Project Summary Drug overdoses now kill more Americans per year than vehicle crash and gun deaths combined. Overdose is a common outcome of opioid use disorder (OUD), which itself has been suspected to evolve from routine prescription treatment of pain with opioid medications. This public health crisis has created a need for the discovery of non-opioid medications to treat pain without eliciting rewarding effects that lead to addiction. New molecular targets for pain therapies offer the best route towards elusive non-opioid medications to treat certain kinds of pain, a fact that is recognized by the present FOA, for which this project is proposed. The ATP- sensitive inward rectifier potassium channel 8 (Kir6.1/KCNJ8) has been linked to the control of postoperative and cancer pain, but little else is known about this ion channel in the context of pain. Specifically, Kir6.1 openers/agonists may be novel analgesics, but drug-like, selective openers/agonists of any KATP family channel have never been identified. Kir6.1 is also considered an understudied protein in terms of its validation as a druggable target generally and is on the list of qualified drug targets for this FOA. We recently investigated the regulation by palmitoylation of Kir6 channels. Our studies identified a druggable site utilized by the palmitoylation mechanism, which provides an avenue to discover drug-like, small molecule opener/agonists of these channels. We proved the principle that we could find selective openers of Kir6.2 by targeting this site using in silico/virtual library screening (VLS) coupled with electrophysiological testing in engineered cell lines. Here, we propose to use the same approach to screen for selective, drug-like openers of Kir6.1 and establish preliminary structure-activity relationships (SAR) between channel opening and specific chemical determinants in the ligands. Specifically, we will build highly accurate homology models of alternative conformations of the Kir6.1/SUR2B complex and use ultra-large VLS to find the maximum number of diverse chemical entities that fit with the target pocket in its agonist, antagonist and unknown conformations (Aim 1). These conformations will be refined based on the observed activity of hit ligands. We will also develop three orthogonal assays to test hit compound identified by VLS: a) a thallium flux cellular primary screening assay, b) patch clamping of Kir6.1 and c) an LPS-stimulated, human astrocyte MCP-1/p-JNK elevation phenotypic assay (Aim 2). Finally, SAR will be derived for each of the three assays, and a meta-SAR across the three, which can serve as an advanced starting point for future drug discovery projects, will be inferred using cheminformatics methods (Deliverable). A specific 3D structural agonist site on KATP channels has not previously been identified, so this proposal is highly innovative.