Discovery and Development of Broad-spectrum Protease Inhibitors of Flaviviruses of Significant Public Health Threats

具有重大公共卫生威胁的黄病毒广谱蛋白酶抑制剂的发现和开发

• 批准号: 9907267
• 负责人: Sridhar G Prasad
• 金额: $ 30万
• 依托单位: PLEX PHARMACEUTICALS, INC.
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-01 至 2022-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9907267
• 关键词: Adult; Affect; American; Animal Model; Animals; Antiviral Agents; Arbovirus Infections; Arboviruses; Arthrogryposis; Aspartic Endopeptidases; Binding; Biochemical; Biological Assay; Biological Availability; Bite; Categories; Cell Line; Cells; Cerebral Calcification; Cessation of life; Child; Clinical; Country; Culicidae; Dengue Vaccine; Dengue Virus; Development; Disease; Disease Outbreaks; Drug Kinetics; Encephalitis; Enzymes; Epidemic; Etiology; Evaluation; Evolution; Exhibits; Family; Family member; Fever; Flaviviridae; Flavivirus; Frequencies; Genome; Geography; Guillain-Barré Syndrome; HIV-1; Hepatitis C virus; Human; In Vitro; Incidence; Infant; Infection; Infection prevention; Japanese encephalitis virus; Lead; Libraries; Ligands; Link; Liver; Luciferases; Measures; Meningitis; Metabolism; Microcephaly; Microsomes; Modeling; Mosquito-borne infectious disease; National Institute of Allergy and Infectious Disease; National Security; Neurons; Nonstructural Protein; Paralysed; Pathology; Patients; Peptide Hydrolases; Performance; Pharmaceutical Chemistry; Pharmaceutical Preparations; Phase; Plaque Assay; Polyproteins; Population; Pregnant Women; Prevention; Preventive Intervention; Process; Property; Protease Inhibitor; Public Health; Rattus; Replicon; Reporter; Risk; Safety; Series; Serine Protease; Serotyping; Severities; Solubility; Structure; System; Testing; Therapeutic; Therapeutic Index; Therapeutic Intervention; Thrombocytopenia; Translating; Vaccines; Vector-transmitted infectious disease; Vero Cells; Viral; Viral Encephalitis; Virus; West Nile viral infection; West Nile virus; World Health Organization; Yellow fever virus; ZIKV infection; Zika Virus; abortion; absorption; acute toxicity; analog; anti-viral efficacy; base; calcification; channel blockers; chemotherapeutic agent; congenital zika syndrome; cytotoxicity; design; effective therapy; efficacy study; efficacy trial; global health; in vitro activity; in vivo; inhibitor/antagonist; innovation; lead optimization; member; mortality; mosquito-borne; novel; pathogen; screening; small molecule; small molecule inhibitor; stillbirth; success; targeted agent; three dimensional structure; vaccine candidate

Project Summary Mosquito-borne members of the Flavivirus family including Zika virus (ZKV), Dengue virus (DNV) and West Nile virus (WNV), are classified as re-emerging pathogens due to the frequency and severity of recent epidemics. Also known as arboviruses, these viruses are the etiologic agents of many debilitating diseases affecting the human population worldwide. Consequently, vector borne diseases now account for 17% of all infections worldwide. DNV is the fastest growing arboviral disease currently affecting 400 million annually with 96 million cases manifesting into clinical severity and 22,000 deaths, mainly children. WNV is considered the most important causative agent of viral encephalitis worldwide. The recent ZKV infection outbreak has been associated with congenital microcephaly and intracranial calcification and, in adults, with GBS and severe thrombocytopenia. Currently there is no effective treatment for infections caused by these viruses, which highlights the urgent need to find preventive and therapeutic interventions. The Flaviviridae genome is translated into a single polyprotein which is processed to yield 3 structural and 7 nonstructural proteins. The correct processing of the polyprotein is essential for replication of all flaviviruses, which requires both host proteases and the highly conserved viral NS2B-NS3 protease (NS2B-NS3pro). Hence, the viral protease is a rational target for development of small molecule inhibitors that block flavivirus replication. Small molecule antivirals targeting HIV-1-encoded and HCV-encoded proteases have been successfully developed, which supports the concept of developing chemotherapeutic agents targeting the flavivirus NS2B-NS3pro. The innovation of our proposal is: (i) optimization of a highly-sensitive screening assay for the identification of low binding fragment hits; (ii) evolution of fragment and compound hits into broad-spectrum leads; (iii) the use of replicon and plaque assays to test for cellular efficacy and guide optimization. Our preliminary results and the use of multiple cell-based models supports the feasibility of the discovery of broad-spectrum anti-flaviviral therapeutics. The specific aims are: Aim 1: Complete the screening of fragment and compound libraries for the identification of broad-spectrum NS2B-NS3pro hits. Milestone 1: Identify 6-8 structurally distinct broad-spectrum NS2B-NS3pro hits with an IC50 ≤ 25μM. Aim 2a: Iterative 3D-structure and SAR-based discovery of three non-overlapping broad-spectrum NS2B-NS3pro inhibitor series, using a combination of: (i) commercial analogues and (ii) med-chem design and synthesis approach. Milestone 2: Identify three non-overlapping broad-spectrum NS2B- NS3pro inhibitor series with an IC50 ≤ 200nM. Aim 2b: Characterize biochemically potent inhibitors for: (a) mode of inhibition and (b) enzyme:ligand interactions and prioritize compounds with IC50 ≤ 200nM, for cellular efficacy studies. Aim 3a: In vitro evaluation and optimization of biochemically potent compounds for cellular efficacy (EC50) and cytotoxicity (CC50). Milestone 3: Identify 4-6 lead compounds from each series with EC50 ≤ 5μM and CC50 ≥ 200μM. Aim 3b: Conduct in vitro ADME-based lead optimization of compounds with broad spectrum in vitro activity and acceptable therapeutic indices. Phase I Milestone: Identify 2-4 lead compounds exhibiting high bioavailability, weak inhibitor of CYPP450s, optimum stability and are not hERG channel blockers.

