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 lyssavirus属的狂犬病病毒（RABV）和相关病原体引起致命神经系统疾病 每年至少导致55,000人死亡。而当前的狂犬病疫苗是节省和高效的 仅防止因I lyssaviruse门群的感染 也会引起狂犬病的病原体。这个有效的差距同样扩展到人类狂犬病免疫球蛋白， 与疫苗接种一起用于暴露后预防狂犬病。独立于 导致剂的门类型类型，目前没有可靠的治疗选项可用于狂犬病 临床症状的发作。开发一个新型药物发现平台，以识别下一步 对所有临床相关的lyssavirus thylogroups的活性生成抗Lyssavirus候选药物是 因此迫切需要。 我们假设具有宽抗Lyssavirus的货架稳定且具有成本效益的小分子抑制剂 指示光谱将改善全球享受挽救生命的抗兔预防后的抗兔，准备 反对不同门群的新兴裂解病毒病原体，并建立探索的基础 有症状的狂犬病的创新治疗选择。建立在我们高度完整的二年级的基础上 药物发现评估开发，自动化药物筛查，Lyssavirus生物学和RABV方面的专业知识 发病机理，我们成立了一个跨学科团队，以最终满足这种未满足的临床需求。 研究，我们已经产生了一个含有纳米卢西酶代替G的第一类RABV报告菌株 蛋白质编码开放阅读框（RABV-ΔG-NONOLUC），会影响单周感染，可以是 保持了解标准生物安全条件，开发了一种新型的抗RABV药物筛查方案 用于自动化，并将测定法至384孔量表。同时，我们已经建立了直接 正交反屏幕，用于候选候选者确认和lyssavirus指示谱系评估，以及 已经开发了一系列用于早期机械表征的测定面板。基于此 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）协议并将测定法应用于使用HTS设施的药物发现活动 在我们的实验室中建立（特定目标1）。新兴的候选人将在Counterscreens中进行验证， 确定人和鼠的固定细胞系和原代细胞确定的毒性和指示光谱 来源以及评估合成优化的潜力（特定目标2）。缩短名单的子集 在结构和机械上不同的铅候选人中，保证了充分的合成发展，确认了命中 将是机械表征的，确定病毒的逃生轮廓以及脑穿透的潜力 评估（特定目标3）。