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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调节器 (CFTR) 是一种 Cl- 和 HCO3- 通道，会导致囊性纤维化 (CF)。 CF 肺部和肠道疾病是最重要的疾病表现。最常见的突变，苯丙氨酸缺失508 (F508del)，扰乱 CFTR 处理并降低通道打开率。 CFTR 活动增加是霍乱毒素引起的腹泻中水和电解质损失的基础。对于这两种疾病都需要使 CFTR 通道功能正常化的更好治疗方法。 CFTR 和其他 ABC 蛋白都具有 ATPase 和腺苷酸激酶活性。 CFTR 函数的传统范式是开闭通道的（“门控”）与 ATP 酶活性耦合。尚不清楚腺苷酸激酶活性是否有助于体内 CFTR 功能，以及该活性是否是治疗 CFTR 相关的有意义的靶标疾病。在初步研究中，我们有两个相关的发现。 1) 我们发现了 CFTR 突变 (Q1291F) 消除了腺苷酸激酶活性，但对 ATP 酶依赖性门控没有显着影响。它人原代气道上皮细胞中氯离子通道活性降低。 2）我们发现腺苷酸激酶抑制剂 Ap5A（P1，P5-二（腺苷-5'）五磷酸） - 与野生型 CFTR 形成鲜明对比 - 通道增加 F508del CFTR 的活性。该应用程序的目的是基于这些初步数据来确定腺苷酸激酶依赖性 CFTR 门控在体内的贡献，并提供了一个概念证明与腺苷酸激酶活性中心相互作用的化合物可能是临床上有用的 F508del 增强剂 CFTR。中心假设是正常的 CFTR 在疾病相关器官、气道和肠道中发挥功能，依赖于其腺苷酸激酶活性，并且由于结构缺陷，Ap5A 会增强 F508del CFTR 通过与残基 Q1291 相互作用来通道活性。在目标 1 中，我们将使用主要气道上皮细胞并检查 Q1291F CFTR 对 HCO3- 分泌和气道表面液体 (ASL) pH 的影响，这两者在 CF 肺病的发展中都发挥着关键作用。我们还将调查是否 1) Q1291F CFTR 的表达挽救了 CFTR-/- 小鼠的肠道表型，2) 该突变减少了霍乱毒素引起的肠道液体流失。在目标 2 中，我们将研究 Ap5A 如何与 F508del CFTR 相互作用使用生化和电生理学方法增强通道活性。这些研究是预计将为 CF 和 CFTR 依赖性腹泻带来新的治疗方法。拟议的研究具有创新性，因为它解决了尚未研究的 CFTR 门控机制、腺苷酸激酶活性和旨在改变 CFTR 在体内如何发挥作用的当前范式。此外，它建立在意想不到的基础上发现 Ap5A 增强 F508del CFTR 通道活性。此外，拟议的研究是与 NIH 寻求有关生命系统的性质和行为的基础知识的使命相关应用这些知识来增强健康、延长寿命、减少疾病和残疾。