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HV1在包括 pH稳态和免疫反应。发现它的活动在缺血性中风，加剧脑外伤和脊髓损伤的影响，并增加不同类型癌症的转移潜力。开发小分子调节器 HV1活性可能导致新的抗炎药和抗癌药。另外，HV1 调节器可以提供有用的药理工具来研究渠道在健康中的功能和疾病。 HV1属于大型电压门控离子通道（VGIC）。大多数这些蛋白质由四个围绕中心孔结构域的电压感应域（VSD）组成。尽管许多类型的药物结合了VGIC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组成，不包含孔域，提供了一个简化的模型，用于研究配体如何与VSD相互作用。我们以前已经发现通过在通道VSD的细胞内前庭结合来抑制HV1活性的小分子处于开放状态（I.1级配体）。使用合理的设计方法，将实验和计算方法，我们确定了能够在封闭状态（I.2级配体）。一些新的配体显示出优质的抑制特性对于I.1级化合物的人，并为进一步发展高亲和力提供了有希望的脚手架 HV1拮抗剂。但是，关于i.2级配体在抑制HV1-的有效有效的效果知之甚少受调节的细胞过程，例如NOX酶产生ROS，或它们针对性其他VGIC的HV1 VSD与VSD。在该项目的目标1中，我们将采用理性设计开发具有提高效力和相应阴性对照的I.2配体的方法。我们将还使用电生理方法研究HV1配体对其他成员的潜在影响 VGIC家族。在AIM 2中，我们将在野生型和HV1上利用各种实时细胞成像测定敲除细胞检查i.2配体如何抑制NOX介导的吞噬细胞中的ROS产生和它们如何以HV1依赖性方式影响癌细胞的增殖和迁移。 HV1通道包含VGIC中独特的VSD-VSD接口。结果，结合这种界面的配体是预计比结合其他跨膜区域的配体更特定的通道调节剂。 HV1二聚体的结构尚未确定，替代二聚体模型已经由具有不同VSD-VSD接口的不同组提出的。在AIM 3中，我们将使用分子动力学仿真与多芯学交联质谱法结合了探测不同的模型并得出共识二聚体接口。