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.

概括 使囊性纤维化跨膜电导调节剂（CFTR）功能有缺陷的突变 导致囊性纤维化，这是一种毁灭性的多系统疾病，影响了全球成千上万的人。 Vertex，Inc。（美国剑桥）的药物发现工作已产生临床高效的药物 组合，将CFTR建立为可访问的目标。所有成功测试的一切 疗法包括ivacaftor，它是“增强剂”，而不是CFTR的激活剂至少依赖于某些 CFTR的磷酸化状态的程度，该度受体内动态激素调节的影响。 此外，积累的证据表明，ivacaftor通过非ATP独立的机制起作用， 这意味着实现稳定CFTR开口的规范路线，即ATP驱动 细胞内结合结构域的二聚化并未由iVacaftor探索。通过更好地理解 CFTR中的磷酸化调节和ATP结合都预计该提案的两个目标将支持 未来开发基于机制的疗法的努力，以提高CFTR功能。我的两个科学目标 建议描述实现这些目标的手段。这些目标中的第一个将使用强大的方法 开发了CFTR通道中特定位点的磷酸化状态的发展 简短（<1秒）光的闪光。这将使我能够观察到通道的固有磷酸化速率， 以及在细胞环境中实时实时的功能后果。鉴于离子的磷酸调节 通道在肺和心脏中描述得很好，在心血管疾病中通常有缺陷，这种训练 预计随之而来的发现可能会直接导致其他离子频道上的其他机会 与人类健康有联系的蛋白质。第二个目的将检查使用的相互作用化学 在核苷酸结合结构域（NBD）和ATP之间，它们的调节目标。 NBD是古代领域 （数十亿年）都在整个生物学中都发现，因此提高其行动方式将只是 影响多个领域。我将使用结构生物学和高级光谱方法来检查 CFTR通道的实体NBD如何与其调节目标ATP结合的机制。可能 这些综合努力的共同产出将是发表多个高价值论文， 现代技术研究离子通道蛋白的高级培训。此外，CFTR的进化 允许它充当离子通道的两种磷酸调节的模型（与许多其他临床上有共同点 肺和心脏中的相关渠道以及基于ATP的其他ABC转运蛋白的激活 肺部生理学的重要作用。彼此之间，该提案的执行将建立一个平台来询问 同样重要的问题，与其他膜蛋白的调节有关。作为训练练习， 努力将为我提供跨越科学，科学沟通和 严格的尖端实验。