Targeting Scap to Lower ApoB-Containing Lipoproteins (ApoBCL) Production

富含甘油三酯的ApoBCL(含ApoB的脂蛋白)水平升高是主要的 接受他汀类药物治疗的患者冠心病(CHD)残余风险的组成部分 和/或PCSK9抑制剂。为了减少ApoBCL风险部分，我们需要更深入地了解 控制ApoBCL生产的机器。这一机制由两种膜蛋白组成， SCAP和Sterol调节元件结合蛋白(SREBPs)。SCAP是一种嵌入在细胞内的蛋白质 内质网(ER)膜由8个跨膜螺旋组成。SCAP与以下物质形成络合物 SREBPs，也通过两个跨膜螺旋与内质网膜结合。SREBP包含 控制脂肪酸、甘油三酯和胆固醇合成的转录因子结构域，它们形成 ApoBCL的脂类成分。SREBPS的激活需要由SCAP将它们运送到高尔基，在那里有两个 蛋白水解酶释放它们的转录因子结构域，现在可以进入细胞核以激活靶基因。 当内质网中的胆固醇上升并与SCAP结合时，它会将SREBPs困在内质网中，从而阻止蛋白质降解 卵裂和核进入。理解SCAP的开关样分子机制及其机制的主要障碍 它控制SREBPs和ApoBCL的产生是因为缺乏一种可溶的类似胆固醇的化合物 特异性地结合和抑制Scap. 这一建议是基于我们实验室最近取得的一项突破，该突破涉及到开发一种新型的高 吞吐量和快速筛选协议，该协议已经确定了第一个与特定结合的小分子 SCAP的胆固醇结合部位并阻断SREBPs的激活。SCAP包含两个大循环(Loop1和Loop1 7)当内质网中的胆固醇较低时，延伸到内质网管腔的环相互结合。当ER 胆固醇上升，它与Loop1结合，导致Loop1与Loop7解离，捕获SCAP/SREBP复合体 并阻断所有SREBP靶基因的转录激活。在目标1中，我们概述了以下研究 提高我们最近发现的模拟胆固醇的SCAP抑制剂的效力，并将使用这些抑制剂 了解Loop1如何与Loop7分离。这些抑制剂也将被用作稳定剂，以使 SCAP的结构测定，这将在原子水平上阐明胆固醇的结合机制。在……里面 目的2，我们概述了我们的方法来优化我们的各种片剂的体内药代动力学特性 抑制剂，然后我们将探讨它们对小鼠肝脏中sCAP和SREBP靶基因的抑制作用 在不同的代谢条件下。我们之前对转基因小鼠的研究表明， 抑制肝脏中的SCAP可阻断SREBPs，并显著减少脂肪酸、甘油三酯和 胆固醇，显著降低这些模型和高脂肪喂养的野生型小鼠的载脂蛋白BCL的产生 饮食和喂食高碳水化合物饮食的仓鼠。如果这些概念验证研究成功，我们的抑制剂 可能会导致公司开发降低血浆载脂蛋白和预防冠心病的新药。

Elevated plasma levels of triglyceride-rich ApoBCLs (ApoB-Containing Lipoproteins) constitute a major component of the residual risk for coronary heart disease (CHD) in patients who have been treated with statins and/or PCSK9 inhibitors. To mitigate the ApoBCL risk component, we need a deeper understanding of the machinery that controls the production of ApoBCLs. This machinery is comprised of two membrane proteins, Scap and Sterol Regulatory Element-Binding Proteins (SREBPs). Scap is a protein embedded in the endoplasmic reticulum (ER) membrane through eight transmembrane helices. Scap forms complexes with SREBPs, which are also bound to the ER membrane through two transmembrane helices. SREBPs contain transcription factor domains that control the synthesis of fatty acids, triglycerides, and cholesterol, which form the lipid component of ApoBCLs. Activation of SREBPs requires their transport by Scap to the Golgi where two proteases release their transcription factor domain that can now enter the nucleus for target gene activation. When cholesterol in the ER rises and binds to Scap, it traps SREBPs in the ER, thus preventing proteolytic cleavage and nuclear entry. A major hurdle in understanding Scap’s switch-like molecular mechanism and how it controls SREBPs and ApoBCL production is the lack of a soluble cholesterol-mimetic compound that specifically binds and inhibits Scap. This proposal is based on a recent breakthrough in our laboratory involving the development of a novel high- throughput and rapid screening protocol, which has identified the first small molecule that binds specifically to Scap’s cholesterol-binding site and blocks activation of SREBPs. Scap contains two large loops (Loop1 and Loop7) that extend into the lumen of the ER bind each other when cholesterol in the ER is low. When ER cholesterol rises, it binds to Loop1, causing Loop1 to dissociate from Loop7, trapping the Scap/SREBP complex in the ER and blocking the transcriptional activation of all SREBP target genes. In Aim 1, we outline studies to improve the potency of our recently discovered cholesterol-mimetic Scap inhibitor and will use these inhibitors to understand how Loop1 dissociates from Loop7. The inhibitors will also be used as stabilizing agents to enable structural determination of Scap, which will elucidate the cholesterol binding mechanism at an atomic level. In Aim 2, we outline our approach to optimizing the in vivo pharmacokinetic properties of our various Scap inhibitors, after which we will explore their effects in inhibiting Scap and SREBP target genes in livers of mice under different metabolic conditions. Our previous studies involving genetically altered mice revealed that inhibition of Scap in the liver blocks SREBPs and markedly reduces synthesis of fatty acids, triglycerides, and cholesterol, dramatically lowering ApoBCL production in these models and also in wild-type mice fed a high fat diet and in hamsters fed a high carbohydrate diet. If these proof-of-concept studies are successful, our inhibitors might lead companies to develop new drugs to reduce plasma ApoBCLs and prevent coronary heart disease.