulate Sirt[6] deacetylation activity[14,15] They inhibit Sirt6-dependent hydrolysis of long chain acylations, implicating the acyl binding channel in this effect[14]. Pharmacophore models[22,26] and docking calculations[23] failed to rationalize the limited and partially contradictory experimental data on Sirt[6] modulation

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