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

• 批准号: 9383695
• 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
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-03 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9383695
• 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; Development; Diarrhea; Diphosphates; Disasters; Disease; Disease Outbreaks; Electrolytes; Electrophysiology (science); Employee Strikes; Enzymes; Epithelial; Epithelial Cells; Goals; Health; Human; Hydrolysis; Intestinal Diseases; Intestines; Ions; Knowledge; Lead; Life; Liquid substance; Medical; Mission; Molecular; Mus; 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; 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; 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; Development; Diarrhea; Diphosphates; Disasters; Disease; Disease Outbreaks; Electrolytes; Electrophysiology (science); Employee Strikes; Enzymes; Epithelial; Epithelial Cells; Goals; Health; Human; Hydrolysis; Intestinal Diseases; Intestines; Ions; Knowledge; Lead; Life; Liquid substance; Medical; Mission; Molecular; Mus; 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; 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

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的功能丧失 CL-和HCO3通道调节剂（CFTR）引起囊性纤维化（CF）。 CF肺和肠道疾病是 最后果的疾病表现。最常见的突变是苯丙氨酸508的缺失 （F508DEL），破坏CFTR处理并降低通道打开率。 CFTR活性增加 霍乱毒素诱导的腹泻中的水和电解质损失是基础。对于两种疾病，都需要 更好的治疗方法将CFTR通道函数归一化。 CFTR和其他ABC蛋白具有ATPase和 腺苷酸激酶活性。 CFTR功能的传统范式是开放和关闭 （通道的“门控”）与ATPase活性耦合。尚不清楚腺苷酸激酶活性是否 在体内有助于CFTR功能，以及该活动是否是治疗CFTR相关的有意义的目标 疾病。在初步研究中，我们做出了两个相关发现。 1）我们确定了CFTR突变 （Q1291F）消除了腺苷酸激酶活性，但对ATPase依赖性门的影响没有显着影响。它 原发性人类气道上皮中的CL通道活性降低。 2）我们发现腺苷酸激酶抑制剂 AP5A（P1，P5-DI（腺苷-5'）五磷酸） - 与野生型CFTR形成鲜明对比 - 增加的通道 F508DEL CFTR的活动。该应用程序的目的是建立在这些初步数据的基础上，以确定 腺苷酸激酶依赖性CFTR门控在体内的贡献，并提供概念验证 与腺苷酸激酶活性中心相互作用的化合物可能是F508DEL的临床上有用的增强剂 CFTR。中心假设是，与疾病相关的器官，气道和肠道中的正常CFTR功能， 依靠其腺苷酸激酶活性，并且由于结构缺陷而导致AP5A增强 F508DEL CFTR通道活性通过与残基Q1291相互作用。在AIM 1中，我们将使用主要气道 上皮并检查Q1291F CFTR对HCO3分泌和气道表面液体（ASL）pH的影响， 这两者在CF肺部疾病的发展中起着关键作用。我们还将调查1）是否 Q1291F CFTR的表达营救了CFTR - / - 小鼠的肠表型，2）突变减少 霍乱毒素诱导的肠液损失。在AIM 2中，我们将研究AP5A如何与F508DEL CFTR相互作用 使用生化和电生理方法增强通道活性。这些研究是 预计将导致CF和CFTR依赖性腹泻的新治疗方法。拟议的研究 之所以创新，是因为它解决了CFTR门控，腺苷酸激酶活性和 试图改变当前CFTR在体内功能的范式。此外，它建立在意外的 发现AP5A增强F508DEL CFTR通道活动。此外，拟议的研究是 与NIH有关的使命涉及有关生活系统的性质和行为的基本知识和 这些知识的应用来增强健康，延长寿命并减少疾病和残疾。