Sirtuins are protein deacylases regulating metabolism and stress responses, and are implicated in aging-related diseases. Small molecule activators for the human sirtuins Sirt1-7 are sought as chemical tools and potential therapeutics, such as for cancer. Activators are available for Sirt1 and exploit its unique N-terminus, whereas drug-like activators for Sirt2-7 are lacking. We synthesized and screened pyrrolo[1,2-a]quinoxaline derivatives, yielding the first synthetic Sirt6 activators. Biochemical assays show direct, substrate-independent compound binding to the Sirt6 catalytic core and potent activation of Sirt6-dependent deacetylation of peptide substrates and complete nucleosomes. Crystal structures of Sirt6/activator complexes reveal that the compounds bind to a Sirt6-specific acyl channel pocket and identify key interactions. Our results establish potent Sirt6 activation with small molecules and provide a structural basis for further development of Sirt6 activators as tools and therapeutics.

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.

Protein lysine deacylases comprise three zinc-dependent families and the NAD + -dependent sirtuins Sirt1−7, which contribute to aging-related diseases. Few Sirt6-specific inhibitors are available. Trichostatin A, which belongs to the potent, zinc-chelating hydroxamate inhibitors of zinc-dependent deacylases, was recently found to potently and isoform-specifically inhibit Sirt6. We solved a crystal structure of a Sirt6/ADP-ribose/trichostatin A complex, which reveals nicotinamide pocket and acyl channel as binding site and provides interaction details supporting the development of improved deacylase inhibitors.

Sirtuins are NAD + -dependent protein lysine deacylases that are considered attractive drug targets for agingrelated diseases. Sirt6 deacetylates, e.g., transcription factors and histone H3, and regulates metabolic processes and stress responses. It has been implicated in lifespan extension and tumor suppression. Sirt6 deacetylase activity can be stimulated with small molecules, and fluvastatin, an FDA-approved synthetic statin, was recently described as a novel Sirt6 activator. We studied the molecular details of this effect on Sirt6 in deacylation assays and by solving a crystal structure of a Sirt6/fluvastatin complex. We find that fluvastatin inhibits Sirt1−3 at higher concentrations but has a unique, activating effect on Sirt6. The complex structure reveals that fluvastatin occupies the Sirt6 substrate acyl channel exit, similar to other, unrelated activator families, providing interaction details that will support the development of potent, druglike Sirt6 activators.

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