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

摘要 在过去的十年中，我们的实验室一直在研究细胞激酶在细胞内运输的作用， RNA病毒和作为广谱抗病毒药物的靶点。此外，我们还提供了概念验证 通过证明宿主靶向广谱抗病毒方法的潜在可行性， 通过新的或批准的抗癌药物舒尼替尼抑制两种细胞激酶AAK 1和GAK， 埃罗替尼，保护小鼠免受登革热和埃博拉病毒的侵害，具有高抵抗屏障。自从治疗 这种药物组合的指数（TI）对于SARS-CoV-2感染是窄的，在这里，我们专注于一个独立的 一类化合物，异噻唑并[4，3-B]吡啶基RMC-113系列，从我们先前的工作中出现， 但不抑制AAK 1或GAK。我们发现RMC-113和25个相关类似物具有有效的广泛的 广谱抗病毒活性，具有高耐药性屏障。令人兴奋的是，RMC-113将SARS-CoV-2滴度降低至 在无毒浓度下无法检测到水平，并结合PIKFYVE，PIKFYVE是一种调节内体 贩卖人口我们假设RMC-113类似物抑制SARS-CoV-2生命中的多个不同步骤 周期和炎症反应，这种病毒，部分通过靶向PIKFYVE，从而提供有吸引力的， 安全的候选抑制剂，以对抗SARS-CoV-2、其他大流行性冠状病毒和其他新兴病毒。 在目标1中，我们将使用多维药物化学方法来优化 引导RMC-113类似物，并将其体外治疗潜力确定为广泛的病毒抑制剂。目的2 将确定优先类似物和阿吡莫德的效果，阿吡莫德是一种重新利用的COVID-19候选药物， 抑制PIKFYVE，对来自切除的类器官中的病毒复制、细胞因子应答和组织损伤 补充有来自20个人类供体和两种啮齿动物模型的PBMC的正常肺组织。目标3将 生成ADME毒性和安全药理学数据集，以选择IND前候选药物。在目标4中，我们 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 进行公正的CRISPRi筛选，以识别RMC-113的靶点并分析其化学遗传格局。在 同时，我们将设计一个可点击的RMC-113探针，通过基于活性的蛋白质来确认分子靶标 侧写并监控目标的交战情况最后，我们将探讨的功能相关性和具体作用， PIKFYVE和其他通过这些方法在SARS-CoV-2感染中出现的候选物，并验证它们作为 介导抗病毒作用的分子靶标。预测的直接影响是，该项目将 提供了对阿吡莫德的治疗潜力和MOA的洞察，阿吡莫德是一种重新用途的候选药物（超越了 报道的对病毒进入的影响），并将建立一个独特的人类肺类器官模型，用于研究SARS-CoV- 2发病机制和对治疗的反应在更自然的条件下。从长远来看，成功 完成我们的研究将提供一种药物样的小分子候选人，旨在防止复发的 COVID-19，并为未来冠状病毒和其他新兴病毒的爆发做好准备。

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.