Drug discovery against lyssaviruses by high thoughput screening

通过高通量筛选发现抗狂犬病病毒药物

• 批准号: 9218526
• 负责人: Richard K. Plemper
• 金额: $ 39.84万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-11-15 至 2019-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9218526
• 关键词: Address; Aggressive behavior; Animal Diseases; Antibodies; Antiviral Agents; Automation; Behavioral; Biological Assay; Biology; Blood - brain barrier anatomy; Brain; Cell Line; Cells; Cessation of life; Chemicals; Chiroptera; Classification; Clinical; Cold Chains; Coma; Communicable Diseases; Complement; Computer Simulation; DNA Viruses; Data; Development; Disease; Dose; Eligibility Determination; Failure; Family; Family member; Foundations; Future; G-substrate; GTP-Binding Proteins; Goals; Human; Human Cell Line; Hyperactive behavior; Immunoglobulins; Improve Access; Infection; Influenza A virus; Joints; Laboratories; Lead; Libraries; Life; Lyssavirus; Modeling; Mokola virus; Molecular Target; Molecular Virology; Mus; Muscle Weakness; Open Reading Frames; Orthomyxoviridae; Paralysed; Penetration; Permeability; Pilot Projects; Preclinical Drug Evaluation; Prophylactic treatment; Protocols documentation; Quantitative Structure-Activity Relationship; RNA Viruses; RNA-Directed RNA Polymerase; Rabies; Rabies Vaccines; Rabies virus; Reading Frames; Recombinants; Regimen; Reporter; Resistance; Respiratory syncytial virus; Rhabdoviridae; Savings; Structure-Activity Relationship; Symptoms; Testing; Therapeutic; Therapeutic Intervention; Toxic effect; Treatment Cost; Treatment Efficacy; Vaccination; Vaccines; Validation; Viral; Virus; Virus Inhibitors; Virus Replication; assay development; base; bioactive natural products; clinically relevant; clinically significant; cost effective; counterscreen; cytotoxicity; design; drug candidate; drug development; drug discovery; established cell line; functional gain; high throughput screening; human disease; immunoprophylaxis; improved; inhibitor/antagonist; innovation; insight; member; miniaturize; nanoluciferase; nervous system disorder; next generation; novel; novel therapeutics; pathogen; prevent; response; reverse genetics; risk minimization; scaffold; screening; small molecule; small molecule inhibitor; virus pathogenesis

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).

抽象的 狂犬病病毒（RABV）和狂犬病病毒属相关病原体可引起致命的神经系统疾病 每年至少导致 55,000 人死亡。尽管目前的狂犬病疫苗既安全又有效，但 只能防止系统群 I 狂犬病病毒感染，但对新出现的系统群 II 无效 也引起狂犬病的病原体。这种功效差距同样延伸到人类狂犬病免疫球蛋白， 它与疫苗接种结合使用，用于狂犬病暴露后预防。独立于 由于病原体的系统群类型，目前没有可靠的治疗方案可用于治疗狂犬病 出现临床症状。开发用于识别下一代的新药物发现平台 产生对所有临床相关狂犬病病毒系统群具有活性的抗狂犬病病毒候选药物 因此迫切需要。 我们假设具有广泛抗狂犬病病毒作用的货架稳定且具有成本效益的小分子抑制剂 适应症谱将改善全球获得挽救生命的抗狂犬病暴露后预防的机会，准备 针对不同系统群的新出现的狂犬病病毒病原体，并为探索奠定基础 针对症状性狂犬病的创新治疗方案。以我们高度互补的多年合作为基础 药物发现分析开发、自动药物筛选、狂犬病病毒生物学和 RABV 方面的专业知识 发病机制，我们组建了一个跨学科团队来最终解决这一未满足的临床需求。试点中 研究中，我们已经生成了一流的 RABV 报告菌株，其含有纳米荧光素酶代替了 G 编码开放阅读框（RABV-ΔG-nanoLuc）的蛋白质，可诱导单周期感染，并且可以 在标准生物安全条件下，开发了适合的新型抗RABV药物筛选方案 自动化，并将测定小型化至 384 孔规模。与此同时，我们建立了直接和 用于候选候选物确认和狂犬病病毒指示谱评估的正交反筛选，以及 开发了一套用于早期机械表征的综合分析方法。在此基础上 有了坚实的基础，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) 方案并使用 HTS 设施将该测定应用于药物发现活动 在我们的实验室建立（具体目标1）。新兴热门候选人将在柜台上得到验证， 对人和鼠的已建立细胞系和原代细胞测定的毒性和指示谱 起源，以及评估合成优化的潜力（具体目标 2）。列出一个子集 结构和机械上独特的先导候选物，需要全面的合成开发，已确认成功 将进行机械表征，确定病毒逃逸特征，以及大脑渗透的潜力 评估（具体目标 3）。