Crystallographic Study Of The AR-V7/ARE Complex

Androgen receptor(AR,NR3C4),which belongs to the steroid receptor subfamily of nuclear receptors(NRs),is a classical ligand-dependent transcription factor.AR is of great significance in the human development,metabolism and reproduction,especially in the development of prostate.AR disorders may lead to a variety of diseases including prostate cancer(PCa).PCa is the second most frequently diagnosed cancer among men worldwide.Androgen,as an endogenous ligand of AR,has been considered as an attribution to the excessive activation of intracellular AR activity,which directly promotes the occurrence and deterioration of PCa.Although blocking androgenactivated AR with drugs or surgery can shortly inhibit the growth of PCa cells,unfortunately,such treatment which called androgen deprivation therapy(ADT)is not a complete cure for PCa.Studies have found that activated AR signals can still be detected in these patients,and these activated signals often make patients eventually develop castration-resistant prostate cancer(CRPC)after 2-3 years.CRPC is a highly aggressive PCa with an overall survival period of less than 3 years.Current research shows that AR splicing-variants(AR-Vs)are most related to the occurrence and development of CRPC,especially the androgen receptor splicing-variant 7(AR-V7),which significantly shortens the time of progression from PCa to CRPC.AR-V7 is produced by alternative splicing of the full-length AR gene,containing exons 1,2,3 and cryptic exon(CE)3.In contrast to the full-length AR(AR FL),there is no ligand binding domain(LBD)for AR-V7,thus depriving PCa drugs targeting the LBD of their expected efficacy.Even in the absence of androgen,AR-V7 can be localized in the nucleus,where it is transcriptionally constitutive and activates AR downstream signaling,thereby contributing to ADT resistance.The absence of LBD in AR-V7 indicates that drug molecules should target regions outside of LBD,such as the DNA binding domain(DBD).Although more and more attention has been paid to the treatment of CRPC by targeting AR splicing-variants,the relevant drugs for clinical therapeutics remain unavailable.This is not only due to the complexity of drugs targeting non-LBDs,but also due to the lack of splicing-variants structures required to guide drug design.Based on the method of structural biology,this project mainly studies the binding mode of AR-Vs with androgen response elements(AREs),analyzes the structural mechanism of AR-Vs promoting ADT drug resistance compared with wildtype AR,with the aim to provide theoretical guidance and structural tools for the design of drugs targeting AR-Vs,especially AR-V7,for the treatment of CRPC.The main work of this paper includes:plasmids construction of eukaryotic androgen receptor splicing-variants,site-directed mutagenesis,expression and purification of prokaryotic proteins,protein crystallization and crystals optimization.The low-resolution complex crystals of ARV7 DBD combined with ARE were finally obtained through several hanging-drop crystallization experiments based on crystallographic studies.The optimized highresolution structure of post-analysis will provide a good foundation for the structurefunction elucidation of the DNA binding specificity and affinity by AR-V7 and a structural template for assisting in the development of drugs targeting AR-V7 DBD to treat CRPC-related diseases.The implementation of this project has theoretical guidance value and drug development significance for targeted AR-Vs in the treatment of CRPC-related diseases.