PROJECT SUMMARY Hormonal therapies have been revolutionary in the treatment of women with estrogen receptor positive (ER+) breast cancers, greatly enhancing survival. ER-targeted hormonal therapies include selective estrogen receptor modulators SERMs, such as tamoxifen, and selective estrogen receptor downregulators (SERDs), such as fulvestrant, whereas aromatase inhibitors block the biosynthesis of estrogen. These therapies have significant side effects, and their prolonged use can lead to chemoresistance in about one- third of treated women, often resulting in more aggressive cancers. Furthermore, tamoxifen use is associated with increased incidence of endometrial thickening and hyperplasia, polyps and cancer. Recent studies have identified novel pathways involving the 7-transmembrane spanning G protein-coupled receptor GPER in patients undergoing SERM/SERD therapies. Cross-reactive pharmacological agonism of GPER contributes to both hormonal resistance and off-target effects in the uterus as all clinically approved anti- estrogens act as agonists for GPER and stimulate these pathways. This critical issue of ER/GPER selectivity has not been addressed in the development of SERM and SERD drugs. In our previous work, we discovered and characterized novel selective ligands for GPER that do not bind ERα or ERβ.Recently, we discovered a novel small molecule that for the very first-time targets ER without interference of GPER. As current anti-estrogens do not discriminate between ERα/β and GPER, our newly identified small molecule affords the opportunity to create novel ligands and therapeutic agents to selectively target ERα. Through chemical modifications to this first-generation ERα-selective compound, we have improved the affinity, receptor-selectivity and antagonist/agonist profile with the ultimate goal of creating truly ERα- selective antagonists. The specific aims of this proposal are to: 1. Synthesize and resolve the bioactive enantiomer of optimized ERα-targeted AB-SERD compounds with GPER anti-selectivity; 2. Prioritize compounds through in vitro methods including receptor binding, cell signaling, proliferation and toxicity and 3. Determine in vivo anti-tumor and ADMET properties of lead compounds, employing ER-dependent and anti-hormone-resistant transgenic, xenograft and PDX tumors. The successful completion of these studies will identify innovative enantiospecific compounds as unique pharmacological tools for delineating the individual functions of ER and GPER, and also initiating the development of first-in-class therapeutic agents, with the goal of reducing anti-hormone resistant recurrence of breast cancer, enhancing survival and the quality of life for the greater than 200,000 women annually diagnosed with ER-positive breast cancer.