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Probing adenylate kinase-dependent CFTR gating in vivo and as therapeutic target

体内探索腺苷酸激酶依赖性 CFTR 门控并作为治疗靶点

基本信息

批准号：
10004609
负责人：
Christoph Oskar Randak
金额：
$ 38.13万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-08-03 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10004609
关键词：
ATP phosphohydrolase ATP-Binding Cassette Transporters Address Adenosine Adenosine Diphosphate Adult Affect Amino Acids Anions Area Behavior Bicarbonates Binding Binding Sites Biochemical Child Cholera Toxin Clinical Consequentialism Coupled Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator Data Delta F508 mutation Development Diarrhea Diphosphates Disasters Disease Disease Outbreaks Electrolytes Electrophysiology (science)Enzymes Epithelial Cells Goals Health Human Hydrolysis Inbred CFTR Mice Intestinal Diseases Intestines Ions Knowledge Lead Life Liquid substance Medical Mission Molecular Mutation Nature Nucleotides Organ Organism Pathogenesis Pathologic Permeability Pharmaceutical Preparations Pharmacology Phenotype Phenylalanine Physiological Play Proteins Pulmonary Cystic Fibrosis Reaction Research Role Side Site Structural defect Structure Testing Time Transgenic Organisms Transmembrane Domain United States United States National Institutes of Health Water Work adenylate kinase airway epithelium airway surface liquid base design disability drug development in vivo innovation inorganic phosphate kinase inhibitor loss of function mortality mutant novel strategies protein function public health relevance therapeutic target

项目摘要

Loss of function of the ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR), a Cl- and HCO3- channel, causes cystic fibrosis (CF). CF lung and intestinal disease are the most consequential disease manifestations. The most common mutation, deletion of phenylalanine 508 (F508del), disrupts CFTR processing and reduces the rate of channel opening. Increased CFTR activity underlies water and electrolyte losses in cholera toxin-induced diarrhea. For both diseases there is a need for better treatments that normalize CFTR channel function. CFTR and other ABC proteins have both ATPase and adenylate kinase activity. The traditional paradigm of CFTR function has been that opening and closing ("gating") of the channel is coupled to ATPase activity. It is not known whether adenylate kinase activity contributes to CFTR function in vivo, and whether this activity is a meaningful target to treat CFTR-related diseases. In preliminary studies we made two pertinent discoveries. 1) We identified a CFTR mutation (Q1291F) that abolished adenylate kinase activity but had no significant effect on ATPase-dependent gating. It reduced Cl- channel activity in primary human airway epithelia. 2) We found that the adenylate kinase inhibitor Ap5A (P1,P5-di(adenosine-5') pentaphosphate) - in striking contrast to wild-type CFTR - increased channel activity of F508del CFTR. The objective of this application is to build on these preliminary data to ascertain a contribution of adenylate kinase-dependent CFTR gating in vivo and to provide a proof-of-concept that a compound interacting with the adenylate kinase active center might be a clinically useful potentiator of F508del CFTR. The central hypothesis is that normal CFTR function in disease-relevant organs, airways and intestine, relies on its adenylate kinase activity and that - as a consequence of a structural defect - Ap5A potentiates F508del CFTR channel activity through interactions with residue Q1291. In aim 1 we will use primary airway epithelia and examine the effects of Q1291F CFTR on HCO3- secretion and airway surface liquid (ASL) pH, which both play a pivotal role in the development of CF lung disease. We will also investigate whether 1) expression of Q1291F CFTR rescues the intestinal phenotype of CFTR-/- mice and 2) the mutation reduces cholera toxin-induced intestinal fluid losses. In aim 2 we will investigate how Ap5A interacts with F508del CFTR to potentiate channel activity using biochemical and electrophysiological approaches. These studies are expected to lead to new treatment approaches for CF and CFTR-dependent diarrheas. The proposed research is innovative because it addresses an understudied mechanism of CFTR gating, adenylate kinase activity, and seeks to shift the current paradigm of how CFTR functions in vivo. Furthermore, it builds on the unanticipated discovery that Ap5A potentiates F508del CFTR channel activity. Furthermore, the proposed research is relevant to NIH's mission to seek fundamental knowledge about the nature and behavior of living systems and the application of that knowledge to enhance health, lengthen life, and reduce illness and disability.

ATP结合盒（ABC）转运蛋白囊性纤维化跨膜电导功能丧失 C1-和HCO 3-通道调节因子（CFTR）引起囊性纤维化（CF）。CF肺和肠道疾病是最重要的疾病表现。最常见的突变，苯丙氨酸508缺失（F508 del），破坏CFTR加工并降低通道打开速率。CFTR活性增加是霍乱毒素引起的腹泻中水和电解质流失的基础。对于这两种疾病，使CFTR通道功能正常化的更好的治疗。CFTR和其他ABC蛋白质具有ATP酶和ATP酶。腺苷酸激酶活性。CFTR功能的传统范式是打开和关闭通道的门控（“门控”）与ATP酶活性偶联。腺苷酸激酶活性是否有助于CFTR在体内的功能，以及这种活性是否是治疗CFTR相关的疾病在初步研究中，我们有两个相关的发现。1)我们发现了一个CFTR突变（Q1291 F），其消除腺苷酸激酶活性，但对ATP酶依赖性门控没有显著影响。它降低了原代人气道上皮细胞中Cl-通道的活性。2)我们发现腺苷酸激酶抑制剂 Ap 5A（P1，P5-二（腺苷-5 '）五磷酸）-与野生型CFTR形成鲜明对比-增加通道 F508 del CFTR的活性。本申请的目的是在这些初步数据的基础上确定腺苷酸激酶依赖性CFTR门控在体内的贡献，并提供一个概念验证，与腺苷酸激酶活性中心相互作用的化合物可能是F508 del的临床有用的增强剂 CFTR。中心假设是，正常的CFTR功能在疾病相关器官，气道和肠道，依赖于其腺苷酸激酶活性，并且-作为结构缺陷的结果-Ap 5A增强通过与残基Q1291相互作用的F508 del CFTR通道活性。在目标1中，我们将使用主气道并检查Q1291 F CFTR对HCO 3分泌和气道表面液体（ASL）pH的影响，这两者在CF肺病的发展中起关键作用。我们还将调查1） Q1291 F CFTR的表达拯救了CFTR-/-小鼠的肠表型，并且2）突变降低了CFTR-/-小鼠的肠表型。霍乱毒素引起的肠液流失。在目的2中，我们将研究Ap 5A如何与F508 del CFTR相互作用使用生物化学和电生理学方法来增强通道活性。这些研究有望为CF和CFTR依赖性哮喘带来新的治疗方法。拟议研究是创新的，因为它解决了CFTR门控，腺苷酸激酶活性，旨在改变目前CFTR在体内如何发挥作用的范式。此外，它建立在意想不到的发现Ap 5A增强F508 del CFTR通道活性。此外，拟议的研究是与NIH的使命有关，即寻求有关生命系统的性质和行为的基本知识，应用这些知识来增进健康、延长寿命、减少疾病和残疾。