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.

Previously, we have demonstrated the presence of a protein factor [tubulin polymerization perturbing protein (TPPP)] in brain and neuroblastoma cell but not in muscle extract that uniquely influences the microtubule assembly. Here we describe a procedure for isolation of this protein from the cytosolic fraction of bovine brain and present evidence that this protein is a target of both tubulin and microtubules in vitro. The crucial step of the purification is the cationic exchange chromatography; the bound TPPP is eluted at high salt concentrations, indicating the basic character of the protein. By IDA-nanoLC-MS analysis of the peptides extracted from the gel-digested purified TPPP, we show the presence of a single protein in the purified fraction that corresponds to p25, a brain-specific protein the function of which has not been identified. Circular dichroism data have revealed that, on one hand, the alpha-helix content of p25 is very low (4%) with respect to the predicted values (30-43%), and its binding to tubulin induces remarkable alteration in the secondary structure of the protein(s). As shown by turbidimetry, pelleting experiments, and electron microscopy, p25 binds to paclitaxel-stabilized microtubules and bundles them. p25 induces formation of unusual (mainly double-walled) microtubules from tubulin in the absence of paclitaxel. The amount of aberrant tubules formed depends on the p25 concentration, and the process occurs at substoichiometric concentrations. Our in vitro data suggest that p25 could act as a unique MAP in vivo.