Drug discovery against lyssaviruses by high thoughput screening

摘要 狂犬病病毒(RABV)和溶酶病毒属的相关病原体可引起一种致命的神经系统疾病 这导致每年至少5.5万人死亡。鉴于目前的狂犬病疫苗是安全有效的，它 仅预防第一类溶血病毒的感染，但对新出现的第二类病毒无效 也会导致狂犬病的病原体。这种疗效差距同样延伸到人类狂犬病免疫球蛋白， 它与暴露后预防狂犬病的疫苗一起使用。独立于 病原体的种系类型，目前没有可靠的治疗方案可用于狂犬病后 出现临床症状。一种用于鉴定NEXT的新型药物发现平台的开发 产生对所有临床相关的溶血病毒类群具有活性的抗溶血病毒候选药物 因此迫切需要。 我们假设货架稳定且经济实惠的小分子抑制剂具有广泛的抗溶病病毒作用 适应症谱将改善全球获得暴露后预防狂犬病救命的机会，准备 针对新出现的不同种系群的溶病病毒病原体，并为探索奠定基础 针对症状性狂犬病的创新治疗方案。在我们高度互补的多年 在药物发现、化验开发、自动药物筛选、溶酶病毒生物学和RABV方面的专业知识 在发病机制方面，我们已经成立了一个跨学科团队，最终解决这一未得到满足的临床需求。在试行中 研究表明，我们已经产生了一种一流的RABV报告株，它含有纳米荧光素酶来代替G 编码开放阅读框的蛋白质(RABV-ΔG-NanLuc)，可诱导单循环感染，并可 在标准生物安全条件下保持，开发了一种新的抗RABV药物筛选方案，适用于 用于自动化，并将化验微型化到384孔级。同时，我们已经建立了直接和 用于确认命中候选和溶酶病毒指示谱评估的正交反筛选，以及 已经开发出了一套全面的分析方法，用于早期的机械特性表征。以此为基础 有形的基础，我们将在这个为期3年的治疗前发现项目中充分验证高通量 筛选(HTS)方案并将该分析应用于使用HTS设施的药物发现活动 在我们实验室建立(具体目标1)。新出现的热门候选人将在反屏幕上进行验证， 人和小鼠已建立的细胞系和原代细胞的毒性和指示谱测定 来源和评估的综合优化潜力(具体目标2)。入围以下内容的子集 在结构和机械上截然不同的候选人，保证全面合成开发，确认命中 将被机械地描述，病毒逃逸特征被确定，以及大脑渗透的可能性 评估(具体目标3)。

Abstract Rabies virus (RABV) and related pathogens of the lyssavirus genus cause a fatal neurological disease that leads to at least 55,000 deaths annually. Whereas the current rabies vaccine is save and efficacious, it protects only against infection by phylogroup I lyssaviruses but is ineffective against emerging phylogroup II pathogens that also cause rabies. This efficacy gap equally extends to the human rabies immunoglobulin, which is used in conjunction with vaccination for post-exposure prophylaxis against rabies. Independent of the phylogroup type of the causing agent, no reliable treatment option is currently available for rabies after the onset of clinical symptoms. The development of a novel drug discovery platform for the identification of next- generation anti-lyssavirus drug candidates with activity against all clinically relevant lyssavirus phylogroups is therefore urgently needed. We hypothesize that shelf-stable and cost-effective small molecule inhibitors with broad anti-lyssavirus indication spectrum will improve global access to life-saving anti-rabies post-exposure prophylaxis, prepare against emerging lyssavirus pathogens of different phylogroups, and establish a foundation to explore innovative treatment options for symptomatic rabies. Building on our highly complementary multiple-year expertise in drug discovery assay development, automated drug screening, lyssavirus biology, and RABV pathogenesis, we have formed an interdisciplinary team to ultimately address this unmet clinical need. In pilot studies, we have generated a first-in-class RABV reporter strain harboring nano-luciferase in place of the G protein encoding open reading frame (RABV-ΔG-nanoLuc) that induces a single-cycle infection and can be maintained under standard biosafety conditions, developed a novel anti-RABV drug screening protocol suitable for automation, and miniaturized the assay to 384-well scale. In parallel, we have established direct and orthogonal counterscreens for hit candidate confirmation and lyssavirus indication spectrum assessment, and have developed a comprehensive panel of assays for early-stage mechanistic characterization. Building on this tangible foundation, we will in this 3-year pre-therapeutic discovery project fully validate the high-throughput screening (HTS) protocol and apply the assay to a drug discovery campaign using the HTS facilities established in our laboratory (specific aim 1). Emerging hit candidates will be verified in counterscreens, toxicity and indication spectrum determined on established cell lines and primary cells of human and murine origin, and the potential for synthetic optimization assessed (specific aim 2). To short-list a subset of structurally and mechanistically distinct lead candidates that warrant full synthetic development, confirmed hits will be mechanistically characterized, viral escape profiles determined, and the potential for brain penetration evaluated (specific aim 3).