Chemical Biology of CFTR Regulation

CFTR 调节的化学生物学

• 批准号: 10425532
• 负责人: Daniel T Infield
• 金额: $ 0.25万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-15 至 2021-07-21
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10425532
• 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.

摘要 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，这是一种毁灭性的多系统疾病，影响着全球数万人。 Vertex，Inc.(马萨诸塞州剑桥市)的药物发现工作美国)生产出了临床上有效的药物 联合，将CFTR确立为治疗上可获得的靶点。到目前为止，所有成功测试的 治疗方法包括异烟肼，它作为一种“增强剂”，而不是CFTR的激活剂，至少对一些人来说是依赖的。 在体内受到激素动态调节的CFTR的磷酸化状态的程度。在……里面 此外，越来越多的证据表明，异烟肼通过一种不依赖于ATP的机制发挥作用， 这意味着实现稳定的CFTR开口的规范路线，即由ATP驱动 细胞内结合结构域的二聚化，不是Ivacaftor所利用的。通过旨在更好地理解 在CFTR中，磷酸调节和ATP结合，这项提案的两个目标预计将支持 未来的努力是开发基于机制的疗法，以增强CFTR的功能。我的两个科学目标 提案描述了实现这些目标的方法。这些目标中的第一个将使用一种强大的方法 已经发展出CFTR通道中特定位点的磷酸化状态由一种 短暂(&lt；1秒)的闪光。这将使我能够观察到通道的固有磷酸化速率， 以及在蜂窝环境中实时的功能结果。考虑到离子的磷调节 经络在肺脏和心脏都有很好的描述，而且经常在心血管疾病中有缺陷，这就是训练 而预期中的后续发现可能会直接在其他离子通道上带来更多机会 与人类健康有关的蛋白质。第二个目标将检查所利用的相互作用化学 在核苷酸结合域(NBD)和它们的调节靶标ATP之间。NBD是古老的领域 (数十亿年前)在整个生物学中被发现，因此它们的行动模式将同时 影响多个领域。我将使用结构生物学和先进的光谱方法来研究 Cftr通道中的可溶性NBD如何与其调节靶标ATP结合的机制。可能的 这些联合努力的共同成果将是出版多篇高价值论文和 研究离子通道蛋白的现代技术方面的高级培训。此外，CFTR的演变 允许它作为离子通道的磷酸化调节的模型(与临床上的许多其他 肺和心脏中的相关通道)，以及基于ATP的其他ABC转运体的激活 在肺生理学中的重要作用。因此，执行这项提案将建立一个平台，以询问 与其他膜蛋白调控有关的同样重要的问题。作为一次训练演习，这 奋进将为我提供更深层次的技能，包括学术、科学交流和 严谨、尖端的实验。

项目成果

Real-time observation of functional specialization among phosphorylation sites in CFTR.

• DOI: 10.1085/jgp.202213216
• 发表时间: 2023-04-03
• 期刊: JOURNAL OF GENERAL PHYSIOLOGY
• 影响因子: 3.8
• 作者: Infield, Daniel T.;Schene, Miranda E.;Fazan, Frederico S.;Galles, Grace D.;Galpin, Jason D.;Ahern, Christopher A.
• 通讯作者: Ahern, Christopher A.