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

冠状病毒病2019(新冠肺炎)代表由严重急性呼吸系统疾病引起的持续大流行 综合症冠状病毒2(SARS-CoV-2)，目前死亡人数约为500万人。只有有限的抗病毒药物 为感染这种疾病的患者提供治疗选择。2020年3月，我们发起了合作小规模 分子药物开发包括Tuschl博士的实验室(高通量筛选(HTS)分析 发展)，格利克曼博士(HTS操作)，帕特尔博士(结构生物学)，赖斯博士(病毒学)，和三个- 机构治疗发现研究所(药物化学)，专注于两种SARS-CoV-2蛋白酶， 病毒多蛋白加工所需的NSP3/PLPro和NSP5/3CLPro，CAP N7-G甲基转移酶 (MTase)NSP14是有效的病毒蛋白质合成和逃避宿主细胞天然免疫反应所必需的， 以及对病毒复制至关重要的RNA解旋酶NSP13。 我们开发了基于高温超导荧光底物的可靠检测方法，用于监测PLPro、3CLPro和 NSP13活性以及基于发光的检测NSP14 N7-G-MTase活性的方法。由HTS Of 洛克菲勒大学(RU)提供了包含43万种化合物的多样化库，我们在所有分析中都确定了匹配， 验证HITS，并在药物化学计划中追求最有前途的非共价HITS。我们的领先优势 化合物对NSP14和PLPro的IC50值分别为2 nM和100 nM，并基于细胞抑制 对SARS-CoV-2复制的EC50值分别为80毫微米和7微米，无细胞毒性。 在这里，我们建议进一步的先导优化，以提高抑制剂的效力和类药物的性质 AS应用我们的HTS专业知识来靶向具有大流行的其他RNA病毒的蛋白酶、MTase和解旋酶 潜在的，包括中东呼吸综合征、黄病毒和甲型病毒。我们的具体目标是：(1)SARS-CoV-2的发展 NSP14和PLPro临床候选。由抑制剂PLPro和NSP14的结构见解指导 对于复合体，我们将以行业质量的药品进一步优化抑制剂的效力和类药物特性 来自TDI和该计划的化学支持，以及开发3-5条高级线索的目标 利用所有用于Hit-to-Lead和Lead的科学核心进行的体外和体内DMPK/ADME-Tox研究 优化(2)开发针对特定成员的MTase、蛋白酶和解旋酶的抗病毒先导系列 属于冠状病毒科、黄病毒科和鹦鹉病毒科。我们将调整和开发HTS检测方法，使用 重组蛋白和鉴定RU药物收集和伴随中的共价和非共价抑制剂 药物化学领先发展，为综合DMPK/ADME-Tox提供每个靶点3-5个先导 分析。(3)在生化分析中监测新型小分子抗病毒药物的选择性(使用 越来越遥远的病毒和人类重组蛋白)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开发其他项目的分析方法 以及确定病毒酶的特异性、复制抑制和病毒突变逃逸的科学核心。

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.