Advancing the development of a novel class of small molecules for treating pan-coronavirus infections

摘要 在过去的十年里，我们的实验室一直在研究细胞激酶在细胞内转运中的作用。 核糖核酸病毒和广谱抗病毒药物的靶标。此外，我们还提供了概念证明 通过证明宿主靶向广谱抗病毒方法的潜在可行性 新的或批准的抗癌药物舒尼替尼和舒尼替尼对两种细胞激酶AAK1和GAK的抑制作用 埃洛替尼保护小鼠免受登革热和埃博拉病毒的侵袭，具有很高的抵抗力。因为治疗性的 对于SARS-CoV-2感染，这种药物组合的指数(TI)较窄，在这里，我们重点介绍一种独立的 一类化合物，基于异噻唑[4，3-b]吡啶的RMC-113系列，从我们先前的工作中出现， 但不抑制AAK1或GAK。我们发现RMC-113和25个相关类似物具有很强的广谱活性。 光谱抗病毒活性，具有高抵抗力。令人兴奋的是，RMC-113将SARS-CoV-2滴度降低到 在无毒浓度下无法检测到的水平，并与PIKfyve结合，PIKfyve是一种调节内体的细胞激酶 贩卖人口。我们假设RMC-113类似物抑制SARS-CoV-2生命中的两个不同步骤 循环和对该病毒的炎症反应，部分通过靶向PIKfyve，从而提供有吸引力的和 用于对抗SARS-CoV-2、其他大流行性冠状病毒和其他新出现病毒的安全候选抑制剂。 在目标1中，我们将使用多维药物化学方法来优化TI和PK谱 引导RMC-113类似物，并将其体外治疗潜力确定为广泛的抗冠状病毒抑制剂。目标2 将确定优先考虑的类似物和阿皮莫德的效果，阿皮莫特是新冠肺炎的一种重新调整用途的候选药物 抑制PIKfyve，对病毒复制，细胞因子反应和组织损伤 用来自20名人类供者和两种啮齿动物模型的PBMCs补充正常肺组织。目标3将 生成ADME毒性和安全性药理学数据集，以选择IND前候选药物。在目标4中，我们将 探讨RMC-113的抗病毒作用机制。我们将验证PIKfyve作为候选目标并使用 无偏CRISPRI筛选以识别RMC-113‘S目标(S)并描述其化学成因景观。在……里面 同时，我们将设计一种可点击的RMC-113探针，通过基于活性的蛋白质来确认分子靶点 分析并监控目标参与情况。最后，我们将探讨功能相关性和具体角色 PIKfyve和其他通过这些方法出现的候选方法在SARS-CoV-2感染中的应用，并验证它们为 介导抗病毒作用的分子靶点S。预计的立竿见影的效果是，这个项目将 提供对阿皮莫特的治疗潜力和MOA的洞察，阿皮莫特是一种重新定位的候选药物(超越 已报道的对病毒进入的影响)，并将建立一个独特的人类肺器官模型来研究SARS冠状病毒- 2发病机制和治疗反应在更自然的条件下。从长远来看，成功 我们的研究完成将提供一种类似药物的小分子候选药物，旨在防止卷土重来 新冠肺炎并为未来爆发冠状病毒和其他新出现的病毒做好准备。

Abstract For the past decade, our laboratory has been studying the role of cellular kinases in intracellular trafficking of RNA viruses and as targets for broad-spectrum antivirals. Furthermore, we have provided a proof of concept for the potential feasibility of the host-targeted broad-spectrum antiviral approach by demonstrating that the inhibition of two cellular kinases, AAK1 and GAK, by novel or the approved anticancer drugs, sunitinib and erlotinib, protects mice from dengue and Ebola viruses with a high barrier to resistance. Since the therapeutic index (TI) of this drug combination is narrower for SARS-CoV-2 infection, here, we focus on an independent class of compounds, the isothiazolo[4,3-b]pyridine-based RMC-113 series, that emerged from our prior work, but does not inhibit AAK1 or GAK. We showed that RMC-113 and 25 related analogs have potent broad- spectrum antiviral activity with a high barrier to resistance. Excitingly, RMC-113 reduces SARS-CoV-2 titer to undetectable levels at non-toxic concentrations and binds PIKFYVE, a cell kinase that regulates endosomal trafficking. We hypothesize that RMC-113 analogs inhibit both multiple distinct steps in the SARS-CoV-2 life cycle and the inflammatory response to this virus, in part by targeting PIKFYVE, thereby offering attractive and safe candidate inhibitors to combat SARS-CoV-2, other pandemic coronaviruses and other emerging viruses. In Aim 1, we will use a multi-dimensional medicinal chemistry approach to optimize the TI and PK profile of lead RMC-113 analogs and define their in vitro therapeutic potential as broad anticoronavirus inhibitors. Aim 2 will determine the effect of prioritized analogs and apilimod, a repurposed drug candidate for COVID-19 that inhibits PIKFYVE, on viral replication, cytokine response and tissue injury in organoids derived from excised normal lung tissue supplemented with PBMCs from 20 human donors and in two rodent models. Aim 3 will generate ADME-toxicity and safety pharmacology datasets to select pre-IND candidates. In Aim 4, we will probe the mechanism of antiviral action of RMC-113. We will validate PIKFYVE as a candidate target and use an unbiased CRISPRi screen to identify RMC-113’s target(s) and profile its chemical-genetic landscape. In parallel, we will design a clickable RMC-113 probe to confirm the molecular target via activity-based protein profiling and to monitor target engagement. Lastly, we will probe functional relevance and specific roles of PIKFYVE and other candidates emerging via these approaches in SARS-CoV-2 infection, and validate them as the molecular target(s) mediating the antiviral effect. The predicted immediate impact is that this project will provide insight into the therapeutic potential and MOA of apilimod, a repurposed drug candidate (beyond the reported effect on viral entry), and will establish a unique human lung organoid model for studying SARS-CoV- 2 pathogenesis and response to treatment under more natural conditions. In the longer term, successful completion of our study will deliver a drug-like small molecule candidate designed to protect against resurge of COVID-19 and to provide readiness for future outbreaks with coronaviruses and other emerging viruses.