Chemical Biology to Modulate PCSK9 and Treat Atherosclerosis

家长助学金项目摘要/摘要 血清低密度脂蛋白(LDL)会导致动脉粥样硬化性心脏病。作为低密度脂蛋白受体(LDLR) 肝脏清除血液中的低密度脂蛋白，上调肝脏低密度脂蛋白受体可减少低密度脂蛋白和心血管事件。这个 自裂解酶PCSK9(前蛋白转换酶枯草杆菌/可信9型)是一个被证实的治疗靶点；它 伴随LDLR降解溶酶体，下调其功能。抗PCSK9抗体降低 低密度脂蛋白和改善临床结果，但成本和给药要求说明需要替代品。 以肝脏为靶点的siRNA也能有效降低低密度脂蛋白，但与耐受性良好的基因变体不同的是，它去除了所有 PCSK9从牢房中释放，引发了安全担忧。黄连9号对肝脏S作用的机制基础 LDLR很好地理解了，但它在这个经典途径之外的功能还不太清楚。因此，总体来说， 我们建议的目标是开发生化工具来机械地剖析PCSK9的生物学，从而 1)预测当前抗PCSK9疗法的长期效果；2)为 新的PCSK9-靶向策略。为此，我们已经确定了三种有希望的先导化合物 特异性靶向细胞内PCSK9。这些化合物既能抑制PCSK9的自身蛋白分解，又能 上调LDLR的表达。在目标1中，我们将对这些化合物进行化学优化，确认其作用机理 并在体内模型中验证其对PCSK9和LDLR的作用。这将是 建立新的化学探针，可用于研究细胞内鲜为人知的PCSK9的功能， 以及作为替代小分子疗法上调低密度脂蛋白受体的起点。我们 还发现，包括硫酸乙酰肝素蛋白多糖(HSPGs)在内的其他因子与PCSK9相互作用 以调节其向溶酶体的运输，这表明在这一途径中有更多的药物靶点。在……里面 目的2，我们将评估热休克蛋白G作为PCSK9内吞运输所需的介体的贡献 LDLR下调，在体外和在人类中都是如此。这些结果将阐明肝素是不是 已经批准的治疗方法可以改变用途，以抑制PCSK9的功能。在目标3中，我们将确定和 通过无偏见的全基因组CRISPR验证PCSK9内吞运输的未知调节因子 干扰屏。这些结果将有助于从机械上确定PCSK9是如何被运输到溶酶体的， 回答为什么PCSK9只影响某些组织，并提供潜在的新治疗靶点 预计将具有类似于PCSK9本身的治疗窗口。总体而言，我们预计我们的研究将 提供有关PCSK9长期抑制的安全性的关键信息，为新的治疗方法奠定基础， 并确定针对动脉粥样硬化的新治疗靶点。

PARENT GRANT PROJECT SUMMARY/ABSTRACT Serum low-density lipoprotein (LDL) causes atherosclerotic heart disease. As the LDL receptor (LDLR) on the liver clears LDL from the blood, upregulating hepatic LDLR reduces both LDL and cardiovascular events. The self-cleaving protease PCSK9 (proprotein convertase subtilisin/kexin type 9) is a validated therapeutic target; it chaperones the LDLR for lysosomal degradation, downregulating its function. Antibodies against PCSK9 lower LDL and improve clinical outcomes, but cost and administration requirements illustrate a need for alternatives. Liver-targeted siRNA also robustly lowers LDL, but unlike the well-tolerated genetic variants, it removes all PCSK9 from the cell, raising safety concerns. Though the mechanistic basis for PCSK9's effect on the hepatic LDLR is well understand, its functions outside this canonical pathway are less clear. Therefore, the overall goal of our proposal is to develop biochemical tools to mechanistically dissect the biology of PCSK9, so as to 1) anticipate the long-term effects of current anti-PCSK9 therapies and 2) develop the proofs-of-principle for novel PCSK9-targeting strategies. To this end, we have identified three promising lead compounds that specifically target intracellular PCSK9. These compounds both inhibit PCSK9 self-proteolysis and also upregulate LDLR expression. In Aim 1, we will chemically optimize these compounds, confirm the mechanistic basis of their effects, and validate their function on PCSK9 and the LDLR in an in vivo model. This will establish novel chemical probes which can be used to study the poorly known function of intracellular PCSK9, as well as serve as the starting point for an alternative small molecule therapeutic to upregulate the LDLR. We have also found that additional factors, including heparan sulfate proteoglycans (HSPGs), interact with PCSK9 to modulate its trafficking to the lysosome, suggesting that there are additional drug targets in this pathway. In Aim 2, we will evaluate the contribution of HSPGs as mediators of the PCSK9 endocytic trafficking required for LDLR downregulation, both in vitro and in the human. These results will elucidate whether heparins, which are already approved therapies, could be repurposed to inhibit PCSK9 function. In Aim 3, we will identify and validate as yet unknown regulators of PCSK9 endocytic trafficking via an unbiased, genome-wide CRISPR interference screen. These results will help mechanistically define how PCSK9 is trafficked to the lysosome, provide answers to why PCSK9 only affects certain tissues, and offer potential novel therapeutic targets which would be anticipated to have similar therapeutic windows to PCSK9 itself. Overall, we expect our study to provide key information on the safety of long-term PCSK9 inhibition, lay the groundwork for novel treatments, and identify new therapeutic targets against atherosclerosis.