Cyclic Tripeptide-based Potent and Selective Human SIRT5 Inhibitors

Background:: SIRT5 is one of the seven members (SIRT1-7) of the mammalian sirtuin family of protein acyl-lysine deacylase enzymes. In recent years, important regulatory roles of SIRT5 in (patho)physiological conditions (e.g. metabolism and cancer) have been increasingly demonstrated. For a better biological understanding and therapeutic exploitation of the SIRT5- catalyzed deacylation reaction, more effort on identifying potent and selective SIRT5 inhibitors beyond those currently known would be rewarding. Objective:: In the current study, we would like to see if it would be possible to develop potent and selective SIRT5 inhibitory lead compounds with a novel structural scaffold than those of the currently known potent and selective SIRT5 inhibitors. Methods: : In the current study, six N-terminus-to-side chain cyclic tripeptides (i.e. 8-13) each harboring the thiourea-type catalytic mechanism-based SIRT5 inhibitory warhead Nε-carboxyethylthiocarbamoyl- lysine as the central residue were designed, synthesized by the Nα-9- fluorenylmethoxycarbonyl (Fmoc) chemistry-based solid phase peptide synthesis (SPPS) on the Rink amide 4-methylbenzhydrylamine (MBHA) resin, purified by the semi-preparative reversedphase high performance liquid chromatography (RP-HPLC), characterized by the high-resolution mass spectrometry (HRMS); and were evaluated by the in vitro sirtuin inhibition assay and the in vitro proteolysis assay. Results:: Among the cyclic tripeptides 8-13, we found that 10 exhibited a potent (IC50 ~2.2 μM) and selective (≥60-fold over the SIRT1/2/3/6-catalyzed deacylation reactions) inhibition against the SIRT5-catalyzed desuccinylation reaction. Moreover, 10 was found to exhibit a ~42.3-fold stronger SIRT5 inhibition and a greater proteolytic stability than its linear counterpart 14. Conclusion:: With a novel and modular structural scaffold as compared with those of all the currently reported potent and selective SIRT5 inhibitors, 10 could be also a useful and feasible lead compound for the quest for superior SIRT5 inhibitors as potential chemical/pharmacological probes of SIRT5 and therapeutics for human diseases in which SIRT5 desuccinylase activity is upregulated.