PROJECT SUMMARY/ABSTRACT The self-cleaving protease PCSK9 induces the lysosomal degradation of the hepatic low-density lipoprotein receptor (LDLR). Thus, PCSK9 raises serum LDL and promotes atherosclerotic heart disease. Human genetics show that secreted PCSK9 is dispensable, making PCSK9 an important drug target. Both therapeutic anti-PCSK9 antibodies and liver-specific anti-PCSK9 siRNA impressively lower LDL and improve cardiovascular outcomes, even when added on top of statins. Yet despite this clinical success, problems remain. First, the role of PCSK9 beyond downregulating the hepatic LDLR remains unclear, and so there are gaps in the knowledge of the site effects from or other indications for the current available therapies. Second, currently approved therapies are not orally available, expensive, and poorly covered by insurance (antibodies), or phenocopy genetic models that associate with liver disease (siRNA). In this proposal, we target a novel mechanism for PCSK9 inhibition: the disruption of PCSK9 processing. PCSK9 is a self-cleaving protease, and this auto-proteolysis is required for its secretion and full effect on the LDLR. However, because PCSK9’s cleavage is both intramolecular and terminal, no other group has discovered how to disrupt it. Our group, however, has solved this problem, and we have discovered a series of molecules that allosterically modulate PCSK9 proteolysis to disrupt PCSK9 function. Importantly, this mechanism phenocopies the well-tolerated human PCSK9 variants who have no adverse phenotypes, only lower LDL and cardiovascular protection. In the R61 phase of this proposal, we will pursue a medicinal chemistry campaign to identify, validate, and screen compounds for activity. We will generate a diversity library to establish novel intellectual property, confirm target engagement with PCSK9, and improve the efficacy, potency, and pharmacologic properties of our compounds. In the R33 phase, we will establish in vivo efficacy and pharmacologically validate a developmental candidate to identify a lead compound for IND-enabling studies. We anticipate that our proposal will have a high impact on cardiovascular disease by enabling the development of an oral small molecule inhibitor of PCSK9 that truly phenocopies the cardioprotective genetic variants found in humans.