Mechanisms of Permeation and Gating of Voltage-Sensing Domains

电压传感域的渗透和门控机制

• 批准号: 10521947
• 负责人: Francesco Tombola
• 金额: $ 47.04万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10521947
• 关键词: Address; Affect; Affinity; Alberta province; Albumins; Anti-Inflammatory Agents; Antineoplastic Agents; Binding; Binding Sites; Biological Assay; Biological Process; Brain Injuries; Breast Cancer Cell; Calcium Channel; Cell Proliferation; Cell physiology; Cells; Collaborations; Complement; Computing Methodologies; Consensus; Development; Disease; Docking; Drug Targeting; Electrophysiology (science); Evaluation; Family; Genetic; Goals; HL-60 Cells; Health; Human; Immune response; Ion Channel Gating; Ischemic Stroke; Knock-out; Lead; Ligand Binding; Ligands; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Methods; Modeling; Molecular; Neuroprotective Agents; Nox enzyme; Organic Synthesis; Peptides; Phagocytes; Pharmaceutical Preparations; Pharmacology; Play; Potassium Channel; Preparation; Production; Property; Proteins; Protons; Recovery; Reporting; Respiratory Burst; Role; Side; Sodium Channel; Specificity; Spinal cord injury; Structural Models; Structure; Study models; System; Toxin; Transmembrane Domain; Traumatic Brain Injury; Universities; Vestibule; analog; antagonist; anti-cancer; cancer cell; cancer type; cell motility; crosslink; design; dimer; extracellular; improved; infancy; inhibitor; live cell imaging; loss of function; member; migration; molecular dynamics; neuroinflammation; neutrophil; novel therapeutics; pH Homeostasis; rational design; response; scaffold; simulation; small molecule; tool; voltage

Project Summary The voltage-gated proton channel Hv1 plays important roles in numerous biological processes, including pH homeostasis and the immune response. Its activity has been found to worsen brain damage after ischemic stroke, to exacerbate the effect of traumatic brain injury and spinal cord injury, and to increase the metastatic potential of different types of cancer. The development of small-molecule modulators of Hv1 activity could lead to new anti-inflammatory agents and anticancer drugs. In addition, Hv1 modulators can provide useful pharmacological tools for studying the function of the channel in health and disease. Hv1 belongs to the large family of voltage-gated ion channels (VGICs). The majority of these proteins consist of four voltage-sensing domains (VSDs) surrounding a central pore domain. While many types of drugs bind the pore domain of VGICs, the number of organic molecules known to bind VSDs is limited. The Hv1 channel is made of only two VSDs and does not contain a pore domain, providing a simplified model for studying how ligands interact with VSDs. We have previously discovered small molecules that inhibit Hv1 activity by binding within the intracellular vestibule of the channel VSD in the open state (class I.1 ligands). Using a rational design approach that combines experimental and computational methods, we identified related compounds that are able to bind the channel also in the closed state (class I.2 ligands). Some of the new ligands display inhibitory properties that are superior to those of class I.1 compounds and provide a promising scaffold for further development of high-affinity Hv1 antagonists. However, little is known about how effective class I.2 ligands are at inhibiting Hv1- regulated cellular processes, such as ROS production by NOX enzymes, or how specifically they target the Hv1 VSD versus VSDs of other VGICs. In aim 1 of this project, we will apply our rational design approach to develop I.2 ligands with improved potency and corresponding negative controls. We will also use electrophysiological methods to investigate potential effects of Hv1 ligands on other members of the VGIC family. In aim 2, we will utilize a variety of live cell imaging assays on wild type and Hv1 knockout cells to examine how I.2 ligands inhibit NOX-mediated ROS production in phagocytes and how they affect proliferation and migration of cancer cells in a Hv1-dependent manner. The Hv1 channel contains a VSD-VSD interface unique among VGICs. As a result, ligands that bind such interface are expected to be more specific channel modulators than ligands that bind other transmembrane regions. The structure of the Hv1 dimer has yet to be determined, and alternative dimer models have been proposed by different groups with different VSD-VSD interfaces. In aim 3, we will use molecular dynamics simulations combined with multichemistry cross-linking mass spectrometry to probe the different models and derive a consensus dimer interface.

项目摘要 电压门控质子通道Hv1在许多生物过程中发挥重要作用，包括 PH值动态平衡和免疫反应。它的活性被发现在以下情况下会加剧大脑损伤 缺血性卒中，加重创伤性脑损伤和脊髓损伤的影响，并增加 不同类型癌症的转移潜能。化学发光小分子调制剂的研究进展 HV1的活性可能导致新的抗炎药和抗癌药物的出现。此外，Hv1 调节剂可以为研究通道在健康中的功能提供有用的药理学工具 和疾病。HV1属于电压门控离子通道(VGIC)大家族。大多数人 这些蛋白质由四个电压敏感结构域(VSD)组成，围绕着一个中心孔区。而当 许多类型的药物结合VGICs的孔域，即已知结合的有机分子的数量 VSD是有限的。Hv1通道仅由两个VSD构成并且不包含孔区， 为研究配体与VSD的相互作用提供了简化的模型。我们之前已经发现 通过结合在VSD通道的细胞内前庭而抑制Hv1活性的小分子 处于开放状态(I.1类配体)。使用合理的设计方法，将实验和 计算方法，我们确定了相关的化合物，能够结合通道也在 闭合状态(I.2类配体)。一些新的配体表现出更好的抑制性能 为进一步开发高亲和力化合物提供了一个有前途的支架 HV1拮抗剂。然而，关于I.2类配体在抑制Hv1-的有效性方面知之甚少。 受调控的细胞过程，如由NOX酶产生的ROS，或它们针对的具体程度 Hv1 VSD与其他VGIC的VSD。在本项目的目标1中，我们将应用我们的合理设计 开发效力更高的I.2配体和相应的阴性对照的方法。我们会 也使用电生理方法来研究Hv1配体对其他成员的潜在影响 VGIC家族的成员。在目标2中，我们将使用多种针对野生型和hv1的活细胞成像分析。 检测I.2配体如何抑制吞噬细胞中NOX介导的ROS的产生 它们如何以依赖于Hv1的方式影响癌细胞的增殖和迁移。Hv1频道 包含在VGIC中独一无二的VSD-VSD接口。因此，与这种界面结合的配体是 与结合其他跨膜区的配体相比，有望成为更特异的通道调节剂。 Hv1二聚体的结构尚未确定，另一种二聚体模型已经确定 由具有不同VSD-VSD接口的不同团体提出。在目标3中，我们将使用分子 动力学模拟与多元化学交联质谱学相结合探索 不同的模型，并衍生出一致的二聚体界面。