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.

概括 导致囊性纤维化跨膜电导调节因子 (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，影响全世界数万人。 Vertex, Inc.（美国马萨诸塞州剑桥）的药物发现工作已经产生了临床有效的药物 组合，将 CFTR 确立为可治疗的靶点。到目前为止，所有已成功测试的 疗法包括 Ivacaftor，它作为 CFTR 的“增强剂”而不是 CFTR 的激活剂，至少依赖于某些 CFTR 磷酸化状态的程度，受体内动态激素调节。在 此外，越来越多的证据表明 Ivacaftor 通过不依赖 ATP 的机制发挥作用， 意味着实现稳定 CFTR 开口的规范途径，即 ATP 驱动 Ivacaftor 不利用细胞内结合域的二聚化。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 未来将努力开发基于机制的疗法，以增强 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。第一个目标将使用我们的强大方法 已经开发出 CFTR 通道中特定位点的磷酸化状态由 短暂（<1 秒）的闪光。这将使我能够观察通道的内在磷酸化率， 以及在细胞环境中实时产生的功能结果。鉴于离子的磷酸调节 肺和心脏中的通道被很好地描述，并且在心血管疾病中经常存在缺陷，这种训练 预期的后续发现可能会直接带来其他离子通道的更多机会 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 核苷酸结合域 (NBD) 和 ATP（它们的调节目标）之间的结构。 NBD 是古老的领域 （数十亿年前）在整个生物学中被发现，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方法来检查 CFTR 通道中的可溶性 NBD 如何与其调节目标 ATP 结合的机制。可能的 这些共同努力的共同成果将是发表多篇高价值论文和 研究离子通道蛋白的现代技术的高级培训。此外，CFTR 的演变 使其能够作为离子通道磷酸化调节的模型（与许多其他临床应用相同） 肺和心脏中的相关通道）以及基于 ATP 的其他 ABC 转运蛋白的激活 在肺生理学中发挥重要作用。因此，本提案的执行将建立一个平台来询问 与其他膜蛋白的调节有关的同样重要的问题。作为一项训练练习，这 努力将为我提供更深入的技能，涵盖学术、科学传播和 严格、前沿的实验。

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.