Project 1: Small Molecule Entry Inhibitors of Pandemic Viruses

项目1-大流行病毒的小分子进入抑制剂 摘要 冠状病毒(CoV)、ArenaVirus(ARV)、黄病毒(Fress)和丝状病毒(FiV)被包被 病毒。在病毒进入过程中，融合蛋白的受体结合和复性，以及随后的脂质混合，分为三个阶段 释放病毒基因组的基本步骤。这三个步骤中的任何一个步骤的抑制剂都可以被开发为有效的 抗病毒药物。目标1.筛选。(A)用纯化的SARS2 S蛋白的胞外结构域进行DEC-Tec筛选， 马丘波病毒糖蛋白(MACV)和寨卡病毒包膜蛋白(ZIKV)。(B)将进行HTS 以SARS2的6-螺旋束(6HB)为靶标的荧光分析及其与化合物的竞争 ALD-1.2与ZIKV E蛋白结合。(C)通过AutoDock或其他计算方法生成的虚拟屏幕也将 因为所有目标蛋白质的3D结构都是可用的。目标2.优化。的作用机制 命中化合物。(SARS2)来自Aim 1A的HIT化合物将通过基于添加时间的抑制试验进行验证 SARS2假型对细胞培养感染的影响将筛选有效的进入抑制剂以抑制受体 与S蛋白结合，形成6HB。受体结合试验通过使用ACE2-Fc的ELISA法进行 蛋白。6HB检测是通过将荧光标记的HR2多肽与5HB结合而建立的。其他药物的进入抑制物 病毒将遵循同样的研究方法。脂类混合的抑制剂。在初步努力中，我们已经确定 三种抑制SARS2感染的药物对细胞培养的EC50值低至190 nM。机械学研究 确认这些抑制物与融合蛋白的跨膜区相互作用并阻断膜 病毒进入过程中的融合。给出了优化的实验设计。埃博拉病毒进入抑制剂。 这些抑制物处于高级阶段，是我们战略的概念验证范例。医学化学 优化。缓蚀剂化合物的结构和QSAR优化将在#年进行 与核心C和D合作，符合进一步评价标准的候选人将被提升至 DMPK/毒性研究(核心C)。逃离变种人。开发了最先进的方法来评估 逃避抑制物抗病毒活性的突变体，以帮助抑制物优化。目的3.体内疗效。 对于SARS2，将对主要候选药物，特别是前药进行广泛的抗病毒活性评估，以对抗 SARS2和SARS分离株。有潜力的候选者将在仓鼠和老鼠模型中进行评估。对于ARV，状态1-/- 以Ifnar1/Ifnar2双基因敲除小鼠为感染模型。对于埃博拉病毒，我们已经确定了一种 针对埃博拉GP的系列小分子抑制剂具有新的作用机制。我们将评估和适应 用于广谱抗其他重要丝状病毒的活性，并评估它们在体内的疗效 动物模型在ABSL4运行。顶级ZIKV抑制剂将在动物模型中针对多种ZIKV进行测试 感染。所有技术工作都在E核心进行。

Project 1 – Small Molecule Entry Inhibitors of Pandemic Viruses ABSTRACT Coronaviruses (CoVs), arenaviruses (Arv), flaviviruses (FLAVs) and filoviruses (FiVs) are enveloped viruses. During virus entry, receptor binding and refolding of the fusion protein, followed by lipid mixing, are three essential steps to release the viral genome. Inhibitors of any one of the three steps may be developed as effective antiviral drugs. Aim 1. Screen. (A) DEC-Tec screen using the purified ectodomain of the SARS2 S protein, glycoprotein of Machupo virus (MACV) and envelope (E) protein of Zika virus (ZIKV). (B) HTS will be carried out using fluorescence assays by targeting the six-helix bundle (6HB) of SARS2 and by competition with compound ALD-1.2 that binds ZIKV E protein. (C) Virtual screens by AutoDock or other computation methods will also be conducted since 3D structures of all target proteins are available. Aim 2. Optimization. Mechanism of action by hit compounds. (SARS2) Hit compounds from aim 1A will be validated by time of addition inhibition assays based on cell culture infection of SARS2 pseudotype. Validated entry inhibitors will be screened for inhibition of receptor binding by the S protein, and formation of 6HB. The receptor binding assay is by ELISA using an ACE2-Fc protein. The 6HB assay is set up by binding a fluorescently labeled HR2 peptide to 5HB. Entry inhibitors of other viruses will follow the same study approach. Inhibitors of lipid mixing. In preliminary efforts, we have identified three inhibitors that have EC50 values as low as 190 nM for SARS2 infection of cell culture. Mechanistic studies confirm that these inhibitors interact with the transmembrane domain of the fusion protein and block membrane fusion during virus entry. The experimental design for optimization is presented. Ebola virus entry inhibitors. These inhibitors are at an advanced stage and serve as a proof-of-concept example for our strategy. Med Chem Optimization. Structure and QSAR-based optimization of the inhibitor compounds will be carried out in collaboration with Cores C and D. Candidates that meet the criteria for further evaluation will be advanced to DMPK/toxicity studies (Core C). Escape mutants. The state-of-the-art approach is developed to evaluate mutants that escape the antiviral activities of the inhibitors, to aid inhibitor optimization. Aim 3. In vivo efficacy. For SARS2, lead candidates, especially prodrugs, will be evaluated for broad antiviral activities against multiple SARS2 and SARS isolates. Potent candidates will be evaluated in hamster and mouse models. For Arv, Stat1-/- and Ifnar1/Ifnar2 double knockout mice will be used as infection models. For Ebola virus, we have identified a series of small molecule inhibitors targeting the Ebola GP with a novel mechanism. We will evaluate and adapt these for the broad-spectrum activity against other significant filoviruses and evaluate their in vivo efficacy in the animal model operating at ABSL4. Top ZIKV inhibitors will be tested in animal models against multiple ZIKV infections. All technical work is carried out in Core E.