Discovery of Bunyaviral Endonuclease Inhibitors for Antiviral Therapy

摘要 包括布尼亚病毒在内的节段性负义单链RNA病毒(SNSVs)是致病病毒 人类疾病的媒介。裂谷热病毒(RVFV)是一种布尼亚病毒，可引起人类出血热 出血热患者病死率约为50%，并已 被NIAID归类为A类优先病原体。RVFV是由蚊子传播的，但也能够使用 广泛的昆虫媒介，有可能蔓延到欧洲和美洲。RVFV有可能 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，医学上迫切需要更强大的 为RVFV量身定做的治疗学。这个项目的总体目标是识别和开发小分子 预防和/或治疗RVFV感染，最好也用于其他高度相关的感染 布尼亚病毒。该策略是通过识别小分子抑制剂来解决未得到满足的医疗需求。 靶向RVFV基本内切酶的酶活性，该酶具有显著的结构 与其他sNSV内切酶相似。方法是利用团队在BunyaVirus上的经验 核酸内切酶和基于均相FRET的生化分析来识别抑制 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 RVFV核酸内切酶活性(FRET-EA)测定法及其在低/中通量格式中的应用 Z‘因子≥为0.9.FRET检测证实了FDA批准的用于治疗流感的抗病毒药物的抑制作用 病毒(IAV)，巴拉沙韦酸(BXA)，抗IAV内切酶。在第一阶段，对于AIM 1，FRET-EA检测将是 针对高通量筛选进行了优化。生化和细胞二级分析，包括FRET、热分析 SHIFT和基于细胞的感染分析将得到优化，以进一步评估确认的命中。在《目标2》中，烦心事- EA高温超导将应用于≥250,000个小分子的不同化学文库的鉴定和 确定抑制RVFV核酸内切酶活性的小分子。在目标3中，确定了命中 在AIM 2中，将在二次分析中验证与内切酶的结合、抗sNSV谱和效力 对抗传染性RVFV。还将根据它们的药物相似性和ADME特性对它们进行优先排序。 在第二阶段，我们将进行基于结构的机理研究，并进一步从化学上优化优先抑制剂 效价、选择性、体内外药代动力学特性及动物感染评价 模特们。

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.