This application is to test a novel strategy for reversing the effects of heritable mutations in the von HippelLindau protein that confer an increased risk for cancer. The proposed studies are highly relevant to 5U54CA272686-02, Cancer prevention-interception targeted agent discovery program at Fox Chase Cancer Center (PI Clapper, Margie L). The project originates from previous work in the Karanicolas laboratory (Project 1), which focused on using computational, structure-based methods to develop compounds that bind and stabilize the folded (active) conformation of tumor suppressors bearing missense mutations. Germline or somatic mutations reducing the expression or function of the von Hippel-Lindau (VHL) tumor suppressor are the most common genetic features of clear cell renal cell carcinoma (ccRCC), with many of these mutations being missense. Beyond ccRCC, individuals with germline VHL mutations are at elevated risk for numerous other cancers, including most commonly pancreatic cancer, hemangiomas and hemangioblastomas of the central nervous system and retina, and cysts in the kidney, pancreas, and other tissues. Evidence in the scientific literature suggests that pathogenic missense mutations impact VHL function by: 1) Increasing the ratio of partially unfolded or misfolded (dysfunctional) protein to functional protein; or 2) disrupting specific contacts between VHL and essential protein partners. Using computational modeling approaches, we identified a specific, novel binding pocket on the VHL protein surface, and generated small molecules compounds predicted to bind this site and stabilize the ratio of folded to unfolded VHL, boosting protein function. Preliminary experiments using NMR established the ability of a lead compound, CP4.29, to bind to the predicted site. We also showed that this compound was active in stabilizing VHL in ccRCC cell lines bearing 3 discrete somatic VHL mutations (P86S, S65W, and I180N). Critically, the compound also restored a critical VHL function - formation of an E3 ligase complex that causes the degradation of hypoxia-inducible transcription factors (HIFs, which include HIF-1 and HIF-2). We have also generated a number of structurally modified variants of CP4.29, with the goal of potentially increasing potency and solubility. The goal of this supplement is to specifically determine whether these compounds are effective in reversing the dysfunction induced by germline mutations in VHL that occur in discrete protein domains.