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

CFTR校正器在拯救Delta F508-CFTR中的作用机制研究

• 批准号: 10454293
• 负责人: Anjaparavanda P Naren
• 金额: $ 42.37万
• 依托单位: CEDARS-SINAI MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10454293
• 关键词: ABCB1 gene; Address; Affect; Affinity; Alkynes; Alleles; Anabolism; Binding; Biological Models; Cell membrane; Cell surface; Chemistry; Chronic lung disease; Clinical; Clinical Trials; Complex; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Defect; Delta F508 mutation; Development; Diazomethane; Disease; Electron Transport Complex III; Endoplasmic Reticulum; Exhibits; Exposure to; Functional disorder; Genes; Grant; Half-Life; Health; Hereditary Disease; Impairment; Individual; Knowledge; Life; Lung diseases; Macromolecular Complexes; Mission; Molecular; Morbidity - disease rate; Mutation; Names; Outcome; Pathway interactions; Pharmaceutical Preparations; Phase; Photoaffinity Labels; Prevention; Process; Property; Proteins; Proteomics; Public Domains; Quality of life; Recycling; Signal Pathway; Structure; Study Subject; Temperature; Testing; United States National Institutes of Health; VX-770; VX-809; analog; cold temperature; combat; cystic fibrosis patients; disease-causing mutation; human disease; improved; mortality; mutant; new therapeutic target; novel; trafficking

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.

摘要 囊性纤维化（CF）是一种缩短寿命的遗传性疾病所造成的损失或功能障碍的CF 跨膜传导调节因子（CFTR）通道活性由突变引起。临床上，慢性肺 疾病是CF患者发病和死亡的主要原因。在2000多个致病 ΔF508是最常见的突变，与严重形式的CF疾病相关。理想 治疗与ΔF508相关的CF需要增加血浆中ΔF508-CFTR蛋白的量， 膜，增强受损的通道门控特性，并提高其稳定性。到这个概念 两种CFTR调节药物Orkambi®（VX-809 + VX-770）和Symdeko®（VX-809 + VX-770）获得批准， 661 + VX-770），治疗ΔF508纯合子CF患者，并通过三重组合试验（VX-445 + VX-770）， VX-661 + VX-770; VX-659 + VX-661 + VX-770）。值得注意的是，批准药物的临床益处是 这些CFTR校正剂的作用机制知之甚少。在这一补助金中，我们计划 研究CFTR校正剂促进ΔF508-CFTR成熟和稳定突变体的机制 蛋白质在细胞膜上。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的大分子复合物的角度对该课题进行了研究。假设， 将被测试的是：（i）CFTR校正器（例如，VX-CFTR校正剂）直接与ΔF508-CFTR结合以发挥其拯救作用 方面的影响.高亲和力结合将产生更好的拯救结果。(ii)ΔF508-CFTR的不稳定性（ 短半衰期）至少部分是由于其与结合相互作用的能力降低。 因此不能形成稳定的大分子复合物，这导致其快速内化 并成为降解的目标。(iii)CFTR校正剂不仅有助于在ER中折叠ΔF508-CFTR，以促进其表达。 成熟，而且还通过增强其与蛋白质的相互作用来稳定质膜上的突变蛋白质。 结合伴侣并促进稳定的大分子复合物的形成。和（iv）通过分离 校正剂相关的和ΔF508-CFTR的复合物在不同条件下和使用蛋白质组学， 我们就能确定在拯救过程中重要的效应器和途径。点击化学和光亲和 标记将用于研究各种CF中的相互作用和大分子复合物的形成 模型系统这项研究将帮助我们（i）更好地了解CFTR校正剂的作用机制，（ii） 在含有ΔF508-CFTR的复合物中鉴定新的靶点，（iii）开发更有效的药物来对抗CF，以及 (iv)了解其他人类疾病的分子基础，这些疾病是由于折叠和加工不足而导致的。 蛋白质（例如，P-glycoprotein），并找到治疗这些疾病的方法。