Establishment Of Screening Platform For Small Molecular Inhibitors Of KDM5B And Screening And Researching Mechanism Of Inhibitors

Histone lysine demethylase 5B(KDM5B)is a member of a family containing the Jmjc domain.KDM5 B uses Fe(II)and ?-KG as cofactors to remove the mono-,di-,and tri-methylated of the fourth lysine of histone H3.Some studies have found that KDM5 B is involved in the formation of inhibitory transcription complexes around multiple target gene promoters,and plays a broad regulatory role in chromatin structure,which is closely related to the occurrence and development of tumors.Through investigation and research of reported literature,we found that KDM5 B is expressed in normal adult tissues with low expression,mainly high expression in testis and brain tissue;while it has high expression in leukemias and kinds of solid tumors,such as breast cancer,prostate cancer,gastric cancer,lung cancer and oral cancer.Moreover,there are numerous studies that KDM5 B is associated with tumor growth,angiogenesis,invasion,metastasis and tumor-related chemoresistance,and tumor-associated chemoresistance.Therefore,KDM5 B is considered to be a potential drug target for cancer therapy.At the same time,many researchers have designed many inhibitors with KDM5 B as a drug target,but there are some problems such as low selectivity or poor cell permeability.So,there are still no KDM5 B inhibitors appearing on the market.Therefore,the development of inhibitors of KDM5 B with independent intellectual property rights,high efficiency,high selectivity and low toxic side effects is of great significance.Based on the previous experience,our team has designed and synthesized a series of new inhibitors against KDM5 B by means of high-throughput virtual screening and structure-based drug design and structural activity relationship studies.Simultaneously,according to the fact that gastric cancer is a malignant tumor that seriously endangers the health of Chinese people,we choose to use gastric cancer cells as our main research object.Therefore,this paper mainly evaluates the anti-tumor activity of the novel KDM5 B inhibitors synthesized by our research group,and selects representative compounds to study the mechanism of action in gastric cancer cells.The research contents are as follows:1.First,we established a KDM5 B small molecule inhibitor screening platform based on the HTRF principle,and optimized the reaction conditions.Then we evaluated the reaction system using the positive compound CPI-455.On the basis of determining the stability and applicability of the reaction system.After this work,a high-throughput screening platform for KDM5 B small molecule inhibitors was successfully established,which laid a foundation for the subsequent discovery of KDM5 B inhibitors.2.After using the KDM5B-inhibitors-screening platform to evaluate the enzyme activity in vitro with the different compounds,we selected the representative compound ZB-16 for subsequent mechanism studies.And the less active compound LQQ-345 was selected as its negative control compound,and CPI-455 as a positive compound.3.After the expression level of KDM5 B in gastric cancer cell lines was examined,we selected the MKN45 cell line with the highest KDM5 B expression for studying the mechanism of KDM5 B and compound ZB-16.4.We studied the inhibitory activity and mechanism of compound ZB-16 on gastric cancer cells.The results suggested that ZB-16 can inhibit the proliferation of MKN45 cells in a dose-and time-dependent manner,and up-regulate the expression of H3K4me3 by inhibiting the activity of KDM5 B protein.ZB-16 also inhibited the metastasis and invasion ability of MKN45 cells in a dose-dependent manner,and further inhibited the EMT process of gastric cancer cells,proving that the compound ZB-16 has antitumor activity.In summary,this project successfully established a high-throughput screening platform for KDM5 B small molecule inhibitors,and have screened 264 new KDM5 B small molecule inhibitors.The best enzyme inhibitory activity compound is the compound LQQ-466,which the IC50 of the enzyme level is 40.03 nM.Among them,we selected a representative compound,ZB-16,with an IC50 of 292 nM at the enzyme level.Subsequently,the mechanism of ZB-16 on KDM5 B in vivo was studied in MKN45 gastric cancer cells.We have found that ZB-16 can inhibit the ability of proliferation,metastasis and invasion of cancer cells by inhibiting the action of KDM5 B.This paper provides a new research direction for the development and optimization of such novel KDM5 B inhibitors,and develops research ideas for the subsequent biological function research of KDM5 B.