Structure-based Drug Screening And Activity Verification For ETS Transcription Factor ERG

Purpose:ERG is a member of ETS(E-26 transformation specific)transcription factor family.In prostate cancer,a TMPRSS2:ERG fusion is very common(about 50%of patients in European and American countries).After fusion with the androgen driven promoter of TMPRSS2,the persistent high expression of ERG promotes the progression of prostate cancer.Not only that,ERG is also associated with other cancers,including Ewing’s sarcoma and leukemia.Development of ERG-targeting inhibitors will provide a new treatment for these diseases.I attempted to identify and block the interaction between ERG and other proteins with repurposed drugs.The ETS domain of ERG was selected as the targeted domain,and active compounds targeting ERG were screened by computer-aided drug design combined with in vitro experiments for drug activity selection.Methods:In this study,the ETS domain of ERG was selected as the binding site of targeted drugs.The ETS domain binds with DNA,functioning in the regulation of gene expression.In order to focus on clinical applicability,my study selected listed drugs and clinical trial drugs,identified from small molecules listed in DrugBank as the docking library and using Ledock docking software to dock about 10000 drugs(nearly 2 million different conformations)to screen for binding to the ETS domain.This approach can explore new uses for "old medicine",and also shorten the development process of the drugs.Combined with the knowledge of the pharmaceutical chemistry,appropriate small molecules were selected,from the docking results,for inhibition of cell proliferation in in vitro inhibition experiments.Nifuroxazide,the most active small molecule,was selected as the main research object.Transcriptome data was measured,and the pathway maps of nifuroxazide in the ERG fusion-positive prostate cancer cell line,VCap,was obtained by KEGG enrichment analysis.KD,the affinity constant of nifuroxazide with ERG was measured by surface plasmon resonance(SPR).Combined with the virtual docking analysis,the interactional mode of nifuroxazide in the ETS domain was studied.Western blot,RT-PCR,immunoprecipitation and immunocytochemistry were used to test the function of nifuroxazide.Results:According to the virtual docking and cell proliferation inhibition experiments,the highly active compound,nifuroxazide,was obtained.The IC50 value was 2.56 μmol/L for the ERG fusion-positive cell line.Transcriptional data and KEGG enrichment analysis indicated that nifuroxazide can down-regulate a large number of inflammatory factors(IL-1,IL-6,CCL2,etc)in VCap cells and upregulate several genes(BRCA1,PARP,FANCA,etc)related to DNA damage repair.Surface plasmon resonance indicated that the affinity constant was 10.8 μmol/L.The docking results showed that nifuroxazide was bound to the ETS domain of ERG,in the pore formed by ASN319 and PHE375.The binding energy was-4.73 kJ/mol.Studies have confirmed that the interaction of nifuroxazide with ERG changed the spatial conformation of ERG,making the interaction between ERG and PARP1 and other proteins difficult.Activation of this parthanatos pathway may eventually lead to cell death.Conclusion:Nifuroxazide,a mature clinical drug,has been used as an antiparasitic drug for a long time.We found that nifuroxazide can block the interaction of ERG with PARP1 by targeting the ETS domain,which may lead to PARP1 overactivation,thereby activating the parthanatos pathway.This not only lays the structural foundation for the development of ERG-targeted drugs,but also provides a new strategy for the treatment of prostate cancer.