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:
  • 金额:
    $ 38.13万
  • 依托单位:
  • 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

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.
三磷酸腺苷结合盒(ABC)转运体的功能丧失囊性纤维化跨膜电导 调节剂(CFTR)是一种氯离子和HCO3离子通道,可引起囊性纤维化(CF)。Cf肺和肠道疾病是 最严重的疾病表现。最常见的突变是苯丙氨酸508的缺失 (F508del),会中断CFTR处理并降低通道开通率。CFTR活性增加 霍乱毒素引起的腹泻中的水分和电解质损失。对于这两种疾病,都需要 使CFTR通道功能正常化的更好的治疗方法。Cftr和其他ABC蛋白既有ATPase又有 腺苷酸激酶活性。CFTR函数的传统范式一直是开放和封闭 通道的(“门控”)与ATPase活动相联系。目前尚不清楚腺苷酸激酶的活性 在体内对CFTR功能的贡献,以及这种活动是否是治疗CFTR相关的有意义的靶点 疾病。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)抑制腺苷酸激酶活性,但对ATPase依赖的门控无明显影响。它 降低原代人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-通道增加形成鲜明对比 F508del CFTR的活性。本应用程序的目标是在这些初步数据的基础上确定 腺苷酸激酶依赖的CFTR门在体内的作用,并提供了一种概念验证 与腺苷酸激酶活性中心相互作用的化合物可能是F508del的临床有用的增强剂 CFTR。中心假设是与疾病相关的器官、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增强了 F508del通过与残基Q1291相互作用激活CFTR通道。在目标1中,我们将使用主气道 并检测Q1291F CFTR对HCO3分泌和呼吸道表面液体(ASL)pH的影响。 二者在慢性阻塞性肺疾病的发生发展中均起着关键作用。我们还将调查1) Q1291F cftr的表达挽救了cftr-/-小鼠的肠道表型,2)突变减少 霍乱毒素引起的肠道液体丢失。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 预计将导致新的治疗方法,以治疗CF和CFTR依赖的腹泻。拟议的研究 是创新的,因为它解决了CFTR门控、腺苷酸激酶活性和 寻求改变目前CFTR在体内如何发挥作用的范式。此外,它还建立在意想不到的 发现Ap5A增强F508del CFTR通道活性。此外,拟议的研究是 与美国国立卫生研究院寻求生命系统的性质和行为的基本知识的使命有关 应用这些知识来增进健康、延长寿命、减少疾病和残疾。

项目成果

期刊论文数量(1)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
An elusive adenylate cyclase complicit in cholera is exposed.
一种与霍乱同谋的难以捉摸的腺苷酸环化酶被暴露。
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Christoph Oskar Randak其他文献

Christoph Oskar Randak的其他文献

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{{ truncateString('Christoph Oskar Randak', 18)}}的其他基金

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
  • 财政年份:
    2017
  • 资助金额:
    $ 38.13万
  • 项目类别:
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
  • 财政年份:
    2015
  • 资助金额:
    $ 38.13万
  • 项目类别:
Mechanisms and Binding-Sites in Adenylate Kinase-Dependent CFTR Gating
腺苷酸激酶依赖性 CFTR 门控的机制和结合位点
  • 批准号:
    8115794
  • 财政年份:
    2009
  • 资助金额:
    $ 38.13万
  • 项目类别:
Mechanisms and Binding-Sites in Adenylate Kinase-Dependent CFTR Gating
腺苷酸激酶依赖性 CFTR 门控的机制和结合位点
  • 批准号:
    8517797
  • 财政年份:
    2009
  • 资助金额:
    $ 38.13万
  • 项目类别:
Mechanisms and Binding-Sites in Adenylate Kinase-Dependent CFTR Gating
腺苷酸激酶依赖性 CFTR 门控的机制和结合位点
  • 批准号:
    7915409
  • 财政年份:
    2009
  • 资助金额:
    $ 38.13万
  • 项目类别:
Mechanisms and Binding-Sites in Adenylate Kinase-Dependent CFTR Gating
腺苷酸激酶依赖性 CFTR 门控的机制和结合位点
  • 批准号:
    8308495
  • 财政年份:
    2009
  • 资助金额:
    $ 38.13万
  • 项目类别:
Mechanisms and Binding-Sites in Adenylate Kinase-Dependent CFTR Gating
腺苷酸激酶依赖性 CFTR 门控的机制和结合位点
  • 批准号:
    7713688
  • 财政年份:
    2009
  • 资助金额:
    $ 38.13万
  • 项目类别:

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