Design,Synthesis And Activity Study Of Novel Selective Epigenetic Targets SIRT2 Inhibitors

Epigenetic modifications refer to that the DNA expression level and function can be stably inherited without changing the DNA sequence,which play an important role in the process of gene expression.Epigenetic modifications include DNA methylation,histone modification,chromatin remodeling,gene imprinting,ribosome switching,and so on.Among them,histone modification is one of the most important modifications,such as acetylation,methylation,and phosphorylation.Histone acetylation is regulated by histone acetyltransferase(HATs)and histone deacetylase(HDACs).Usually,HATs and HDACs are in the state of dynamic balance,unless one of them become abnormal expression,this balance will be broken,and then cause a series of diseases.At present,there are 18 kinds of human histone deacetylase reported.Sirtuins(SIRTs),including SIRT1-7,are atypical histone deacetylases,which are completely different from HDAC1-11.They catalyze substrate deacetylation with the direct engagement of NAD+.Specifically,SIRT2 is mainly distributed in the cytoplasm and travels to the nucleus during mitosis.It not only catalyzes the deacetylation of histones,but also catalyzes some non-histones such as ?-tubulin,P53,P65,FOXO1 / 3a,etc.SIRT2 plays pivotal roles in multiple biological processes such as cell cycle regulation,autophagy,immune and inflammatory responses.Recently,many studies have found that SIRT2 is a new potential target for diseases such as cancer and Parkinson's disease,and its small-molecule inhibitors may provide an effective strategy for treatment of related diseases.Many pharmaceutical companies and research institutions around the world put much efforts in developing highly potent and selective small-molecule SIRT2 inhibitors.Up to date,there are no inhibitors used in clinical studies,and still,a lack of such high-quality inhibitors for the development of candidate drugs.In this paper,422 potent SIRT2 inhibitors were obtained by using the automatic lead optimization tool LEADOPT to screen a number of compound libraries after analyzing the reported SIRT2 inhibitor SirReal2 and SIRT2 composite crystal structures.Then,we selected two compound for further structural optimization according to computational scores and related experiences,as well as synthetic feasibility.A total of 33 new target compounds were synthesized,all these compounds were characterized by 1H-NMR,13C-NMR,HRMS and HPLC.All the target compounds were tested against SIRT2 in vitro.The results showed that many compounds had a good inhibitory activity at both 50 ?M and 5 ?M.Compound 28 e exhibits the most excellent inhibitory activity in these compounds,which exhibited 98% and99% inhibition rates at 5 ?M and 50 ?M,respectively.We further tested compound 28 e against SIRT1 and SIRT3,which are close analogues to SIRT2,with the aim of testing the selectivity of 28 e.The results showed that the IC50 values of 28 e on SIRT2 was up to 42 nM,while the IC50 values ??of SIRT1 and SIRT3 were significantly higher than 300 ?M,which indicated that 28 e was a potent active and selective SIRT2 inhibitor.Molecular docking simulations indicate that the SIRT2 protein active site is fully occupied by compound 28 e and interacts with multiple amino acid residues,which further explains why the compound 28 e has high activity.In view of the above experimental results,the inhibitory activity of compound 28 e and negative control compound 28 b on breast cancer cell MCF-7 was further tested.The results showed that 28 e could inhibit the activity of MCF-7 in a dose-dependent manner by suppressing the SIRT2 activity.Finally,the effects of compound 28 e on the level of ?-tubulin acetylation in MCF-7 were tested by immunoblotting.The results showed that28 e could increase the acetylation level of ?-tubulin in a dose-dependent manner.In conclusion,the highly active and selective SIRT2 inhibitor 28 e was obtained by rational design and synthesis.In vitro antitumor experiments confirmed that the compound had good anti-breast cancer effect.It is believed that this study could lay a foundation for developing new selective SIRT2 inhibitors for anti-cancer therapeutics...