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.