The Activity Evaluation Of LSD1 Inhibitors And Its Anti-Tumor Mechanism In Gastric Cancer

After LSD1(Histone Lysine Specific Demethylase 1)was identified as the first histone lysine specific demethylase in 2004,the molecular biological development of reversible protein methylation has been in a crazy stage.LSD 1 can affect the activation,inhibition and chromosome inactivation of gene transcription by removing the monomethylation and dimethylation of histones and other non-histone substrates.Many researchers believe that changes in LSD1 expression have become a major feature of tumorigenesis.At the same time,LSD1 has been reported as a key regulatory factor for a variety of tumors,and inhibition of LSD1 can inhibit the proliferation,differentiation,metastasis and invasion of tumor cells.Therefore,this project mainly takes LSD1 as the research object,screens its small molecule inhibitors with high efficiency and high selectivity,and studies the regulation mechanism of LSD 1.In recent years,small molecule inhibitors of LSD1 have been designed and synthesized based on the phenylcycloalanine structure of 2-PCPA,an MAO-A/B inhibitor.Although some active compounds have been obtained and some have entered the clinical detection period.the clinical data show that the compounds have certain limitations and side effects.Therefore,it is still a serious challenge to find effective and highly selective compounds.The main research contents and results of this project:(1)Expression and purification of LSD1 recombinant protein and screening of small molecule inhibitorsThis project applied and optimized the LSD 1 expression purification method constructed by the research group in the early stage.According to the literature research and experimental experience,the concentration of the imidazole buffer solution on the equilibrium column was increased,the LSD 1 recombinant protein with higher purity was obtained by one-step purification method.In addition,this project applied the LSD1 small molecule inhibitor screening method constructed earlier by the research group to screen small molecule compounds designed and synthesized in the research group and extracted from natural products.(2)The antitumor effect of natural product-like LSD1 inhibitors on the level of LSD1 recombinant protein and the level of gastric cancer cellsThis project screened the natural product library in the research group,and obtained two LSD1 inhibitors with better activity,MS-1 and MS-2.Through further mechanism studies,we found that at the recombinant protein level,compounds MS-1 and MS-2 were competing for LSD1 substrate binding site to inhibit LSD1 activity,and both of them interacted with LSD1 recombinant protein in the manner of fast binding and slow dissociation.At the cellular level,compounds MS-1 and MS-2 can bind to LSD1 and inhibit EMT process,metastasis and migration ability of MGC-803 cells by targeting LSD1.In addition,we preliminarily explored the regulation of LSD1 inhibitors on the immune checkpoint PD-L1,and found that MGC-803 cells treated with compounds MS-1 and MS-2 could increase the expression level of PD-L1.(3)The antitumor effect of novel biphenyl LSD1 inhibitors on LSD1 recombinant protein and gastric cancer cellsThe research group has been studying the targeted design and synthesis of LSD 1 small-molecule inhibitors for a long time.In this project,the compound library in the research group was screened and compound HML-390 was obtained.Compound HML-390 is an irreversible inhibitor of LSD1 with IC50=68.299±1.834 nM.Compound HML-390 has a strong affinity with LSD1 recombinant protein,KD-=3.338×10-5M.At the cellular level,compound HML-390 can bind LSD1 in cells to maintain its stability under high temperature conditions,indicating that the compound HML-390 can target LSD1 and play a role in regulating its biological activity.Cell scratch healing experiments and transwell experiments have shown that compound HML-390 can inhibit the metastasis and migration ability of MGC-803 cells.The clone formation experiment showed that compound HML-390 inhibited the colony formation rate and colony size of MGC-803 cells.In addition,we also detected the effect of compound HML-390 on the expression of PD-L1,and the results showed that the expression of PD-L1 could be up-regulated after compound treatment of MGC-803 cells.To sum up,this project mainly screened the small molecule compound library in the research group,and provided a new active skeleton for researchers of LSD1 inhibitors.At the same time,we explored the antitumor activity of some LSD1 inhibitors,which provided a biological basis for clinical application of LSD 1 targeted therapy for tumors.In addition,this study for the first time explored the regulation of LSD1 on PD-L1 in gastric cancer,providing a biological basis for the clinical use of LSD1 inhibitors.