Targeting the serine protease PCSK9 via covalent complementarity

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DESCRIPTION (provided by applicant): Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease which regulates LDL cholesterol levels and, by extension, the development of atherosclerosis. Epidemiologic and genetic studies have provided strong evidence that mutations in PCSK9 are related to cardiovascular outcomes. Interestingly, the catalytic activity of the PCSK9 is not directly required for the PCSK9/LDL-receptor interaction. As such, this protein has become an exciting target for development of therapeutics to block this PCSK9/LDL-R interaction. This study proposes to evaluate the relationship between PCSK9 catalytic activity and the PCSK9/LDL-R interaction on a biochemical basis. In Aim 1, the study will engineer "electrophile-sensitive" PCSK9 analogs with rationally designed active site modifications that will allow for specific and covalent binding of small molecule inhibitors with electrophilic handles. The catalytic activity of the ES-PCSK9 analogs, along with the ability of the small molecules to achieve specific inhibition, will be assessed in vitro. The ES-PCSK9 will be introduced in a cellular system, and the effects on LDL processing will be assessed. In Aim 2, the structure activity relationship of the specific inhibitors will be assessed. The study will test the hypothess that binding of inhibitor to the PCSK9 active site will cause allosteric inhibition of the PCSK9/LDLR interaction. In Aim 3, the role of intracellular and extracelullar PCSK9 to LDL regulation will be evaluated. The mechanisms of clinically significant polymorphisms will be assessed by introducing GOF and LOF mutations into ES-PCSK9. The possibility of dominant negative and dominant positive effects will be evaluated. In addition to the training in basic science and translational research through the conduct of the study, the applicant will undertake didactic instruction in synthetic chemistry and chemical biology techniques through auditing graduate courses offered at the University of California, San Francisco.