PROJECT SUMMARY Ovarian cancer is among the five leading causes of cancer death in the US with estimated 20,000 new cases and more than 13,000 deaths in 2023. High-grade serous ovarian cancer (HGSOC) is the most common and deadly subtype and accounts for ~75% of ovarian cancer deaths. Amplification and overexpression of CCNE1, the gene encoding cyclin E1, leads to hyperactivation of the cognate kinase CDK2 and occurs in ~50% of HGSOC patients. Dysregulated CCNE1 expression is also frequently observed in aggressive subtypes of endometrial cancer and triple-negative breast cancer. In ovarian cancer. CCNE1 amplification is associated with poor prognosis and resistance to chemotherapy, and most patients with advanced HGSOC (>80%) experience recurrence and ultimately succumb to their disease. The paucity of therapeutic options for CCNE1-driven tumors makes the development of novel drugs that counteract this aberration a critical unmet need. CDK2 is a validated drug target, and studies by many laboratories in academia and the pharmaceutical industry have shown that targeting CDK2 is a viable and perhaps ideal approach to combat cancers characterized by CCNE1 amplification. However, conventional ATP-site (type I) inhibitors have largely failed in the clinic due to insufficient selectivity, particularly off target inhibition of the closely related and common essential kinase CDK1. We have previously identified an allosteric site unique to CDK2 and demonstrated that small molecule ligands occupying this site stabilize a CDK2 conformational state that is less favorable for interaction with cyclins. While such monovalent allosteric (type III) ligands lack the high binding affinity required to effectively disrupt the interaction of CDK2 with cyclin E1, our recent structural and computational studies provide strong evidence that joining an allosteric ligand with a high-affinity ATP-site (type I) inhibitor presents a new means to suppress the hyperactivation of CDK2 caused by elevated cyclin E1 levels. The overall objective of this project is to develop first-in-class bifunctional allosteric inhibitors that stabilize CDK2 in a conformational state incompatible with cyclin E1 binding. We hypothesize that such inhibitors potently and selectively disrupt the CDK2-cyclin E1 complex, thereby counteracting the oncogenic activity of elevated CCNE1 expression. The rationale underlying this research is that it will provide general insights into the feasibility of bivalent allosteric CDK2 inhibitors in the context of CCNE1-driven cancers and build a strong scientific framework whereby new therapeutic strategies can be developed.