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Mechanisms of Permeation and Gating of Voltage-Sensing Domains

电压传感域的渗透和门控机制

基本信息

批准号：
10672274
负责人：
Francesco Tombola
金额：
$ 49.88万
依托单位：
UNIVERSITY OF CALIFORNIA-IRVINE
依托单位国家：
美国
项目类别：
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/10672274
关键词：
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 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 Proliferating 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 pharmacologic rational design response scaffold simulation small molecule spinal cord and brain injury tool voltage

项目摘要

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Hv 1在许多生物过程中起着重要作用，包括 pH稳态和免疫反应。它的活性被发现会加重脑损伤，缺血性中风，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力。小分子药物的研究进展 hv 1的活性可能导致新的抗炎药和抗癌药。此外，Hv 1 调节剂可以提供有用的药理学工具，用于研究通道在健康中的功能和疾病Hv 1属于电压门控离子通道（VGIC）大家族。大多数这些蛋白质由围绕中心孔结构域的四个电压敏感结构域（VSD）组成。而许多类型的药物结合VGIC的孔域，已知结合的有机分子的数量 VSD是有限的。Hv 1通道仅由两个VSD组成，不含孔结构域，为研究配体如何与VSD相互作用提供了一个简化的模型。我们之前发现通过结合在VSD通道的细胞内前庭内抑制Hv 1活性的小分子在开放状态下（I.1类配体）。使用结合实验和计算方法，我们确定了相关的化合物，能够结合通道，也在封闭状态（I.2类配体）。一些新的配体显示出上级的抑制特性 I. 1类化合物的那些，并为进一步开发高亲和力的 Hv 1拮抗剂。然而，关于I.2类配体在抑制Hv 1-Hv 2方面的有效性知之甚少。受调控的细胞过程，如NOX酶的ROS产生，或它们如何特异性靶向 Hv 1 VSD与其他VGIC的VSD。在本项目的目标1中，我们将应用我们的理性设计开发具有改进的效力的I.2配体和相应的阴性对照的方法。我们将我还使用电生理学方法研究Hv 1配体对其他成员的潜在影响 VGIC家族的成员。在目标2中，我们将利用野生型和Hv 1的多种活细胞成像测定，敲除细胞以检查I.2配体如何抑制吞噬细胞中NOX介导的ROS产生，它们如何以Hv 1依赖的方式影响癌细胞的增殖和迁移。Hv 1频道包含VGIC中唯一的VSD-VSD接口。因此，结合这种界面的配体是预期是比结合其它跨膜区的配体更特异的通道调节剂。 Hv 1二聚体的结构尚未确定，替代的二聚体模型已经被证实。由不同的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结合多化学交联质谱来探测不同的模型，并得出一个共识二聚体接口。