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.