Discovery of new antiviral methylase, protease, and helicase inhibitors of corona-, flavi-, and alphaviruses

发现冠状病毒、黄病毒和甲病毒的新型抗病毒甲基化酶、蛋白酶和解旋酶抑制剂

• 批准号: 10513923
• 负责人: THOMAS TUSCHL
• 金额: $ 570.11万
• 依托单位: HACKENSACK UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-16 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10513923
• 关键词: 2019-nCoV; Aerosols; Alphavirus; Animal Model; Animals; Antiviral Agents; Biochemical; Biological Assay; Biological Availability; COVID-19; COVID-19 outbreak; Caspase; Cell Membrane Permeability; Cells; Cessation of life; Characteristics; Chemicals; Chikungunya virus; China; Collection; Combined Modality Therapy; Complex; Consultations; Contracts; Coronavirus; Development; Disease; Disease Outbreaks; Distant; Drug Kinetics; Enzymes; Family; Flaviviridae; Future; Goals; Human; In Vitro; Individual; Industry; Infection; Innate Immune Response; Institutes; Laboratories; Lead; Libraries; Medicine; Metabolic; Methyltransferase; Middle East Respiratory Syndrome Coronavirus; Monitor; Mutation; Names; Oral; Patients; Peptide Hydrolases; Pharmaceutical Chemistry; Pharmaceutical Preparations; Pharmacology; Polyproteins; Preparation; Program Development; Property; Protein Biosynthesis; RNA Helicase; RNA Viruses; Recombinant Proteins; Recording of previous events; Reporting; Resources; Rice; SARS-CoV-2 protease; SH2D3A gene; SH2D3C gene; Series; Solubility; Specificity; Structure; Surface; Therapeutic; Togaviridae; Toxic effect; Universities; Viral; Viral Pneumonia; Viral Proteins; Virus; Virus Replication; ZIKA; Zoonoses; assay development; base; clinical candidate; clinical development; combat; design; drug development; drug resistant virus; falls; helicase; high throughput screening; improved; in vivo; inhibitor; insight; lead optimization; lead series; luminescence; member; metropolitan; mosquito-borne; novel; operation; pandemic disease; programs; resistance mutation; small molecule; structural biology; virology

Coronavirus Disease 2019 (COVID-19) represents an ongoing pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and a current death toll of about 5 million. There are only limited antiviral treatment options for patients who contracted the disease. In March 2020, we initiated collaborative small molecule drug development including the laboratories of Dr. Tuschl (high-throughput screening (HTS) assay development), Dr. Glickman (HTS operations), Dr. Patel (structural biology), Dr. Rice (virology), and the Tri- Institutional Therapeutics Discovery Institute (medicinal chemistry) focusing on the two SARS-CoV-2 proteases, NSP3/PLpro and NSP5/3CLpro, required for viral polyprotein processing, the cap N7-G methyltransferase (MTase) NSP14, required for efficient viral protein synthesis and escape from host cell innate immune response, and the RNA helicase NSP13 important for viral replication. We developed robust, HTS fluorescent-substrate-based assays for monitoring PLpro, 3CLpro, and NSP13 activities as well as a luminescence-based assay for monitoring NSP14 N7-G-MTase activity. By HTS of the diverse library of 430,000 compounds available at Rockefeller University (RU), we identified hits in all assays, validated hits, and pursued the most promising non-covalent hits in medicinal chemistry programs. Our lead compounds for NSP14 and PLpro show IC50 values of 2 nM and 100 nM, respectively, and cell-based inhibition of SARS-CoV-2 replication with EC50 values of 80 nM and 7 µM, without cellular toxicity. Here, we propose further lead optimization to improve inhibitor potency and drug-like properties, as well as apply our HTS expertise to target proteases, MTases, and helicases of other RNA viruses with pandemic potential, including MERS, flavi- and alphaviruses. Our specific aims are: (1) Development of SARS-CoV-2 NSP14 and PLpro clinical candidates. Guided by structural insights from inhibitor-PLpro and NSP14 complexes, we will further optimize inhibitor potency and drug-like characteristics with industry-quality medicinal chemistry support from TDI and this program, and the goal to develop 3-5 advanced leads that are subjected to in vitro and in vivo DMPK/ADME-Tox studies utilizing all scientific cores designated for hit-to-lead and lead optimization (2) Develop antiviral lead series against MTases, proteases and helicases of select members of the Coronaviridae, Flaviviridae and Togaviridae families. We will adapt and develop HTS assays using recombinant proteins and identify covalent and non-covalent inhibitors in the RU drug collection and accompany the medicinal chemistry lead development to provide 3-5 leads per target for comprehensive DMPK/ADME-Tox analysis. (3) Monitor the selectivity of novel small molecule antivirals in biochemical assays (using increasingly distant viral and human recombinant proteins) and cell-based viral propagation, as well as resistance mutation identification. We will express recombinant proteins or exploit assays from other projects and scientific cores to define specificity of viral enzyme and replication inhibition and viral mutational escape.

