Discovery And Mechanism Studies Of Protein Lead Compounds Targeting Related Regulation Of Transcription

Transcriptional regulation is a specific form of gene regulation,and a necessary link to the expression of biomacromolecules.It sometimes involves complete termination of transcription to switch off the expression of specific genes;sometimes it might activate transcription to activate genes that are used only in certain circumstances.Transcriptional regulation is effective,because it's the first step in gene expression.By preventing translation or regulation that affects the performance of the translated protein.Transcription regulation is essential for maintaining the health of an organism and involves many different factors.Transcriptional regulation is critical to maintenance of the health of the organism.It involves many different factors and also a wide range of biological processes,including methylation,fatty acylation and ubiquitination,alternative splicing,DNA repair,immunoreaction and autophagy—all related to transcriptional regulation.In return,problems with any of these links,transcription factors,for example,cause a variety of disease.Therefore,targeted transcriptional regulation of key protein is a current research focus of life science.Hippo signaling pathway is the first discovered pathway in D.melanogaster that is highly conservative and is related to cell growth,differentiation and development.Hippo pathway regulates tissue growth and cell fate,and is regarded as the central regulator for tissue homeostasis and organ size.It was first discovered through mosaic gene screening in D.melanogaster.The pathway shares the name with one of its kinase components(Hippo,or MST1/2 in mammals),which was named after the overgrowth phenotype observed in the pathway mutation experiment.It functions mainly by transcriptional regulation,recruitment and co-activation of upstream and downstream protein among its members.According to previous research.YAP is the key protein in Hippo signaling pathway,which has no DNA binding domain.In the event of Hippo pathway anomalies,transcriptional enhancer associated domain family members(TEADs)to activate the transcription of downstream genes.TEADs therein are the key regulatory protein at the end of Hippo signaling pathway,which can recruit YAP/TAZ,VGLL and other co-activators,regulate the expression of cancer-promoting genes such as CTGF,Cyr61,AXL,Myc,Gli2 and BIRC5,and promote activation of the tumor marker mesothelin(MSLN).According to clinical research,TEAD is an important tumor marker of abnormally high expression in multiple disease and significantly related to poor clinical outcome.Histone acetylation modification is an important mechanism for epigenetic transcriptional regulation.Posttranslational modification(PTM)of histone can trigger chromatin remodeling in eukaryotic organisms.Therefore,it has a key role in regulation of gene expression.The process of histone acetylation is roughly as follows:lysine acetylation catalyzed by histone acetylases(HATs)—Writer,gene transcription activated,and deacetylation by histone deacetylases(HDACs)—Eraser.The imbalance state of histone acetylation is closely related to tumorigenesis.Based on the research and development status of drugs for targeted transcriptional regulation of key protein,starting from two pharmaceutical and innovative targets,i.e.TEAD and BRD protein family(identify lysine acetylation of histone),the research is unfolded using such methods as drug design,screening of high throughput compounds in vitro,chemical synthesis and alteration and pharmacological means.In the first part of this thesis,based on existing research reports on TEAD-YAP inhibitor,an Alpha Screen technology-supported targeted high throughput screening(HTS)platform is built and optimized based on targeted TEAD-YAP interaction;in addition,such physical and chemical conditions as pH,salinity and detergent are also optimized to detect the small molecule inhibitor of TEAD-YAP.On top of that,we run HTS of the natural product library in this laboratory,by which DCTEAD06 is recognized as a new hit for TEAD4-YAP,IC50:19.9±3.0?M.Then,the binding reaction pair TEAD4-YAP and DCTEAD06 is further verified with FP assay,experiment of protein thermal shift,microscale thermophoresis test(MST),SPR and other technology,and false positives excluded.We also notice that DCTEAD06 can bind with TEAD4.After verification with in vitro experiment,the compound DCTEAD06 is further studied on the cell context.This proves that it can inhibit the proliferation.Luciferase reporter gene experiment help verify that