ABSTRACT Breast cancer is the most frequently diagnosed cancer and the second leading cause of death in American women. The majority of breast cancer (~75%) expresses estrogen receptor α (ERα) protein and therefore inhibition of this protein function by endocrine therapy is the mainstay of treatment in ER-positive breast cancer patients. Nevertheless, acquired resistance has developed in nearly half of women treated with endocrine therapy that is associated with poor patient survival. Specifically, in a substantial number of cases, prolonged treatment with endocrine therapy creates the development of resistant tumor cells and, consequently, tumor relapse, which manifests as metastatic disease that is extremely difficult to manage. Recently, deep DNA sequencing has identified somatic mutations at specific sites in the ERα gene (ESR1) in a large subset of patients with breast cancers that have spread. Specifically, Y537S and D538G mutations make ERα resistant to current endocrine therapy and therefore, it is important to develop a novel targeted therapy to address these clinical challenges by inhibiting ERα mutant proteins using next-generation ERα antagonist. Our long-term goal is to develop more effective and safer small molecule drugs that block constitutively active mutant ER for treating endocrine resistant metastatic breast cancer and have the potential to significantly improve current therapeutic targeting strategies. The short-term goal of this R21 application is to identify and develop specific drug-like probes and nominate preclinical candidates to target ER mutant protein by using a DNA-encoded chemical library (DEL) screening platform. The objective of this proposal will be evaluated by addressing the following specific aims: 1. We will perform DNA-encoded chemical libraries screen to identity small-molecule binders to the ligand-binding domain of mutant ERα protein. 2. We will validate and prioritize lead compounds by using biochemical and functional studies. Our central hypothesis is that small molecule antiestrogens with high affinity and specificity for ERα mutants can inhibit ERα mutant function in breast cancer. This research will lay the groundwork for more detailed follow up studies for future applications. After successful execution of this proposed research plan, we expect to have identified lead compounds for mutant ER and that can be used for future pre- clinical and clinical studies.