Hit evaluation results in 5-benzyl-1,3,4-thiadiazole-2-carboxamide based SIRT2-selective inhibitor with improved affinity and selectivity.

Sirtuin 2 (SIRT2), member of sirtuin family, belongs to class III histone deacetylases (HDACs) and is majorly cytosolic with occasional nuclear translocation. The enzymatic activity of SIRT2 is dependent on nicotinamide adenine dinucleotide (NAD+) and SIRT2 regulates post-translational modifications that are responsible for deacetylation of lysine residues in histone and non-histone substrates. SIRT2, thus affects most likely multiple cellular processes, such as signaling, gene expression, aging, autophagy, and has been identified as potential drug target in relation to inflammation, neurodegenerative diseases and cancer. Therefore, probing potential selective inhibitors is essential for the accurate understanding of enzyme functions. Here, we report a series of heteroaryl-2-carboxamide hybrids bearing substituted benzyl or substituted phenoxy group at the 5-position of the central heterocyclic ring. The synthesized compounds were screened against SIRT1-3 and MCF-7 human breast cancer cell line to evaluate their biological activity. The best SIRT2 inhibition profiles were displayed by ST29 (SIRT2 IC50 = 38.69 µM) and ST30 (SIRT2 IC50 = 43.29 µM) with excellent selectivity against SIRT2 over SIRT1 and SIRT3. Molecular docking study of the synthesized compounds into SIRT2 active site was performed to rationalize the remarkable SIRT2 inhibitory activity. Furthermore, we performed all-atom, explicit-solvent molecular dynamics (MD) simulations and end-point binding free energy calculations using molecular mechanics/generalized Born surface area (MM/GBSA) method to evaluate whether this design strategy was successfully deployed. The results implied that the binding poses and ligand affinities were predicted without significant loss of accuracy. Conclusively, the developed chemotypes were advocated as promising leads for SIRT2 inhibition and required further investigation for SIRT2-targeted drug discovery and development.

Hit-to-lead optimization on aryloxybenzamide derivative virtual screening hit against SIRT.

Sirtuins (SIRTs) are a class of nicotinamide adenine dinucleotide (NAD+)-dependent protein histone deacetylases (HDACs) that are evolutionarily conserved from bacteria to mammals. This group of enzymes catalyses the reversible deacetylation of lysine residues in the histones or non-histone substrates using NAD+ as a cosubstrate. Numerous studies have demonstrated that the aberrant enzymatic activity of SIRTs has been linked to various diseases like diabetes, cancer, and neurodegenerative disorders. Previously, we performed a pharmacophore-based virtual screening campaign and an aryloxybenzamide derivative (1) displaying SIRT1/2 inhibitory effect was identified as a hit compound. In the current study, the hit-to-lead optimization on the hit compound was explored in order to improve the SIRT binding and inhibition. Fourteen compounds, ten of which were new, have been synthesized and subjected to in vitro biological evaluation for their inhibitory activity against SIRT1-3. By the structural modifications performed, a significant improvement was observed in selective SIRT1 inhibition for ST01, ST02, and ST11 compared to that of the hit compound. The highest SIRT2 inhibitory activity was observed for ST14, which was designed according to compatibility with pharmacophore model developed for SIRT2 inhibitors and thus, providing the interactions required with key residues in SIRT2 active site. Furthermore, ST01, ST02, ST11, and ST14 were subjected to in vitro cytotoxicity assay against MCF-7 human breast cancer cell line to determine the influence of the improvement in SIRT1/2 inhibition along with the structural modifications on the cytotoxic properties of the compounds. The cytotoxicity of the compounds was found to be correlated with their SIRT inhibitory profiles indicating the effects of SIRT1/2 inhibition on cancer cell viability. Overall, this study provides structural insights for further inhibitor improvement.