Over 70% of breast cancers are Estrogen Receptor alpha (ERα)-positive, and current ERα- targeted therapies singularly focus on the receptor's hormone-binding domain (HBD) to which the drug tamoxifen binds. While initial treatments with endocrine therapies are successful for these patients, some with advanced diseases later develop resistance, including those who carry constitutively active ERα mutations (e.g., Y537S and D538G) in HBD. Moreover, these drug-resistant mutations bind and respond much more less effectively than the wild-type protein to fulvestrant, an FDA-approved first-line drug for treating ER-positive endocrine-resistant breast cancer. As such, new drugs are needed to target ERα in locations other than the tamoxifen- binding pocket in the HBD to overcome endocrine resistance. We recently reported the presence of a novel functional interface that bridges the HBD and the DNA-binding domain (DBD), two major functional units of the receptor. Disruption of the DBD-HBD bridging interface prevents the two parts of the receptor from communicating. As such, targeting the interface represents a “burning the bridge” strategy in the fight against ERα-positive breast cancer. Drug targeting of the ERα domain-bridging interface is particularly appealing; technologically, however, the ability to directly screen for specific ERα inhibitors requires a way to read out structure variations at the interface. By genetically engineering fluorescent probes to the purified receptor, we are able to determine whether or not HBD and DBD communicate properly. This approach represents a unique assay to screen small molecule inhibitors in vitro for their ability to disrupt the ER domain-interface, while cell-based experiments will further provide functional characterization for selected small molecules. Using this fluorescence assay, we will screen libraries of 2000+ FDA-approved drugs. Because the drug development process is long and slow, this repurposing or recycling approach is poised to jump-start the process; indeed, we have identified several molecules that inhibit the ERα activity by targeting the DBD-HBD interface. Collectively, our proposed “burning the bridge” strategy, armed with the ERα-specific fluorescence assay and cell-based analyses, presents a novel approach for ERα drug targeting. In this proposal, we will conduct in-depth functional and mechanistic studies for repurposed drugs (and their derivatives with similar fingerprints) and further determine their impacts on constitutively active ERα mutants found in drug-resistant ER-positive patients.