Discovery of novel openers of the understudied human drug target Kir6.1

项目摘要 药物过量每年杀死的美国人比车祸和枪支死亡的总和还要多。过量 是阿片类药物使用障碍（OUD）的常见结果，OUD本身被怀疑是从常规的 用阿片类药物治疗疼痛。这场公共卫生危机造成了对 发现非阿片类药物治疗疼痛，而不会引起导致成瘾的奖励效应。新 疼痛治疗的分子靶点为难以捉摸的非阿片类药物治疗某些疾病提供了最佳途径。 这是目前FOA所承认的一个事实，因此提出了这个项目。ATP- 敏感性内向整流钾通道8（Kir6.1/KCNJ 8）与术后 和癌症疼痛，但很少有人知道这个离子通道在疼痛的背景下。特别是Kir6.1 开放剂/激动剂可能是新型镇痛药，但任何KATP家族的药物样、选择性开放剂/激动剂 渠道从未被发现。Kir6.1在其验证方面也被认为是一种研究不足的蛋白质 作为一个可药物化的目标，并在名单上的合格的药物目标，为这个FOA。我们最近调查了 棕榈酰化对Kir 6通道的调节。我们的研究确定了一个可药物化的网站所利用的 棕榈酰化机制，这为发现药物样小分子开放剂/激动剂提供了途径 这些渠道。我们证明了我们可以通过靶向该位点来找到Kir6.2的选择性打开剂的原理 在工程细胞系中使用计算机/虚拟库筛选（VLS）结合电生理学测试。 在这里，我们建议使用相同的方法来筛选选择性的，药物样的Kir6.1开放剂，并建立 通道开放与特定化学物质之间的初步构效关系（SAR） 配体中的决定簇。具体来说，我们将建立高度准确的同源性模型的替代 Kir6.1/SUR 2B复合物的构象，并使用超大VLS来找到最大数量的不同构象。 在其激动剂、拮抗剂和未知构象中与靶口袋相匹配的化学实体（Aim 1）。 这些构象将根据所观察到的命中配体的活性进行细化。我们还将开发三个 正交试验以测试通过VLS鉴定的命中化合物：a）铊通量细胞初步筛选试验， B）Kir6.1的膜片钳和c）LPS刺激的人星形胶质细胞MCP-1/p-JNK升高表型 测定（目的2）。最后，将推导三种检测试剂盒的SAR，以及三种检测试剂盒的荟萃SAR， 可以作为未来药物发现项目的先进起点，将使用 化学信息学方法（可验证）。KATP通道上的特定3D结构激动剂位点没有 这一点，在此前已经有过明确的表述，因此，这一建议具有很强的创新性。

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.