项目摘要 Flavivirus家族的蚊子传播成员，包括寨卡病毒（ZKV），登革热病毒（DNV）和西尼罗河 由于最近发作的频率和严重程度，病毒（WNV）被归类为重新出现的病原体。也已知 作为arbovirus，这些病毒是许多影响人群的衰弱疾病的病因学剂 全世界。因此，媒介疾病现在占全球所有感染的17％。 DNV是最快的 目前每年增加4亿杆病毒疾病，9600万例病例表现为临床严重程度 和22,000人死亡，主要是儿童。 WNV被认为是全球病毒脑炎最重要的灾难性药物。 最近的ZKV感染暴发与先天性小头畸形和颅内钙化有关，在 成人，患有GBS和严重的血小板减少症。目前尚无对这些引起的感染的有效治疗 病毒强调了迫切需要寻找预防性和治疗性干预措施。 Flaviviridae基因组被翻译成单个多蛋白，该多蛋白被加工为产生3个结构和7 非结构蛋白。多蛋白的正确加工对于复制所有黄病毒至关重要，这需要 宿主蛋白酶和高度配置的病毒NS2B-NS3蛋白酶（NS2B-NS3PRO）。因此，病毒蛋白酶是 开发小分子抑制剂的合理靶标，这些抑制剂阻断了黄病毒复制。小分子抗病毒药 靶向HIV-1编码和HCV编码的蛋白酶已经成功开发出来，这支持了 开发针对黄病毒NS2B-NS3Pro的化学治疗剂。我们提案的创新是：（i） 优化高度敏感的筛选测定法，以鉴定低结合片段命中； （ii）进化 碎片和复合命中量为广谱铅； （iii）使用副本和牙菌斑测试细胞效率 和指南优化。我们的初步结果和多个基于单元的模型的使用支持了 发现广谱抗葡萄病毒疗法。具体目的是：目标1：完成片段的筛选 以及用于识别广谱NS2B-NS3Pro命中的复合库。里程碑1：在结构上识别6-8 IC50≤25μm的不同广谱NS2B-NS3PRO命中。 AIM 2A：迭代3D结构和基于SAR的发现 使用：（i）商业类似物组合的三个非重叠的广谱NS2B-NS3PRO抑制剂系列 （ii）Med-Chem设计和合成方法。里程碑2：确定三个非重叠的广谱NS2B- 具有IC50≤200nm的NS3PRO抑制剂系列。 AIM 2B：表征生化潜在抑制剂的特征：（a） 抑制和（b）酶：配体相互作用并优先考虑IC50≤200nm的化合物，用于细胞效率研究。 AIM 3A：在体外评估和优化细胞效率的生化潜在化合物（EC50）和 细胞毒性（CC50）。里程碑3：从每个系列中识别出EC50≤5μM和CC50≥200μm的4-6个铅化合物。目的 3B：在体外基于ADD ADD的铅铅优化，对具有广泛的体外活性和可接受的化合物进行 治疗指标。第一阶段的里程碑：识别2-4个铅化合物，表现出高生物利用度，弱抑制剂 CYPP450，最佳稳定性，不是HERG通道阻滞剂。