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: 9135633
• 负责人: Christoph Oskar Randak
• 金额: $ 17.39万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-20 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9135633
• 关键词: ATP phosphohydrolase; ATP-Binding Cassette Transporters; Address; Adenosine; Adenosine Diphosphate; Adult; Affect; Anions; Area; Behavior; Bicarbonates; Binding; Binding Sites; Biochemical; Cells; Child; Cholera Toxin; Coupled; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Data; Deletion Mutation; Development; Diarrhea; Diphosphates; Disasters; Disease; Disease Outbreaks; Electrolytes; Employee Strikes; Enzymes; Epithelial; Epithelial Cells; Goals; Health; Human; Hydrolysis; Intestinal Diseases; Intestines; Ions; Knowledge; Lead; Life; Liquid substance; Mission; Molecular; Mus; Mutation; Nature; Nucleotides; Organ; Organism; P(1),P(5)-di(adenosine-5' -)pentaphosphate; Pathogenesis; Pharmaceutical Preparations; Phenotype; Phenylalanine; Physiological; Play; Process; Proteins; Pulmonary Cystic Fibrosis; Reaction; Research; Role; Side; Structural defect; Testing; Time; Transgenic Organisms; Transmembrane Domain; United States; Water; Work; adenylate kinase; airway epithelium; airway surface liquid; base; disability; in vivo; innovation; inorganic phosphate; kinase inhibitor; loss of function; mortality; mutant; novel strategies; protein function; small molecule; therapeutic target

 DESCRIPTION (provided by applicant): 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 an adenylate kinase inhibitor 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 adenylate kinase inhibition. 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) transgenic expression of Q1291F CFTR rescues the lethal 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 a not widely studied 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.

 描述（由申请人提供）：ATP结合盒（ABC）转运蛋白囊性纤维化跨膜传导调节因子（CFTR）（一种Cl-和HCO 3-通道）功能丧失导致囊性纤维化（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加工和降低通道开放的速率。CFTR活性增加是霍乱毒素诱导的腹泻中水和电解质损失的基础对于这两种疾病，需要更好的治疗方法来使CFTR通道功能正常化。CFTR和其他ABC蛋白具有ATP酶和腺苷酸激酶活性。CFTR功能的传统范例是通道的打开和关闭（“门控”）与ATP酶活性偶联。目前尚不清楚腺苷酸激酶活性是否有助于体内CFTR功能，以及这种活性是否是治疗CFTR相关疾病的有意义的靶点。在初步研究中，我们有两个相关的发现。1)我们鉴定了一个CFTR突变（Q1291 F），该突变消除了腺苷酸激酶活性，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F508 del CFTR的通道活性。本申请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，并提供腺苷酸激酶抑制剂可能是F508 del CFTR的临床有用的增效剂的概念证明。核心假设是CFTR在疾病相关器官、气道和肠道中的正常功能依赖于其腺苷酸激酶活性 并且-作为结构缺陷的结果-Ap 5A通过腺苷酸激酶抑制增强F508 del CFTR通道活性。在目的1中，我们将使用原代气道上皮细胞并检查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。我们还将研究1）Q1291 F CFTR的转基因表达是否挽救CFTR-/-小鼠的致死肠表型和2）突变是否减少霍乱毒素诱导的肠液损失。在目标2中，我们将研究Ap 5A如何与F508 del CFTR相互作用，以使用生物化学和电生理方法增强通道活性。这些研究有望为CF和CFTR依赖性哮喘带来新的治疗方法。拟议的研究是创新的，因为它解决了一个未被广泛研究的CFTR门控机制，腺苷酸激酶活性，并寻求改变CFTR在体内如何发挥作用的当前模式。此外，它建立在Ap 5A增强F508 del CFTR通道活性的意外发现之上。此外，拟议的研究与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，以及这些知识的应用，以增强健康，延长寿命，减少疾病和残疾。