The Research On Inhibitor And Inhibitory Strategy Against Protein Lysine Demodification Enzyme

Post-translational modifications(PTMs) have increasingly proved to be important in regulating crucial cellular processes, such as gene transcription, DNA repair, chromatin formtion, and metabolism. These PTMs and their reversal include lysine side chain Nε-acylation/deacylation, Nε-methylation/demethylation, Nε-ubiquitination/deubiquitination, and arginine side chain Nη-methylation /demethylation. The dynamic equilibrium between PTMs and their reversal maintain life processes in normal cells. My thesis work was focused on the lysine Nε-deacylation and demethylation catalyzed by the sirtuin family of protein lysine deacylase enzymes and the JMJD2 family of protein lysine demethylase enzymes, respectively.The sirtuin-catalyzed deacylation reaction and the JMJD2-catalyzed demethylation reaction can be two potential therapeutic targets(especially for developing cancer treatments), the research on their catalytic mechanisms and developing their inhibitors has been on spotlight. In this thesis work, highly potent and also selective sirtuin inhibitors of a new chemotype have been developed; moreover, attempts to develop a novel type of JMJD2 inhibitors have also been made.Specifically, we used the peptide chain cyclization strategy to cyclize the straight-chain pentapeptide lead compound 6, and the resulting cyclic peptides with different ring structures were assessed for their inhibitory potencies against the sirtuin-catalyzed deacylation reaction. We found the peptide chain cyclization strategy is a very effective way for the development of sirtuin inhibitors. Six designed cyclic peptides harboring the catalytic mechanism-based SIRT1/2/3 inhibitory warhead N?-thioacetyl-lysine(i.e. compounds 7-12) were found in this work to exhibit very potent SIRT1/2/3 inhibition(nM level); particularly, compound 9 was found to be the most potent SIRT1 inhibitor identified so far(IC50=2.3 ± 0.28nM), and compound 10 was found to be one of the most potent and selective SIRT2 inhibitors ever identified. In this work, four more cyclic peptides harboring the sirtuin catalytic warheads other than N?-thioacetyl-lysine(i.e. compounds 13-16) were also prepared and assessed for their SIRT1/2/3 inhbitory potencies. While they all exhibited potent(nM level) SIRT2 inhibition with different degrees of inhibitory selectivity versus SIRT1/3, compound 13 was the most potent and selective one with ~23.5fold and ~80.3fold stronger SIRT2 inhibition than inhibiting SIRT1/3, respectively, and its SIRT2 inhibitory potency was only ~3.6fold weaker than that of the afore-mentioned inhibitor 10. One more appealing feature of inhibitor 13 is its conceivable lack of a hepatotoxicity potentially associated with the use of the thioamide-type of the SIRT1/2/3 inhibitory warhead N?-thioacetyl-lysine.With the work on the JMJD2-catalyzed demethylation reaction, we succeeded in establishing a high-performance liquid chromatographic(HPLC)-based method for a quantitative analysis of the in vitro JMJD2 C assay. We found that JMJD2 C can accept N?-isopropyl-ornithine as an alternate substare for N?-dimethyl-lysine, suggesting that the side chain of N?-isopropyl-ornithine can also be accommodated by the N?-dimethyl-lysine binding pocket at JMJD2 C catalytic center. However, the substrate activity of N?-isopropyl-ornithine is lower than N?-dimethyl-lysine, which could be due to the sub-optimal positioning of the side chain N and the two side chain methyl groups of N?-isopropyl-ornithine when bound at JMJD2 C active site. Nevertheless, this finding can still be useful for the discovery of novel JMJD2 inhibitors in the future. In this work, we also found that the side chain of N?-difluoroisopropyl-ornithine(with an isosteric replacement of C-F for C-H at one of the two terminal methyl groups on the side chain of N?-isopropyl-ornithine) seemed not to be able to be efficiently accommodated at JMJD2 C active site, whch could be die to the following: 1) the strong electron withdrawing effect of the two fluorine atoms would prevent a favorable binding interaction with JMJD2C; 2) the N?-dimethyl-lysine binding pocket at JMJD2 C catalytic center is too narrow to accommodate difluoroisopropyl which is slightly bulkier than dimethyl-lysine and diisopropyl-ornithine.