Discovery of Bunyaviral Endonuclease Inhibitors for Antiviral Therapy

摘要 节段性负义单链RNA病毒（sNSV），包括布尼亚病毒，是致病的 人类疾病的病原体。裂谷热病毒（RVFV）是一种布尼亚病毒，可引起人类出血热 发生出血热的患者病死率达到约50%， 被NIAID列为A类优先病原体。RVFV是蚊子传播的，但也能够使用 一系列有可能传播到欧洲和美洲的昆虫媒介。RVFV有潜力 造成重大的全球健康和经济影响。不幸的是，没有FDA批准的药物或 用于治疗RVFV感染的疫苗。因此，医学上迫切需要更有效的 为RVFV量身定制的治疗方法。该项目的总体目标是识别和开发小分子 用于RVFV感染和优选还用于其它高度相关的病毒感染的药物和/或治疗剂 布尼亚病毒。该策略是通过识别小分子抑制剂来解决未满足的医疗需求 靶向必需的RVFV内切核酸酶的酶活性，其表现出显著的结构 与其它sNSV内切核酸酶相似。该方法是利用团队对本扬病毒的经验 核酸内切酶和均相FRET为基础的生化测定，以确定小分子，抑制 布尼亚病毒核酸内切酶的酶活性。在初步研究中，我们开发了一种FRET-内切酶， 活性（FRET-EA）测定，并以低/中通量形式应用该测定 Z因子≥0.9。FRET测定证实了FDA批准的治疗流感的抗病毒药物的抑制作用 病毒（IAV），Baloxavir酸（BXA），针对IAV核酸内切酶。在I期，对于目标1，FRET-EA测定将是 优化用于高通量筛选。生化和细胞二级检测，包括FRET、热分析 偏移和基于细胞的感染性测定，将被优化以进一步评价确认的命中。在目标2中，FRET- EA HTS将应用于≥ 250，000个小分子的各种化学库，以进行鉴定和 确认抑制RVFV核酸内切酶的酶活性的小分子。在目标3中， 将在与核酸内切酶结合、抗sNSV谱和效价的二级试验中进行验证 针对传染性RVFV。它们还将根据其药物相似性和ADME特性进行优先排序。 在第二阶段，我们将进行基于结构的机制研究，并进一步化学优化优先抑制剂 效价、选择性、体外和体内药代动力学特性，并在动物感染中对其进行评价 模型

Abstract Segmented negative-sense, single-stranded RNA viruses (sNSVs), which include bunyaviruses, are causative agents of human diseases. Rift Valley Fever Virus (RVFV), a bunyavirus, causes hemorrhagic fever in humans with a case fatality rate of patients developing hemorrhagic fever reaching approximately 50% and has been classified by the NIAID as a Category A Priority Pathogen. RVFV is mosquito-borne, but is also capable of using a wide range of insect vectors with potential to spread to Europe and the Americas. RVFV has the potential to cause significant global health and economic impact. Unfortunately, there are no FDA-approved drugs or vaccines for the treatment of the RVFV infection. Therefore, there is an urgent medical need for more potent therapeutics tailored for RVFV. The overall goal of this project is to identify and develop small molecule prophylactics and/or therapeutics for RVFV infections and preferably also for infections of other highly related bunyaviruses. The strategy is to address the unmet medical need by identifying small molecule inhibitors targeting the enzymatic activity of the essential RVFV endonuclease, which exhibits significant structural similarity to other sNSV endonucleases. The approach is to leverage the team’s experience with bunyaviral endonucleases and a homogeneous FRET-based biochemical assay to identify small molecules that inhibit the enzymatic activity of bunyaviral endonucleases. In Preliminary Studies, we developed a FRET-endonuclease activity (FRET-EA) assay for RVFV endonuclease and applied the assay in a low/medium-throughput format with Z’-factors ≥0.9. The FRET assay confirmed the inhibition of an FDA approved antiviral to treat Influenza virus (IAV), Baloxavir acid (BXA), against IAV endonuclease. In Phase I, for Aim 1, the FRET-EA assay will be optimized for high-throughput screening. Biochemical and cellular secondary assays, including FRET, thermal shift, and cell-based infectious assays, will be optimized to further evaluate confirmed hits. In Aim 2, the FRET- EA HTS will be applied to diverse chemical libraries of ≥250,000 small molecules for the identification and confirmation of small molecules that inhibit the enzymatic activity of RVFV endonuclease. In Aim 3, hits identified in Aim 2 will be validated in secondary assays for binding to endonuclease, anti-sNSV spectrum, and potency against infectious RVFV. They will also be prioritized based on their drug-likeness and their ADME properties. In Phase II, we will perform structural based mechanism studies and further chemically optimize priority inhibitors for potency, selectivity, in vitro and in vivo pharmacokinetics properties and evaluate them in animal infection models.