Evaluation of the sigma-1 receptor as a potential therapeutic target for COVID-19

目标1.通过配体诱导的1 R寡聚化对拮抗剂和激动剂进行分类 对于已经显示出降低SARS-CoV-2感染性的几种1 R拮抗剂，确定其1 R拮抗剂性质的原始研究要么模糊要么不存在（例如，PB 28的那些），没有提及如我们已经开发的使用1 R特异性功能测定的体外水平。因此，我们将使用BRET，结合和蛋白质印迹分析专门开发的1 R在我的实验室，以评估是否已发现具有抗病毒或前病毒活性的配体确实具有相反的功能在1 R，并评估是否1 R是一个有效的治疗靶点COVID-19。 如果我们或其他小组发现1 R是COVID-19的有效治疗靶点，我们将寻求开发一种BRET测定法来表征配体诱导的对1 R-Nsp 6相互作用的影响，类似于我们之前对蛋白质的描述1 R及其客户蛋白之间的蛋白质相互作用。这种测定将用于进一步评估靶向1 R-Nsp 6界面用于治疗目的的可行性，并验证来自我们自己的虚拟筛选的命中化合物（见下文）。 目标二。发现和开发1 R和hERG的配体 发现几种1 R拮抗剂对hERG具有显著的亲和力，当以高剂量使用时可能导致心脏毒性。由于1 R可能是治疗可卡因滥用和COVID-19的潜在药物靶点，我们将鉴定和开发对hERG具有低亲和力的新型高亲和力1 R拮抗剂。具体而言，我们将调整现有的平台，整合化学信息学，分子建模和体外试验，以i）筛选对hERG不具有显著亲和力的新型1 R配体;和ii）合理优化显示有希望的治疗效果的1 R配体的当前支架。 为了进一步增强整合，我们将使用1 R的分子建模和模拟以及网络分析来研究所选配体的特异性结合模式，这些配体可能诱导受体构象变化以影响1 R的均一化状态。因此，通过对这些变化的计算机表征，我们将把1 R（和/或其客户蛋白）的构象变化与我们开发的BRET和蛋白质印迹分析的药理学读数联系起来。

Aim 1. Classify antagonists and agonists by ligand-induced 1R oligomerization For several 1R antagonists that have been shown to reduce SARS-CoV-2 infectivity, the original studies in determining their 1R antagonist properties are either vague or do not exist (e.g., those for PB28), not mentioning at in vitro level using 1R specific functional assays as we have developed. Thus, we will use BRET, binding, and western blot assays specifically developed for 1R in my lab, to evaluate whether the ligands that have been found to have either anti-viral or pro-viral activities indeed have opposite functions at 1R, and to assess whether 1R is a valid therapeutic target for COVID-19. If 1R will be found to be an effective therapeutic target for COVID-19 either by us or by other groups, we will seek to develop a BRET assay to characterize ligand-induced effects on the 1R-Nsp6 interaction, similar to our previous characterizations of the protein-protein interactions between 1R and its client proteins. Such an assay will serve to further evaluate the feasibility of targeting the 1R-Nsp6 interface for the therapeutic purposes and to validate the hit compounds from our own virtual screening (see below). Aim 2. Discover and develop the ligands for 1R and against hERG Several 1R antagonists were found to have significant affinities at hERG, which may result in cardiotoxicity when used at high doses. As 1R can be potential drug targets for the treatment of cocaine abuse and COVID-19, we will identify and develop novel high-affinity 1R antagonists that possess low affinity at hERG. Specifically, we will adapt the existing platform that integrated chemoinformatics, molecular modeling, and in vitro assays to i) screen novel 1R ligands that do not possess significant affinity at hERG; and ii) to rationally optimize current scaffolds of the 1R ligands that show promising therapeutic effects. To further enhance the integration, we will use molecular modeling and simulations and network analysis of 1R to investigate the specific binding modes of selected ligands that may induce receptor conformational changes to affect 1R homomerization state. Thus, by in silico characterizations of these changes, we will connect the conformational changes of the 1R (and/or its client proteins) to pharmacological readouts from the BRET and western blot assays we have developed.