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: 10406127
• 负责人: Anjaparavanda P Naren
• 金额: $ 44.03万
• 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/10406127
• 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.

抽象的 囊性纤维化（CF）是由CF的丧失或功能障碍引起的一种遗传遗传疾病 突变引起的跨膜电导调节剂（CFTR）通道活性。临床上，慢性肺 疾病是CF患者发病率和死亡率的主要原因。在2000多种导致疾病的 突变，ΔF508是最常见的突变，并且与严重的CF疾病形式相关。理想 与ΔF508相关的CF的治疗需要增加等离子体处ΔF508-CFTR蛋白的量 膜，增强了受损的通道门控性能并提高其稳定性。这个想法是 得到两种CFTR调节药物的批准ORKAMBI®（VX-809 + VX-770）和SYMDEKO®（VX-- 661 + VX-770），用于治疗纯合子ΔF508的CF患者，并通过具有三组合的试验（VX-445 + VX-- 661 + VX-770; VX-659 + VX-661 + VX-770）。应该注意的是，批准药物的临床益处是 这些CFTR校正器的谦虚和作用机制知之甚少。在这笔赠款中，我们计划 研究CFTR校正器如何促进ΔF508-CFTR的成熟并稳定突变体的机制 质膜处的蛋白质。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的大分子复合物的角度研究该受试者。假设 进行测试是：（i）CFTR校正器（例如VX-CFTR校正器）直接与ΔF508-CFTR结合以执行其救援 效果。高亲和力结合将产生更好的救援结果。 （ii）ΔF508-CFTR的不稳定性 质膜处的短半衰期）至少部分是由于其与结合相互作用的能力降低 伴侣，因此无法形成稳定的大分子复合物，从而导致其快速内在化 并针对降解。 （iii）CFTR校正器不仅有助于折叠ER中的ΔF508-CFTR以促进其促进 成熟，但也通过增强其与 结合伴侣并支持稳定的大分子复合物的形成。 （iv）通过隔离 在不同条件下并使用蛋白质组学，与校正器相关的 - 和ΔF508-CFTR的复合物， 我们可以确定在救援过程中重要的效果和途径。点击化学和照片亲和力 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校正器的作用机理，（ii） 识别含有ΔF508-CFTR的复合物中的新靶标，（iii）开发出更有效的药物来对抗CF，并且 （iv）了解因折叠和处理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。