Inhibitor Screening Of Drug Targets BACE1,JARID1A And BRD4

The research and development of new drugs is a multidisciplinary system engineering,including the drug target validation,in vitro and in vivo screening model development,leads related design,synthesis process,structure analysis,structure-activity relationship,pharmacokinetics,security and dosage forms studies in preclinical,and the clinical trial finally determines the practical application value and the approval of investigational new drug?IND?.The discovery of lead compounds is the first step in drug discovery,and the discovery approaches can be divided into screening approach and rational drug design.The suitable screening model and method can accelerate the screening of the lead compounds.In this study,Alzheimer's disease?AD?related target BACE1??-site amyloid precursor protein cleaving enzyme 1,also named?-secretase?and cancer related epigenetic targets JARID1A?Jumonji at-rich interactive domain1A,also called KDM5A or RBP2?and BRD4?bromodomain-containing protein4?were selected as the research objects.Firstly,establish the screening methods;then,identify hit compounds from the compound library by using the screening approach or a combination of virtual screening approach;finally,the structure-activity relationship,selectivity,dynamics or celluar function of the new inhibitors were further studied.For BACE1 target,we first developed a new microfluidics-based mobility shift assay?MSA?for the screening of BACE1 inhibitors in both kinetic and endpoint modes,the advantages of which including the potential interference of fluorescent compounds will be minimized and monitored and the ability of monitoring enzymatic reactions in real time facilitates the kinetics study.Then,5 new inhibitors were identified from the screening of 36100 compounds.Finally,based on the novel developed counter screen MSA assays for proteins BACE2,Cathepsin D and Cathepsin E,we found two compounds P46-N22 and P51-O06 showed high selectivities on BACE1 and BACE2 over Cathepsin D and Cathepsin E,and the other two compounds P69-M03 and P70-M22 showed high selectivities on Cathepsin D over BACE1,BACE2 and Cathepsin E.For JARID1A,2-hydroxy-1,3-indandione derivatives were identified as novel potent JARID1A inhibitors from high-throughput AlphaLISA screens using commercial compound libraries,with one compound named CPJA1 being the most potent inhibitor with IC₅₀ at 215±2 nM.Further characterization demonstrated CPJA1 acted as an iron?co-factor?competitive,pan-JARID1inhibitor.Moreover,CPJA1 exhibits anti-proliferation towards JARID1A overexpressive ZR-75-1 human breast cancer cell line with IC₅₀ at 14.6?M.For BRD4,14 inhibitors were identified from high-throughput AlphaScreen screens through structure-based drug design and virtual screening approaches.The IC₅₀ values of these inhibitors were in the range of 0.77³2?M,most of them showed high selectivities over CECR2,JARID1A and JAK3 and some of them displayed cytotoxicity towards Raji,MM.1s.and MV4-11 cancer cell lines.In this work,the developed in vitro assays have the advantages such as low compound interference,high throughput and low cost.Several new inhibitors have been identified by using these assays,which can be lead compounds for further structure activity relationship,pre-clinical or clinical studies.