Novel SIRT5 Inhibitors And Inhibitory Strategies

Post-translational modifications(PTMs)have increasingly been demonstrated to play important roles in regulating protein functions and involved in many physiological and pathological processes.Lysine side chain acylation is one of the most studied modifications and regulates multiple crucial cellular processes including protein degradation,transcriptional activation,and metabolism.Silent information regulator 2(Sir2)proteins or sirtuins are a highly conserved family of ?-nicotinamide adenine dinucleotide(?-NAD+)-dependent acyl-lysine deacylases that regulate a variety of metabolic pathways.Since 2000,mammals have been found to have seven sirtuins,i.e.SIRT1 to SIRT7.Sirtuins are regarded as the contemporary therapeutic targets for cancer,and metabolic/neurodegenerative diseases.Accordingly,their inhibitors have been actively pursued over the past few years.Four of the mammalian sirtuins,i.e.SIRT4 to SIRT7 have undetectable or very weak acetyl-lysine deacetylase activity,rather prefer to remove long-chain fatty acyl groups or succinyl group,etc.In this thesis work,we chose SIRT5 as the subject sirtuin.SIRT5 is found in both mitochondria and cytosol,and is an efficient protein lysine demalonylase,desuccinylase,and deglutarylase.The preference for removing negatively charged acyl groups with terminal carboxyl could be explained by the presence of Arg105 and Tyr102 in the acyl binding pocket of SIRT5.Due to past insufficient research on the inhibitor development for SIRT5,more potent and selective SIRT5 inhibitors should be developed.Employingthe efficientcatalytic mechanism-based sirtuin inhibitory warhead Nε-carboxyethylthiocarbamoyl-lysine,cyclic peptides harboring this warhead were designed.We synthesizedthe designed compounds,performed their SIRT5 inhibitory assay and the relevant structure-activity relationship(SAR)analysis.The obtained results indicated that the peptide chain cyclization was a sound strategy for enhancing the SIRT5 inhibitory potency of the linear peptide inhibitor.Compound 3 was found to be apotent SIRT5 inhibitor(IC50=2.2±0.89μM),which is ~42.3-fold stronger than its linear counterpart 7 harboring the same warhead.Moreover,compound 3 wasfound to be highly proteolytically stable,in contrast to the linear peptide 7,as assessed by the pronase digestion assay,which also proved the superiority of cyclic peptides to their linear counterparts.We also designed novel inhibitory warheads against SIRT5.Specifically,replacing the terminal propionic acid of Nε-carboxyethylthiocarbamoyl-lysine with benzoic acid was intended to occupy SIRT5 acyl binding pocket tighter,however,this warhead was found to be 2-3-fold weaker than Nε-carboxyethylthiocarbamoyl-lysine.We also turned the thiourea-based warhead into selenourea-based one.In this regard,compound 9 was found to be a stronger SIRT5 inhibitor(IC50=1.7±0.1μM)than its thiourea counterpart(IC50=5±1.9μM)with the same tripeptidic scaffold,which suggested that Nε-carboxyethylselenocarbamoyl-lysine is a ~2.9-fold stronger SIRT5 inhibitory warhead than Nε-carboxyethylthiocarbamoyl-lysine.