The Development Of Inhibitors Of Protein Lysine Deacylases SIRT6 And SIRT5

The sirtuin family of enzymes catalyze the ?-nicotinamide adenine dinucleotide(?-NAD+ or NAD+)-dependent side chain deacylation of specific N?-acyl-lysine residues on proteins.Unlike other protein deacylation enzymes,the sirtuin-catalyzed deacylation is not merely an N?-acyl-lysine side chain amide hydrolysis reaction,instead it is coupled to the nicotinamide cleavage from NAD+ with the generation of three enzymatic products,i.e.the deacylated protein species,nicotinamide,and 2-O-acyl-ADP-ribose(2-O-AADPR).The NAD+-dependent N?-acyl-lysine deacylation reaction catalyzed by sirtuin family members has been increasingly demonstrated to be important in regulating multiple crucial cellular processes and has also been proposed to be a therapeutic target for multiple human diseases.SIRT6 is one of the seven sirtuins(SIRT1-7)that have been found in mammals including humans.SIRT6 is predominantly present in nucleus,and can catalyze the N?-acetyl-lysine deacetylation reaction.However,SIRT6 can more proficiently catalyze N?-fatty-acyl-lysine defatty acylation(e.g.demyristoylation).The NAD-dependent deacetylase activity of SIRT6 on lysine 9 of histone H3(H3K9)and lysine 56 of histone H3(H3K56)is responsible for the regulation of gene expression and the maintenance of chromatin telomeres.Moreover,dysregulation of SIRT6 activity has been associated with many pathologies,including cardiovascular diseases(CVD),cancer,neurodegenerative diseases and diabetes.SIRT5 is an enzyme that is predominantly present in mitochondria,and has a stronger desuccinylase,deglutarylase and demalonylase activities than its deacetylase activity.It has been found that lysine succinylation is widely present on mitochondrial energy metabolism regulating enzymes,which are involved in the regulation of a variety of important metabolic pathways including amino acid metabolism,fatty acid metabolism and tricarboxylic acid cycle.So SIRT5 can achieve metabolic regulation by regulating the activity of these metabolic enzymes through catalyzing the N?-acyl-lysine deacylation on them.Since the sirtuin-catalyzed deacylation reaction is intimately related to human health and disease,the development of sirtuin inhibitors has been a great interest in recent years.This enterprise will potentially furnish not only new treatments for humandiseases but also new tools for exploring the unknown world of sirtuin.In this thesis work,cyclic peptides were designed and examined as novel inhibitors for SIRT6 and SIRT5,and the thiourea-type of sirtuin inhibitory warheads were employed in the design.The designed compounds were prepared by solid-phase synthesis.The purified compounds were then tested in in vitro sirtuin inhibition assay,the pronase digestion assay,and the Western blotting assay.We discovered that cyclic pentapeptides harboring a central N?-dodecyl(or tetradecyl)-thiocarbamoyl-lysine residue all behaved as highly potent inhibitors against human SIRT6-catalyzed deacylation reaction.Moreover,one compound was also found to be selective for SIRT6 versus SIRT2/3/5(?20-,?11-,and>940-fold,respectively),despite its modest(?2.3-fold)SIRT6 inhibitory selectivity versus SIRT1.At the same time,we found that this compound was also proteolytically stable,and it was able to inhibit SIRT6 inside cells.In this thesis work,we also found a potent and selective SIRT5 cyclic peptide inhibitor(relative to SIRT1/2/3/6),which was also found to be a stronger SIRT5 inhibitor than its corresponding linear peptide.The findings from the current thesis work have laid a good foundation for the future development of potent and selective,proteolytically stable and metabolically safe sirtuin inhibitors with cell membrane permeability.