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）、沙粒病毒（Arv）、黄病毒（FLAV）和丝状病毒（FiV）被包膜 病毒在病毒进入，受体结合和融合蛋白的重折叠，其次是脂质混合，是三个 释放病毒基因组的关键步骤三个步骤中的任何一个的抑制剂可以被开发为有效的 抗病毒药物目标1。屏幕(A)DEC-Tec使用纯化的SARS 2 S蛋白的胞外域进行筛选， 马丘波病毒（MACV）的糖蛋白和寨卡病毒（ZIKV）的包膜（E）蛋白。(B)将进行HTS 通过靶向SARS 2的六螺旋束（6 HB）并通过与化合物竞争， ALD-1.2结合ZIKV E蛋白。(C)通过AutoDock或其他计算方法的虚拟屏幕也将 因为所有靶蛋白的3D结构都是可用的。目标二。优化.作用机制 击中化合物。（SARS 2）目标1A的命中化合物将通过添加时间抑制试验进行验证， 对SARS 2假型细胞培养感染的影响。将筛选经验证的进入抑制剂对受体的抑制 与S蛋白结合，形成6 HB。受体结合试验是通过ELISA使用ACE 2-Fc 蛋白通过将荧光标记的HR 2肽与5 HB结合来建立6 HB测定。其他进入抑制剂 病毒将遵循相同的研究方法。脂质混合抑制剂。在初步的努力中，我们已经确定 三种抑制剂对SARS 2感染细胞培养物的EC 50值低至190 nM。机制研究 证实这些抑制剂与融合蛋白的跨膜结构域相互作用， 病毒进入时的融合提出了优化的试验设计。埃博拉病毒进入抑制剂。 这些抑制剂处于高级阶段，可作为我们策略的概念验证示例。Med Chem 优化.抑制剂化合物的结构和基于QSAR的优化将在 与Cores C和D合作。符合进一步评估标准的候选人将被提升到 DMPK/毒性研究（核心C）。逃跑变种人开发了最先进的方法来评估 逃避抑制剂的抗病毒活性的突变体，以帮助抑制剂优化。目标3。体内功效。 对于SARS 2，将评估主要候选药物，特别是前药，对多种病毒的广泛抗病毒活性。 SARS 2和SARS分离株。将在仓鼠和小鼠模型中评价有效候选物。对于Arv，Stat 1-/- 将Ifnar 1/Ifnar 2双敲除小鼠用作感染模型。对于埃博拉病毒，我们已经确定了一种 一系列小分子抑制剂靶向埃博拉GP与一种新的机制。我们将评估和适应 这些用于针对其他显著丝状病毒的广谱活性，并评价它们在体内的功效。 在ABSL 4下操作的动物模型。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Core E中进行。

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.