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)：三磷酸腺苷结合盒(ABC)转运体囊性纤维化跨膜电导调节器(CFTR)的功能丧失，这是一种氯和HCO3-通道，导致囊性纤维化(CF)。Cf肺部和肠道疾病是最严重的疾病表现。最常见的突变是苯丙氨酸508(F508del)的缺失，会扰乱CFTR的处理并降低通道开通率。在霍乱毒素引起的腹泻中，CFTR活性的增加是水和电解质损失的基础。对于这两种疾病，都需要更好的治疗方法，使CFTR通道功能正常化。CFTR和其他ABC蛋白同时具有ATPase和腺苷酸激酶活性。传统的cftr功能范式认为，通道的开启和关闭(“门控”)与ATPase的活性有关。目前尚不清楚腺苷酸激酶活性是否参与体内CFTR的功能，以及该活性是否是治疗CFTR相关疾病的有意义的靶点。在初步研究中，我们有两个中肯的发现。1)我们发现了一个cftr突变(Q1291F)，该突变使腺苷酸激酶活性丧失，但对ATPase依赖的门控没有显著影响。降低人原代呼吸道上皮细胞的氯离子通道活性。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--增加了F508del CFTR的通道活动。这项应用的目的是建立在这些初步数据的基础上，以确定体内腺苷酸激酶依赖的CFTR门控的贡献，并提供腺苷酸激酶抑制剂可能是F508del CFTR临床有用增强剂的概念的证据。中心假说是疾病相关器官、呼吸道和肠道中正常的cftr功能依赖于它的腺苷酸激酶活性。 作为结构缺陷的结果，Ap5A通过抑制腺苷酸激酶来增强F508del CFTR通道的活性。在目标1中，我们将使用原代呼吸道上皮细胞，检测Q1291F CFTR对HCO3分泌和呼吸道表面液体(ASL)pH的影响，这两个因素在CF肺部疾病的发生发展中都起着关键作用。我们还将研究1)转基因表达Q1291F cftr是否挽救了cftr-/-小鼠致死的肠道表型，以及2)该突变是否减少了霍乱毒素诱导的肠液损失。在目标2中，我们将使用生化和电生理方法研究Ap5A如何与F508del CFTR相互作用来增强通道活性。这些研究有望为CFR和CFTR依赖型腹泻带来新的治疗方法。这项拟议的研究具有创新性，因为它解决了CFTR门控的一个尚未被广泛研究的机制，即腺苷酸激酶活性，并试图改变目前CFTR在体内如何发挥作用的范式。此外，它建立在一个意想不到的发现之上，即Ap5A增强了F508del CFTR通道的活性。此外，拟议的研究与NIH的使命相关，该使命是寻求关于生命系统的性质和行为的基本知识，并应用这些知识来增强健康、延长寿命、减少疾病和残疾。