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

基本信息

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
项目类别：
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

项目摘要

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) 是一种因囊性纤维化丧失或功能障碍而导致的缩短寿命的遗传性疾病临床上，慢性肺突变导致跨膜电导调节器（CFTR）通道活性。在 2000 多个致病因素中，该病是 CF 患者发病和死亡的主要原因。突变，ΔF508 是最常见的突变，与严重的 CF 疾病相关。治疗与 ΔF508 相关的 CF 需要增加血浆中 ΔF508-CFTR 蛋白的数量膜，增强受损的通道门控特性，并提高其稳定性。两种 CFTR 调节药物 Orkambi® (VX-809 + VX-770) 和 Symdeko® (VX- 661 + VX-770），用于治疗 ΔF508 纯合子 CF 患者，并通过三重组合（VX-445 + 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。我们对这些 CFTR 校正器的作用机制知之甚少。研究CFTR校正子促进ΔF508-CFTR成熟并稳定突变体的机制我们将重点关注 VX-CFTR 校正因子（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课题的假设。被测试的是：（i）CFTR校正器（例如，VX-CFTR校正器）直接结合到ΔF508-CFTR以发挥其救援作用 (ii) ΔF508-CFTR 的不稳定性（具有一定的稳定性）。质膜上的半衰期短，至少部分是由于其与结合相互作用的能力降低伙伴，因此不能形成稳定的大分子复合物，导致其快速内化 (iii) CFTR 校正剂不仅有助于在 ER 中折叠 ΔF508-CFTR，以促进其降解。成熟，而且还通过增强突变蛋白与质膜的相互作用来稳定突变蛋白结合配偶体并促进稳定的大分子复合物的形成，并且（iv）通过分离在不同条件下并使用蛋白质组学，包含校正子相关和 ΔF508-CFTR 的复合物，我们可以识别在拯救过程中重要的效应器和途径。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这项研究将帮助我们 (i) 更好地了解 CFTR 校正器的作用机制，(ii) 确定包含 ΔF508-CFTR 的复合物中的新靶点，(iii) 开发更有效的药物来对抗 CF，以及 (iv) 了解因折叠和加工不充分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。