Inhibitors of virus glycoprotein-LAMP1 receptor binding for Lassa virus therapy

摘要 自1969年发现以来，拉萨病毒(LASV)，一种来自Arenaviridae家族的双节段RNA病毒，已经 被认为是每年影响25万人的疾病原因，导致约5000人死亡 每年在西非。由于全球旅行，已经有许多确诊的LASV感染病例 西非以外的地区，包括美国。它以气雾剂的形式传播的能力和约15%的病死率使 LASV是对人类健康的主要威胁，也是BSL4的病原体。不幸的是，没有FDA批准的药物或 有治疗LASV的疫苗可用。该项目的总体目标是满足这一医疗需求 通过识别和验证LASV感染的小分子抑制剂作为预防和/或治疗药物。 该项目的策略是确定以LASV进入细胞为靶点的小分子抑制剂。存在 病毒感染和发生在细胞外/内体环境中的第一步是病毒进入 抗病毒治疗的易感和易接触的靶点。我们的方法是利用我们的经验 均相、生化、高通量筛选(HTS)方法，AlphaLISA，用于识别小分子 阻止LASV的糖蛋白与其宿主受体LAMP1之间的相互作用。此前，我们 开发并应用了基于AlphaLISA技术的生化高温超导技术来识别封闭的化合物 与宿主受体NPC1结合的埃博拉糖蛋白(GP)。鉴定了两个不同的支架，其中一个 在一项小鼠体内研究中显示了对抗传染性埃博拉病毒的效力。在第一阶段，对于AIM 1，AlphaLISA HTS将被开发和优化，用于识别抑制相互作用的小分子 在LASV GP1及其受体LAMP1之间。在目标2中，生物层干涉(BLI)生化分析和 利用携带阿雷诺病毒的重组水疱性口炎病毒进行细胞二次检测 将构建和优化取代VSV-GP的糖蛋白(GP)以确认来自主屏幕的初始命中， 以确定它们结合了哪个交互作用伙伴，并近似该交互作用的亲和力。在《目标3》中， HTS将应用于不同的化学库，HITS将在二次分析中得到确认。在AIM 4、HITS将在传染性拉萨病毒检测中得到验证，并按类药物结构特征和 体外ADME特性。总之，这些分析将识别和验证抑制LASV的化合物 通过抑制病毒进入感染，将为确定这些抑制剂的优先顺序提供有价值的信息。同相 II，我们将在化学上优化优先抑制剂的效力和选择性，并在动物身上进行评估 感染模型。

Summary Since its discovery in 1969, Lassa virus (LASV), a bi-segmented RNA virus from the family Arenaviridae, has been recognized as the cause of disease affecting a quarter million people per year, resulting in ~5,000 deaths annually in West Africa. Due to global travel, there have been numerous confirmed cases of LASV infection outside West Africa including the US. Its ability to spread as an aerosol and a case fatality rate of ~15% make LASV a major threat to human health and a BSL 4 pathogen. Unfortunately, no FDA-approved drugs or vaccines are available for treatment of LASV. The overall goal of this project is to address this medical need by identifying and validating small molecule inhibitors of LASV infection as prophylactics and/or therapeutics. The strategy of this project is to identify small molecule inhibitors that target the cellular entry of LASV. Being the first step in viral infection and occurring in the extracellular/endosomal environment, viral entry is a susceptible and accessible target for antiviral therapy. The approach is to leverage our experience with a homogeneous, biochemical, high-throughput screening (HTS) method, AlphaLISA, to identify small molecules that prevent interactions between the glycoprotein of LASV and its host receptor LAMP1. Previously, we developed and applied a biochemical HTS based on AlphaLISA technology to identify compounds that block the Ebola glycoprotein (GP) binding to its host receptor NPC1. Two distinct scaffolds were identified and one exhibited potency against infectious Ebola virus in a murine in vivo study. In Phase I, for Aim 1, an AlphaLISA HTS will be developed and optimized for the identification of small molecules that inhibit the interaction between LASV GP1 and its receptor LAMP1. In Aim 2, a biolayer interferometry (BLI) biochemical assay and cell-based secondary assays utilizing recombinant vesicular stomatitis virus (VSV) carrying arenavirus glcyoproteins (GP) in place of VSV-GP will be built and optimized to confirm initial hits from the primary screen, to determine which interacting partner they bind, and to approximate the affinity of that interaction. In Aim 3, the HTS will be applied to diverse chemical libraries, and hits will be confirmed in the secondary assays. In Aim 4, hits will be validated in infectious Lassa virus assays and prioritized by drug-like structural features and in vitro ADME properties. Together, these assays will identify and validate compounds that suppress LASV infection by inhibiting viral entry and will provide valuable information for prioritizing those inhibitors. In Phase II, we will chemically optimize priority inhibitors for potency and selectivity and evaluate them in animal infection models.