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

基本信息

批准号：
10522810
负责人：
MING LUO
金额：
$ 817.71万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-05-16 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10522810
关键词：
2019-nCoV ACE2 Animal Model Antiviral Agents Arenavirus Binding Binding Proteins Biological Assay Biological Availability Bolivian Hemorrhagic Fever Virus COVID-19 treatment Cell Culture Techniques Cell membrane Cells Chimeric Proteins Collaborations Computing Methodologies Coronavirus Data Set Ebola Ebola virus Endocytosis Enzyme-Linked Immunosorbent Assay Escape Mutant Evaluation Experimental Designs Filovirus Flavivirus Fluorescence Genome Glycoproteins Hamsters In Vitro Infection Knockout Mice Label Libraries Lipids Lung Lung infections Mediating Membrane Membrane Fusion Membrane Glycoproteins Methods Midwestern United States Modeling Molecular Conformation Mus Oral Outcome Peptides Prodrugs Proteins Quantitative Structure-Activity Relationship Receptor Cell Receptor Inhibition SARS-CoV-2 infection SARS-CoV-2 spike protein Series Severe Acute Respiratory Syndrome Structure Testing Time Toxic effect Transmembrane Domain Update Variant Viral Viral Genome Viral Proteins Virus Virus Inhibitors Virus Replication Work ZIKV infection Zika Virus analog base design dimer env Gene Products in vivo in vivo Model indexing inhibitor lead candidate lead series mouse model novel pandemic disease receptor receptor binding respiratory small molecule small molecule inhibitor stem three dimensional structure virtual virus envelope

项目摘要

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.

项目1 - 大流行病毒的小分子进入抑制剂抽象的冠状病毒（COVS），体育症病毒（ARV），黄病毒（Flavs）和丝状病毒（FIV）被包裹病毒。在进入病毒期间，融合蛋白的受体结合和重折叠，然后是脂质混合，为三个释放病毒基因组的重要步骤。三个步骤中任何一个的抑制剂均可开发为有效抗病毒药物。目标1。屏幕。（a）使用SARS2 S蛋白的纯化的外域域的DEC-TEC筛选， Machupo病毒（MACV）和Zika病毒（ZIKV）的包膜（E）蛋白的糖蛋白。（b）将进行HTS 通过靶向SARS2的六螺旋束（6hb）和与化合物竞争，使用荧光测定结合ZIKV E蛋白的Ald-1.2。（c）Autodock或其他计算方法的虚拟屏幕也将是由于所有靶蛋白的3D结构都可以使用。目标2。优化。作用机理点击化合物。（SARS2）AIM 1A的命中化合物将通过基于添加抑制测定的时间来验证关于SARS2假型的细胞培养感染。经过验证的进入抑制剂将被筛选以抑制接收器由S蛋白结合，形成6Hb。受体结合测定是通过ELISA使用ACE2-FC的蛋白质。通过将荧光标记的HR2胡椒与5HB结合来建立6HB测定。其他其他抑制剂病毒将遵循相同的研究方法。脂质混合的抑制剂。在初步努力中，我们已经确定三种抑制剂的EC50值低至190 nm，用于细胞培养的SARS2感染。机械研究确认这些抑制剂与融合蛋白的跨膜结构域相互作用病毒进入期间的融合。提出了优化的实验设计。埃博拉病毒进入抑制剂。这些抑制剂处于高级阶段，并作为我们策略的概念验证示例。 Med Chem 优化。基于结构和基于QSAR的抑制剂化合物的优化将在与核心C和D的合作。 DMPK/毒性研究（核心C）。逃避突变体。开发了最先进的方法来评估逃避抑制剂抗病毒活性的突变体有助于抑制剂优化。目标3。体内效率。对于SARS2，将评估主要候选人，尤其是前药，以进行广泛的抗病毒活动。 SARS2和SARS分离株。有效的候选者将在仓鼠和小鼠模型中进行评估。对于ARV，STAT1 - / - IFNAR1/IFNAR2双基因敲除小鼠将用作感染模型。对于埃博拉病毒，我们已经确定了针对埃博拉病毒GP的一系列小分子抑制剂，具有新的机制。我们将评估和适应这些用于针对其他重要的铁病毒的广泛活性，并评估其体内效率在ABSL4运行的动物模型。顶部ZIKV抑制剂将在动物模型中测试针对多个ZIKV 感染。所有技术工作均在Core E中进行。