ABSTRACT About 1 in 8 women in the U.S. will develop invasive breast cancer during their lifetime. Human Epidermal growth factor Receptor-2 (HER2) is a clinically validated target that is upregulated in 25% of these cancers and is associated with poor disease prognosis. Current medications targeting HERs have several limitations because of their inadequate effectiveness and sensitivity to resistance by cancer cell mutations, causing treatment failure. HER receptor family requires an important dimerization step for activation that is essential for cancer cells to maintain growth and division. Inhibition of dimerization is currently clinically achieved with antibodies that cannot target mutated truncated receptor forms and cannot cross the blood-brain barrier, turning the brain into a sanctuary for cancer cells and leading to metastasis and relapse. Our lab discovered a small molecule through virtual screening; molecular modeling algorithms predict it will bind to the HER2 dimer interface surface, which would allosterically inhibit the receptor activation by preventing dimerization. This molecule was shown in cellular assays to inhibit SKBr3 cancer cell lines overexpressing HER2 in a selective and non-toxic manner. This is the first-in-class molecule to target a member of this receptor family using an allosteric inhibition mechanism. If advanced for clinical testing, it can work as a stand-alone therapy for patient populations that resist current medications or in combination therapies to decrease resistance, metastasis, and relapse. The overall objective of this proposal is to characterize and validate analogs of this inhibitor predicted by modeling to share a similar binding pose and the same or higher affinity to the HER2 dimer interface. We will validate their interaction with HER2 using cellular, biophysical, and biochemical assays. We will also study their effect on cancer cells' downstream targets expression and phosphorylation levels and test the top-performing lead compound plasma distribution properties and efficacy in HER2+ breast cancer in vivo models. The proposed study is innovative due to the new mechanism of inhibition proposed by modeling and indicated by the selective toxicity shown in cellular studies. Our long-term goal is to extend this research to related receptors in the same family (EGFR, HER3, and HER4) that are implicated in many solid tumors, some of which lack targeted therapies. The outcomes of this study will offer proof of concept to usher in a new class of anticancer agents targeting HER2 receptors. The project will support research experiences in cancer drug discovery and therapeutics to six PharmD, one graduate, and six undergraduate students in two REAP-eligible institutions.