Project Summary Title: Structural and functional analysis of a novel class of androgen receptor antagonists Androgen deprivation therapy (ADT) and androgen receptor (AR)-targeting agents are the mainstay of metastatic prostate cancer (PCa) treatment. However, PCa on ADT and/or AR-targeting agents usually lead to relapse and castration-resistant prostate cancer (CRPC), which is mainly driven by AR activation under castration conditions. Next-generation AR-targeting agents such as enzalutamide and abiraterone can inhibit AR in CRPC but prolong the survival of CRPC patients for only about 4-5 months on average. The major mechanism responsible for the resistance to next-generation AR-targeting agents is reactivation of AR. Novel strategies to block the development of resistance to the current AR-targeted therapy are an urgent need and a major drug development challenge. Existing FDA-approved AR antagonists compete with dihydrotestosterone (DHT) for binding to the ligand binding domain (LBD) of AR, and patients eventually develop resistance to these treatments. One approach to overcoming resistance is to develop compounds that inhibit AR in alternative ways. We have identified a small molecule, SID 3712502, that is capable of inhibiting AR lacking LBD. To optimize the efficacy of this new class of inhibitors, we developed structural analogues and identified (+)-JJ-74-138 as a promising candidate. Our preliminary data suggest that (+)-JJ-74-138 is a novel AR antagonist capable of degrading nuclear AR and inhibiting enzalutamide-resistant CRPC both in culture and in xenograft tumors. However, it is not yet clear how AR interacts with (+)-JJ-74-138, which makes it difficult to rationally develop more potent analogues. We propose the following 2 specific aims to define how (+)-JJ-74- 138 and its parent compound SID 3712502 bind to AR and develop new analogs with improved potency, aqueous solubility and specificity for inhibition and degradation of nuclear AR. Aim 1 will determine how the SID 3712502 scaffold binds to AR using cryogenic electron microscopy (cryoEM). We will purify full-length AR (AR-FL), characterize interactions of AR-FL with SID 3712502 and (+)-JJ-74-138 for cryoEM structural analysis of a suitable complex of AR-FL2/R1881/ARE/SID 3712502 (or (+)-JJ-74-138) using single particle methods. Aim 2 will design, synthesize and analyze analogues of (+)-JJ-74-138, with the goal to identify small molecules with submicromolar potency, improved aqueous solubility, and high specificity for AR-positive cells. We will systematically prepare analogs of the lead compound(s) in iterative rounds of physicochemical improvements and optimizations based on structure–activity relationship (SAR) analyses from in vitro and cell-based assays. The success of this project will greatly facilitate the development of this class of novel AR antagonists for the treatment of CRPC, including enzalutamide-resistant CRPC.