Abstract Human estrogen receptor alpha (ERα) is a molecular driver of hormone-responsive cell proliferation in breast cancer. Acquired ERα mutations—Y537S and D538G being the two most commonly found—represent a newly recognized mechanism of drug resistance due to their constitutive transcription activity. Our preliminary data and recently published reports indicate that these drug-resistant mutants are non-conventional therapeutic targets for small molecule binding to modulate their activity and inhibit cell proliferation. However, the mechanisms by which drug- resistant mutations act on the receptor to regulate hormonal signaling and the extent to which small molecule inhibitors bind the receptor for intervention are not yet known. The ERα harbors two major functional entities, i.e., the DNA-binding domain (DBD) and the ligand-binding domain (LBD). We recently reported the multi-domain assembly and revealed the mode of interactions between these two domains, through a previously uncharacterized domain-bridging interface. Specifically, mutations at the domain-interface prevent the two domains from communicating and inhibit ERα activity, highlighting the modulation of the domain- interface as an “allosteric” channel with loss/gain of receptor function. This functional significance raises the questions of (a) whether the drug-resistant mutations alter the domain-domain assembly and the mode of DBD-LBD interactions, and (b) whether/how the domain-bridging interface can be targeted by small molecules to disrupt receptor activity. Our preliminary studies show that a repurposed small molecule binds the receptor via the domain-interface and inhibits ERα-mediated cellular function. Based on these findings and other preliminary data, we hypothesize that how the ERα domains interact with one another is influenced by these drug- resistant mutations and this domain-domain interaction is critical for small molecule binding to alter receptor function. To test this hypothesis, we will characterize the multi-domain assemblies of disease-resistant mutants (Y537S/D538G) and examine the molecular and functional correlation of inhibitor-receptor binding. In contrast to the hormone-binding pocket where all current drugs bind, this study will provide novel insights into the ERα domain-interface as a new target site for small molecule binding, and ultimately offer a much-needed molecular understanding of ER-positive breast cancer therapy resistance.