Sirtuins are a highly conserved class of NAD+-dependent lysine deacylases. The human isotype Sirt2 has been implicated in the pathogenesis of cancer, inflammation and neurodegeneration, which makes the modulation of Sirt2 activity a promising strategy for pharmaceutical intervention. A rational basis for the development of optimized Sirt2 inhibitors is lacking so far. Here we present high-resolution structures of human Sirt2 in complex with highly selective drug-like inhibitors that show a unique inhibitory mechanism. Potency and the unprecedented Sirt2 selectivity are based on a ligand-induced structural rearrangement of the active site unveiling a yet-unexploited binding pocket. Application of the most potent Sirtuin-rearranging ligand, termed SirReal2, leads to tubulin hyperacetylation in HeLa cells and induces destabilization of the checkpoint protein BubR1, consistent with Sirt2 inhibition in vivo. Our structural insights into this unique mechanism of selective sirtuin inhibition provide the basis for further inhibitor development and selective tools for sirtuin biology.

Sirtuins are NAD(+)-dependent protein deacylases that cleave off acetyl groups, as well as other acyl groups, from the ɛ-amino group of lysines in histones and other substrate proteins. Dysregulation of human Sirt2 activity has been associated with the pathogenesis of cancer, inflammation, and neurodegeneration, thus making Sirt2 a promising target for pharmaceutical intervention. Here, based on a crystal structure of Sirt2 in complex with an optimized sirtuin rearranging ligand (SirReal) that shows improved potency, water solubility, and cellular efficacy, we present the development of the first Sirt2-selective affinity probe. A slow dissociation of the probe/enzyme complex offers new applications for SirReals, such as biophysical characterization, fragment-based screening, and affinity pull-down assays. This possibility makes the SirReal probe an important tool for studying sirtuin biology.

Sirtuins are NAD + -dependent protein deacylases that cleave off acetyl, but also other acyl groups from the ε-amino group of lysines in histones and other substrate proteins.Dysregulation of human Sirt2 (hSirt2) activity has been associated with the pathogenesis of cancer, inflammation, and neurodegeneration, which makes the modulation of hSirt2 activity a promising strategy for pharmaceutical intervention. The Sirtuin Rearranging Ligands (SirReals) have recently been discovered by us as highly potent and isotype-selective hSirt2 inhibitors.Here, we present a well-defined structure-activity relationship study, which rationalizes the unique features of the SirReals and probes the limits of modifications on this scaffold regarding inhibitor potency. Moreover, we present a crystal structure of hSirt2 in complex with an optimized SirReal derivative that exhibits an improved in vitro activity. Lastly, we show cellular hyperacetylation of the hSirt2 targeted tubulin caused by our improved lead structure.

Controlling the activity of sirtuins is of high biomedical relevance as the enzymes are involved in cancer, neurodegeneration and other diseases.

Naturally occurring anticancer compounds represent about half of the chemotherapeutic drugs which have been put in the market against cancer until date. Computer-based or in silico virtual screening methods are often used in lead/hit discovery protocols. In this study, the "drug-likeness" of ~400 compounds from African medicinal plants that have shown in vitro and/or in vivo anticancer, cytotoxic, and antiproliferative activities has been explored. To verify potential binding to anticancer drug targets, the interactions between the compounds and 14 selected targets have been analyzed by in silico modeling. Docking and binding affinity calculations were carried out, in comparison with known anticancer agents comprising ~1,500 published naturally occurring plant-based compounds from around the world. The results reveal that African medicinal plants could represent a good starting point for the discovery of anticancer drugs. The small data set generated (named AfroCancer) has been made available for research groups working on virtual screening.