Targeting the serine protease PCSK9 via covalent complementarity

描述（由申请人提供）：前蛋白转化酶枯草杆菌蛋白酶/kexin 9型（PCSK 9）是一种丝氨酸蛋白酶，可调节LDL胆固醇水平，进而调节动脉粥样硬化的发展。流行病学和遗传学研究提供了强有力的证据表明，PCSK 9突变与心血管结局相关。有趣的是，PCSK 9的催化活性不是PCSK 9/LDL-受体相互作用的直接需要。因此，这种蛋白质已成为开发治疗剂以阻断这种PCSK 9/LDL-R相互作用的令人兴奋的靶标。本研究拟在生化基础上评价PCSK 9催化活性与PCSK 9/LDL-R相互作用之间的关系。在目标1中，该研究将设计具有合理设计的活性位点修饰的“亲电敏感性”PCSK 9类似物， 允许小分子抑制剂与亲电子柄特异性和共价结合。将在体外评估ES-PCSK 9类似物的催化活性，沿着小分子实现特异性抑制的能力。将在细胞系统中引入ES-PCSK 9，并评估对LDL加工的影响。在目标2中，将评估特异性抑制剂的构效关系。本研究将检验抑制剂与PCSK 9活性位点结合将导致PCSK 9/LDLR相互作用变构抑制的假设。在目的3中，将评估细胞内和细胞外PCSK 9对LDL调节的作用。将通过将GOF和LOF突变引入ES-PCSK 9来评估具有临床意义的多态性的机制。将评价显性负效应和显性正效应的可能性。除了通过研究进行基础科学和转化研究的培训外，申请人还将通过审核加州大学旧金山弗朗西斯科提供的研究生课程进行合成化学和化学生物学技术的教学。

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.