2019年冠状病毒病（COVID-19）是由严重急性呼吸道感染引起的持续大流行病， 冠状病毒2型综合征（SARS-CoV-2），目前死亡人数约为500万。只有有限的抗病毒药物 治疗方案的病人谁感染了疾病。2020年3月，我们启动了协作小 分子药物开发，包括Tuschl博士的实验室（高通量筛选（HTS）测定 开发），Glickman博士（HTS运营），Patel博士（结构生物学），Rice博士（病毒学）和Tri- 机构治疗发现研究所（药物化学），专注于两种SARS-CoV-2蛋白酶， NSP 3/PLpro和NSP 5/3CLpro，病毒多蛋白加工所需，cap N7-G甲基转移酶 （MTase）NSP 14，其是有效的病毒蛋白质合成和逃避宿主细胞先天免疫应答所需的， 以及对病毒复制重要的RNA解旋酶NSP 13。 我们开发了稳健的基于HTS荧光底物的检测方法，用于监测PLpro、3CLpro和 NSP 13活性以及用于监测NSP 14 N7-G-MTase活性的基于发光的测定。通过HTS， 洛克菲勒大学（RU）的430，000种化合物的多样性库，我们在所有测定中鉴定了命中， 验证命中，并追求最有前途的非共价命中药物化学计划。我们的首席 化合物对NSP 14和PLpro的IC 50值分别为2 nM和100 nM， SARS-CoV-2复制的EC 50值为80 nM和7 µM，无细胞毒性。 在这里，我们提出进一步的铅优化，以提高抑制剂的效力和药物一样的性质，以及 将我们的HTS专业知识应用于靶向蛋白酶，MTase和其他RNA病毒的解旋酶， 包括MERS、黄病毒和甲病毒。我们的具体目标是：（1）SARS-CoV-2的开发 NSP 14和PLpro临床候选物。以Alteror-PLpro和NSP 14的结构见解为指导 复合物，我们将进一步优化抑制剂的效力和药物样的特点与工业质量的药用 来自TDI和该计划的化学支持，以及开发3-5个高级电极导线的目标， 体外和体内DMPK/ADME-Tox研究，使用指定用于电极导线和电极导线的所有科学核心 优化（2）开发针对选择成员的MTase、蛋白酶和解旋酶的抗病毒先导系列 冠状病毒科、黄病毒科和披膜病毒科。我们将采用以下方法调整和开发HTS检测方法： 重组蛋白，并确定RU药物收集和伴随中的共价和非共价抑制剂 药物化学领先开发，为综合DMPK/ADME-Tox提供每个靶点3-5个导联 分析. (3)在生物化学测定中监测新型小分子抗病毒药物的选择性（使用 越来越远的病毒和人重组蛋白）和基于细胞的病毒繁殖，以及 抗性突变鉴定我们将表达重组蛋白或利用其他项目的检测方法 以及定义病毒酶的特异性、复制抑制和病毒突变逃逸的科学核心。