Targeting the serine protease PCSK9 via covalent complementarity

描述(申请人提供)：原蛋白转换酶枯草杆菌/可信9型(PCSK9)是一种丝氨酸蛋白酶，调节低密度脂蛋白胆固醇水平，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PCSK9的突变与心血管结果有关。有趣的是，PCSK9的催化活性并不是PCSK9/低密度脂蛋白受体相互作用直接需要的。因此，该蛋白已成为阻止PCSK9/LDL-R相互作用的治疗药物开发的一个令人兴奋的靶点。本研究建议在生化基础上评估PCSK9催化活性与PCSK9/LDL-R相互作用的关系。在目标1中，这项研究将通过合理设计的活性部位修饰来设计“亲电敏感”的PCSK9类似物，这将 允许小分子抑制剂与亲电手柄的特定和共价结合。ES-PCSK9类似物的催化活性以及小分子实现特定抑制的能力将在体外进行评估。ES-PCSK9将被引入蜂窝系统，并将评估其对低密度脂蛋白加工的影响。在目标2中，将评估特定抑制剂的构效关系。这项研究将检验这样的假设，即抑制剂与PCSK9活性部位的结合将导致PCSK9/LDLR相互作用的变构抑制。在目标3中，将评估细胞内和细胞外PCSK9对低密度脂蛋白调节的作用。通过将GOF和LOF突变引入ES-PCSK9，将评估具有临床意义的多态的机制。将评估显性负面影响和显性正面影响的可能性。除了通过这项研究进行基础科学和翻译研究方面的培训外，申请人还将通过审计加州大学旧金山分校提供的研究生课程，进行合成化学和化学生物学技术的教学。 公共卫生相关性：在西方国家，动脉粥样硬化是导致大多数心脏病的潜在疾病实体。PCSK9是最近发现的一种蛋白酶，它在调节低密度脂蛋白胆固醇方面具有重要的生化作用，低密度脂蛋白胆固醇是动脉粥样硬化发展的主要危险因素。这项建议开发工程PCSK9类似物的选择性抑制剂，以进一步确定PCSK9/低密度脂蛋白受体治疗抑制的性质和可能性。

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. PUBLIC HEALTH RELEVANCE: Atherosclerosis is the underlying disease entity causing the majority of heart disease in Western countries. PCSK9 is a recently identified protease which has a major biochemical role in modulating LDL cholesterol, a major risk factor in the development of atherosclerosis. This proposal develops selective inhibitors of engineered PCSK9 analogs to further define the nature and possibility for therapeutic inhibition of the PCSK9/LDL receptor.