Sirtuins are protein deacetylases regulating metabolism, stress responses, and aging processes, and they were suggested to mediate the lifespan extending effect of a low calorie diet. Sirtuin activation by the polyphenol resveratrol can mimic such lifespan extending effects and alleviate metabolic diseases. The mechanism of Sirtuin stimulation is unknown, hindering the development of improved activators. Here we show that resveratrol inhibits human Sirt3 and stimulates Sirt5, in addition to Sirt1, against fluorophore-labeled peptide substrates but also against peptides and proteins lacking the non-physiological fluorophore modification. We further present crystal structures of Sirt3 and Sirt5 in complex with fluorogenic substrate peptide and modulator. The compound acts as a top cover, closing the Sirtuin’s polypeptide binding pocket and influencing details of peptide binding by directly interacting with this substrate. Our results provide a mechanism for the direct activation of Sirtuins by small molecules and suggest that activators have to be tailored to a specific Sirtuin/substrate pair.
The prominent role of Sirtuins in aging and metabolic regulation renders them attractive drug targets . The Sirt1 activator resveratrol extends lifespan , and protects against diseases such as insulin resistance ,. A link of resveratrol effects to Sirtuins, besides other cellular targets ,, is well documented ,. Two types of effects, direct Sirtuin activation and indirect effects on Sirtuin regulating pathways seem to contribute ,. Our data suggest that direct activation and inhibition of Sirt5 and Sirt3 activities, besides Sirt1 activation, might also contribute to physiological effects in mammals, but that remains to be tested. The effects on Sirt3 and Sirt5 also show that resveratrol-like compounds can interact with the conserved Sirtuin catalytic core, instead of the suggested binding to the Sirt1-specific N-terminus , consistent with the finding that piceatannol is a potent Sirt2 inhibitor . The low potency of resveratrol against Sirt5 (this study) and Sirt1 , is in fact consistent with the high compound concentrations needed to induce many of its in vivo effects . Sirtuin activation thus appears an attractive therapeutic approach ,, but more potent, specific, and bioavailable compounds are needed. Our activation model provides a tool for compound development, although it remains to be shown whether the same mechanism applies to the compound effects observed with other Sirtuins and with fluorophore-free substrates. However, our model rationalizes data for several Sirtuin isoforms and the direct Sirtuin activator/substrate contact could resolve a heated debate on the possibility of Sirtuin activation . It explains the requirement for the C-terminal fluorophore for Sirtuin activation against FdL1 substrate , and how other C-terminal extensions, in particular regular amino acids, can also enable activation. Consistently, we and others find resveratrol activation also for longer peptides without fluorophore and for entire substrate proteins (this study and ,). This mechanism also explains how the compound can activate, not affect, or inhibit a Sirtuin depending on the substrate, consistent with assay data (this study and ,,) and the fact that resveratrol shows Sir2-dependent effects in C. elegans overlapping with but not identical to effects of Sir2 overexpression . This sequence dependence suggests that for analyzing effects of this compound class on Sirtuin-dependent deacetylation we have to consider each Sirtuin/substrate pair individually, which on the other hand suggests that drugs can be developed that modulate only deacetylation of few specific Sirtuin substrates. The described resveratrol mechanism is in fact related to an alternative model, which suggested that activator and substrate form a complex in solution that then acts as an improved substrate . However, a lack in correlation between substrate/compound interaction and activation  and the substantial contribution of the protein to binding surface and outcome (Sirt5 activation versus Sirt3 inhibition) suggest that the modulator/substrate contact is preferentially formed in the enzyme/substrate complex.
n summary, our results reveal a mechanism enabling resveratrol-like compounds to directly activate Sirtuins. They indicate that several mammalian Sirtuin isoforms can be activated and reveal the exciting possibility to develop regulators targeting only specific Sirtuin/substrate pairs.