the compound DCTEAD06 can affect the transcriptional activity of TEADs.Part two herein focuses on the discovery of targeted TEAD selective small molecule inhibitors and the mechanism behind.In this part,we screen the covalent compound library in this laboratory with activity-based protein profiling(ABPP)Click assay,and obtain a small molecule inhibitor DC-TEADin1072 with TEAD 1/3 selectivity and a novel skeleton.Through the structure-based reasonable pharmaceutical chemical optimization,we use the difference in the TEADs family protein in residue and design the double-target inhibitor of TEAD 1/3 and the selective inhibitor of TEAD3.The specific molecular mechanism behind its selectivity is also revealed.DC-TEADin 1072 is identified as a new TEAD1/3 selective covalent inhibitor,IC50 being 0.61± 0.02 ?M and 0.58±0.12?M respectively.As verified by proteomics analysis and mutation detection of protein site,the protein binds with cysteine at TEAD1 C359 and TEAD3 C371 sites.And its physiological and pharmacological functions were further verified.It has been verified in the animal phenotype of zebrafish that it can have an impact on the development of animals.Following further studies based on our previous work,we have found dual target inhibitors of TEAD 1 and TEAD3.We obtained more selective and specific TEAD1 or TEAD3 inhibitors through sequence alignment and further structure-oriented chemical optimization.According to the literature,we found that there are few studies focus on the physiological function of TEAD3.At present,there is a lack of a selective compound with high activity as a probe to further explore the function of TEAD3.Further structure oriented chemical optimization produces the TEAD3 inhibitor DC-TEAD3in03 of higher selectivity,which is 100 times the selectivity of other TEAD3 targets.To further verify TEAD3's selective inhibition in cell,the inhibiting effect of TEAD3 on fluorescence signals GAL4-TEAD1/2/3/4 is tested with GAL4-luciferin reporter gene,whose activity in TEAD3 is 1.15?M.According to animal experiment,covalent binding of DC-TEAD3in03 with TEAD3 inhibits the growth of zebrafish.This also demonstrates TEAD3's relation to functional development.In this part of the work,we found TEAD1 and TEAD3 dual target inhibitors based on compound screening,analyzed,and compared the crystal structure of the TEAD family from this starting point,and found the first selective covalence small molecule inhibitor of TEAD3 by chemical modification structure optimization,and proved its cell target effect.Selective inhibition of TEAD3 transcriptional activity can also be achieved in cells.In terms of its function exploration,we took zebrafish as the model and used TEAD1 and TEAD3 dual target inhibitors as control to study its influence on development.The inhibition of TEAD 1/3 was found to inhibit and affect the overall growth rate of zebrafish.The TEAD3 inhibitor specifically affects the growth rate of the caudal fin.This proves the importance of biological development of TEAD3,and reminds us that different members of the TEADS family may regulate different genes.Therefore,selective inhibitors of other members of the TEADS family should be developed,and the specific physiological and pathological functions of different transcription factors should be studied with this probe.Part four of this thesis is the study on.the discovery and mechanism of the selective inhibitor of BD2 in BET family—targeted identifiers of lysine acetylation in histone.Bromodomain,the identifier of lysine acetylation in histone,is an essential target for cancer treatment,which is represented by BET contains two kinds of Bromodomain,BD1 and BD2.In recent years,selective inhibition of BET BD2 has become a promising strategy for drug discovery.Though major advances have been made in this field,research on the ligand/protein complex structure,difference in gene expression changes,and specific mechanism of selective BET BD2 inhibitors is still rarely found.We build an in vitro activity detection platform for selective BET BD2 and discover inhibitors BY27 and BD-224.The specific binding mode of the compound with BD1 and BD2 is explained with their high-resolution eutectic structure,thus providing its chemical alteration with a further basis.In addition,BY2 7 demonstrated a 67%tumor growth inhibition in a mouse tumor model,comparable to the panBET inhibitor I-BET762 currently in phase II clinical trials,which is less toxic to mice at higher doses.Therefore,the development of targeted selective BET BD2 inhibitors has the advantages of strong in vivo antitumor activity and better safety.