Chemical Biology of CFTR Regulation

CFTR 调节的化学生物学

• 批准号: 10001337
• 负责人: Daniel T Infield
• 金额: $ 6.93万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-22 至 2021-07-21
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10001337
• 关键词: ATP-Binding Cassette Transporters; Address; Adenine; Affect; Affinity; Amino Acids; Area; Binding; Binding Sites; Biological; Biology; Cardiovascular Diseases; Chemicals; Chemistry; Chloride Channels; Clinical; Communication; Complex; Consensus; Cryoelectron Microscopy; Cyclic AMP-Dependent Protein Kinases; Cysteine; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Data; Dimerization; Disease; Drug Combinations; Engineering; Environment; Evolution; Exercise; Future; Goals; Health; Heart; Human; Individual; Interferometry; Ion Channel; Ion Channel Protein; Kinetics; Lead; Learning; Ligand Binding; Light; Lung; Mammalian Cell; Measures; Mediating; Membrane Proteins; Methods; Mind; Modeling; Modernization; Molecular; Mutate; Mutation; Nucleotides; Outcome; Output; Paper; Pharmacology; Phosphorylation; Physiological Processes; Physiology; Play; Positioning Attribute; Proteins; Publications; Regulation; Role; Route; Scholarship; Serine; Side; Site; Structure; Techniques; Testing; Therapeutic; Time; Training; Work; analog; base; biophysical techniques; biophysical tools; career; clinically relevant; drug discovery; experimental study; hormone regulation; in vivo; insight; interest; knock-down; milligram; millisecond; mutant; protein structure; receptor; recruit; structural biology; tryptophan analog; voltage clamp

SUMMARY Mutations that render the cystic fibrosis transmembrane conductance regulator (CFTR) defective in function lead to cystic fibrosis, a devastating multisystem disease affecting tens of thousands of people worldwide. Drug discovery efforts by Vertex, Inc. (Cambridge, Mass. USA) have yielded clinically efficacious drug combinations, establishing CFTR as a therapeutically accessible target. Thus far all of the successfully tested therapies include Ivacaftor, which as a “potentiator,” rather than an activator of CFTR relies at least to some degree on the phosphorylation state of CFTR, which is subject to dynamic hormonal regulation in vivo. In addition, accumulating evidence suggests that Ivacaftor works through an ATP-independent mechanism, meaning that the canonical route by which stable CFTR openings are achieved, namely ATP-driven dimerization of the intracellular binding domains, is not exploited by Ivacaftor. By aiming to better understand both phospho regulation and ATP binding in CFTR, the two aims of this proposal are expected to support future efforts to develop mechanism-based therapies that increase CFTR function. Two scientific aims in my proposal describe the means to achieve these goals. The first of these aims will use a powerful method we have developed whereby the phosphorylation state of a specific site in the CFTR channel is controlled by a brief (<1 second) flash of light. This will allow me to observe the intrinsic phosphorylation rates of the channel, and the functional consequence, in real time, in a cellular environment. Given that phosphoregulation of ion channels is well-described in the lung and heart, and often defective in cardiovascular disease, this training and the anticipated ensuing discoveries will likely lead directly to additional opportunities on other ion channel proteins with ties to human health. The second aim will examine the interaction chemistry that is utilized between nucleotide binding domains (NBD) and ATP, their regulatory target. NBDs are ancient domains (billions of years old) that are found throughout biology, thus advancing their mode of action will simultaneously impact multiple areas. I will use structural biology and advanced spectroscopic methods to examine the mechanism of how the soluble NBDs from the CFTR channel bind to their regulatory target, ATP. The likely common output from these combined efforts will be the publication of multiple high value papers and the advanced training in modern techniques for the study of ion channel proteins. Additionally, CFTR’s evolution allows it to serve as a model for both phospho-regulation of ion channels (in common with many other clinically relevant channels in the lung and heart) and for ATP-based activation of other ABC transporters which play important roles in lung physiology. Accordingly, execution of this proposal will establish a platform to ask similarly important questions relating to the regulation of other membrane proteins. As a training exercise, this endeavor will provide me with a deepened skillset spanning scholarship, scientific communication, and rigorous, cutting-edge experimentation.

总结 导致囊性纤维化跨膜传导调节因子（CFTR）功能缺陷的突变 导致囊性纤维化，这是一种影响全世界数万人的毁灭性多系统疾病。 Vertex，Inc.的药物发现工作（马萨诸塞州剑桥）美国）已经产生了临床有效的药物 组合，将CFTR确立为治疗上可及的靶标。到目前为止，所有成功测试的 治疗包括依伐卡托，其作为CFTR的"增效剂"而非活化剂至少依赖于一些细胞因子。 CFTR的磷酸化状态受体内动态激素调节。在 此外，越来越多的证据表明，依伐卡托通过ATP非依赖性机制发挥作用， 这意味着实现稳定CFTR开口的规范路线，即ATP驱动的 细胞内结合结构域的二聚化未被依伐卡托利用。为了更好地理解 CFTR中的磷酸调节和ATP结合，该提案的两个目标预计将支持 未来的努力，以开发机制为基础的疗法，增加CFTR功能。我的两个科学目标 提案描述了实现这些目标的手段。第一个目标将使用一种强大的方法， 已经开发出CFTR通道中特定位点的磷酸化状态由一种 短暂（<1秒）闪光。这将使我能够观察通道的内在磷酸化速率， 和功能性的结果，在真实的时间，在细胞环境中。考虑到离子的磷酸化调节 通道是很好地描述了在肺和心脏，往往有缺陷的心血管疾病，这种训练 预期的后续发现可能会直接导致其他离子通道的额外机会 蛋白质与人类健康的关系。第二个目标将检查相互作用化学， 核苷酸结合结构域（NBD）和ATP之间，它们的调节目标。NBD是古老的领域 （数十亿年前）在整个生物学中发现，因此推进它们的作用方式将同时 影响多个领域。我将使用结构生物学和先进的光谱学方法来检查 来自CFTR通道的可溶性NBD如何与其调节靶标ATP结合的机制。可能的 这些共同努力的共同成果将是出版多篇高价值论文， 离子通道蛋白研究的现代技术的高级培训。此外，CFTR的发展 使其能够作为离子通道磷酸化调节的模型（与临床上许多其他模型相同） 肺和心脏中的相关通道）和其他ABC转运蛋白的基于ATP的激活， 在肺生理学中的重要作用。因此，本建议的实施将建立一个平台， 与其他膜蛋白的调节有关的类似重要问题。作为一项训练， 努力将为我提供一个深化的技能跨越奖学金，科学交流， 严谨的前沿实验