Investigating of the Mechanisms of Action of CFTR Correctors in RescuingDelta F508-CFTR

摘要 囊性纤维化是由于囊性纤维化的缺失或功能障碍引起的一种寿命缩短的遗传性疾病。 突变引起的跨膜电导调节因子(CFTR)通道活性。临床上，慢性肺 疾病是导致CF患者发病和死亡的主要原因。在2000种致病病毒中 突变，ΔF508是最常见的突变，与一种严重的CF病有关。理想 治疗ΔF508相关性CF需要增加血浆中ΔF508-cftr蛋白的含量 膜，加强受损的通道门控性能，并提高其稳定性。这个概念是 由两种CFTR调节药物Orkambi®(VX-809 VX-770)和Symdeko®(VX- 661VX-770)，用于治疗ΔF508纯合子的CF患者，并通过三联试验(VX-445 VX- 661 VX-770；VX-659 VX-661 VX-770)。值得注意的是，经批准的药物的临床益处是 人们对这些《儿童权利公约》校正者的作用机制知之甚少。在这笔赠款中，我们计划 Cftr校正子促进ΔF508-cftr成熟和稳定突变株的机制研究 质膜上的蛋白质。我们将重点介绍VX-CFTR校正器(VX-661、VX-809、VX-445和VX-659) 并从含CFTR的大分子络合物的角度进行研究。假设 要测试的是：(I)cftr校正器(例如，VX-cftr校正器)直接绑定到ΔF508-cftr以施救 效果。高亲和力的结合将产生更好的救援结果。(Ii)ΔF508-CFTR的不稳定性(具有 短的半衰期)至少部分是由于其与结合的相互作用能力降低 因此不能形成稳定的大分子复合体，从而导致其快速内化 并被定为降级目标。(3)cftr校正器不仅帮助在ER中折叠ΔF508-cftr，以促进其 成熟，但也稳定在质膜上的突变蛋白，通过加强它与 结合伴侣，促进形成稳定的大分子复合体。以及(Iv)通过隔离 不同条件下的ΔF508-cftr复合体的蛋白质组学研究 我们可以确定在救援过程中重要的效应器和路径。点击化学和光亲和力 标记法将被用来研究不同碳纤维之间的相互作用和大分子络合物的形成 模型系统。这项研究将帮助我们(I)更好地理解cftr校正器的作用机制，(Ii) 在ΔF508-cftr-含配合物中确定新的靶点，(Iii)开发更有效的药物来对抗cf，以及 (4)了解因折叠和加工不足而导致的其他人类疾病的分子基础 蛋白质(例如，P-糖蛋白)，并找到治疗这些疾病的方法。

ABSTRACT Cystic fibrosis (CF) is a life-shortening inherited disease caused by the loss or dysfunction of the CF transmembrane conductance regulator (CFTR) channel activity resulting from mutations. Clinically, chronic lung disease is the main cause of morbidity and mortality for CF patients. Among the 2000+ disease-causing mutations, ΔF508 is the most common mutation and associates with a severe form of CF disease. The ideal therapy for CF associated with ΔF508 requires increasing the quantity of ΔF508-CFTR protein at the plasma membrane, potentiating the impaired channel gating properties, and improving its stability. This notion was supported by the approval of two CFTR modulating drugs, Orkambi® (VX-809 + VX-770) and Symdeko® (VX- 661 + VX-770), to treat CF patients homozygous for ΔF508, and by trials with triple combinations (VX-445 + VX- 661 + VX-770; VX-659 + VX-661 + VX-770). It is to be noted that the clinical benefits of approved drugs are modest and the mechanisms of action of these CFTR correctors are poorly understood. In this grant, we plan to study the mechanisms of how CFTR correctors promote the maturation of ΔF508-CFTR and stabilize the mutant protein at the plasma membrane. We will focus on VX- CFTR correctors (VX-661, VX-809, VX-445, and VX-659) and study the subject from the perspective of CFTR-containing macromolecular complexes. The hypotheses to be tested are: (i) CFTR correctors (e.g., VX- CFTR correctors) bind directly to ΔF508-CFTR to exert their rescue effects. A high-affinity binding will produce a better rescue outcome. (ii) The instability of ΔF508-CFTR (with a short half-life) at the plasma membrane is, at least in part, due to its reduced ability to interact with binding partners and consequently cannot form a stable macromolecular complex, which leads to its rapid internalization and targeted for degradation. (iii) CFTR correctors not only help fold ΔF508-CFTR in the ER to promote its maturation, but also stabilize the mutant protein at the plasma membrane by enhancing its interaction with binding partners and facilitating the formation of a stable macromolecular complex. And (iv) by isolating the corrector-associated- and ΔF508-CFTR-containing complexes under different conditions and using proteomics, we can identify effectors and pathways important in the rescuing process. Click chemistry and photo-affinity labeling will be used to investigate the interactions and macromolecular complexes formation in various CF model systems. This study will help us (i) better understand the mechanisms of action of CFTR correctors, (ii) identify novel targets in ΔF508-CFTR-containing complexes, (iii) develop more potent drugs to combat CF, and (iv) understand the molecular basis of other human diseases resulting from insufficiently folded and processed proteins (e.g., P-glycoprotein) and find ways to treat these diseases.