Research Project 1: Coronavirus antiviral lead development and combination testing

摘要 严重急性呼吸综合征冠状病毒2型，新冠肺炎的病原体有 深刻影响了全球人类健康，并表明必须开发抗病毒药物来预防和 治疗针对关键要求的冠状病毒复制功能并口服的冠状病毒。高度- 范德比尔特大学医学中心(VUMC)丹尼森实验室的协作CoV研究计划和 30多年来，北卡罗来纳大学教堂山分校的Sheahan和Baric实验室在冠状病毒复制方面一直处于世界领先地位， 演变、发病机制和对策。我们的计划领导了IND-Enabling临床前研究 核苷类似抗病毒药物雷米昔韦(RDV)和莫诺普拉韦(MPV)。对于当前提议的READDI-AC AVIDD计划，项目1(VUMC-Denison Pi)和项目2(UNC-Sheahan Pi)将并行但高度 综合抗病毒发现和开发项目侧重于不同的复制酶功能。项目1(此 项目)将重点介绍两种基本的病毒编码的蛋白水解酶nsp3-木瓜蛋白酶样酶(nsp3-plpro)和 NSP5-3C样蛋白酶或主要蛋白酶(NSP5-3CLPro/MPRO)，而项目2将以抗病毒开发为目标 对于依赖于nsp12-RNA的RNA聚合酶和nsp13-解旋酶。对于项目1，我们组建了一个团队 在最先进的药物设计和化学方面拥有丰富的经验和成就， 冠状病毒蛋白酶生物学和人体冠状病毒(HCoV)感染的体内模型 多程序核心。项目1的总体目标是发现和开发直接作用的、口服的、 针对冠状病毒蛋白水解酶的高效广谱抗病毒药物和设计的组合，可提高活性和 防止出现对SARS-CoV-2和其他新出现的冠状病毒的耐药性。AIM 1中的研究 将发现并验证命中结果，并对先导化合物进行优先排序和优化。对于nsp5-3CLPro，我们将启动 帕德斯生物科学公司已确定的先导化合物的研究。对于nsp3-plpro，我们使用经过验证的HITS和 从基于片段结构的筛选方法中不断发现。我们将确定活动、广度和 高通量病毒学分析中化合物的毒性，并优化铅的摄取和代谢谱。 目标2将使用先导化合物来确定病毒耐药性的遗传基础，耐药性突变体的病毒适合性， 和作用机制。我们将测试项目1中的铅与其他蛋白酶抑制剂的组合， 核苷类似物(RDV，MPV)，以及来自项目2的先导。在目标3，我们将优化在体内的PK/PD 化学引线，测定优化后的引线在体内对SARS-CoV-2和其他冠状病毒的效力，确定 观察抗性对体内药效的影响，并检验联合药效。我们已经有了一个小组 来自合作伙伴的先导化合物和验证的命中，以对抗将进入 目标2和目标3中的流水线，以及多个早期热门候选开发。因此，项目1将具有 化合物的所有阶段，从基础发现到口服可用的高级线索，动物试验和 IND能够在项目开始和整个过程中进行药代动力学研究。

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiologic agent of COVID-19 has profoundly impacted global human health and shown that it is imperative to develop antivirals for prevention and treatment of CoVs that are targeted to key required CoV replication functions and are orally available. The highly- collaborative CoV research programs in the Denison lab at Vanderbilt University Medical Center (VUMC) and the Sheahan and Baric labs at UNC Chapel Hill have been world leaders for over 30 years on CoV replication, evolution, pathogenesis, and countermeasures. Our programs led IND-enabling preclinical studies for the nucleoside analog antivirals remdesivir (RDV) and molnupiravir (MPV). For the current proposed READDI-AC AVIDD program, Project 1 (VUMC-Denison PI) and Project 2 (UNC-Sheahan PI) will pursue parallel but highly integrated antiviral discovery and development projects focused on distinct replicase functions. Project 1 (this project) will focus on the two essential virus encoded proteases nsp3-papain like protease (nsp3-PLPro) and nsp5-3C-like protease or main protease (nsp5-3CLPro / Mpro), while Project 2 will target antiviral development for the nsp12-RNA-dependent RNA polymerase and nsp13-helicase. For Project 1, we have assembled a team with deep experience and achievement in state-of-the-art drug design and chemistry, drug development, coronavirus protease biology, and in vivo models of human CoV (HCoV) infection from academia, industry and multiple-program Cores. The overall goal of Project 1 is to discover and develop direct-acting, orally-available, potent and broad-spectrum antivirals targeting CoV proteases and design combinations that boost activity and prevent the emergence of resistance against SARS-CoV-2 and other emerging coronaviruses. Studies in Aim 1 will discover and validate hits and prioritize and optimize lead compounds. For nsp5-3CLPro, we will initiate studies with established lead compounds from Pardes Biosciences. For nsp3-PLPro we use validated hits and ongoing discovery from a fragment-structure-based screening approach. We will determine activity, breadth, and toxicity of compounds in high-throughput virological assays and optimize uptake and metabolic profile of leads. Aim 2 will use lead compounds to define the genetic basis for viral resistance, viral fitness of resistance mutants, and mechanism of action. We will test leads from Project 1 in combination with other protease inhibitors, nucleoside analogs (RDV, MPV), and leads from Project 2. In Aim 3, we will optimize the in vivo PK/PD of chemical leads, determine the efficacy of optimized leads against SARS-CoV-2 and other CoV in vivo, determine the effect of resistance on in vivo efficacy, and test the efficacy of combinations. We already have a panel of lead compounds and validated hits from partners against both nsp5-3CLPro and nsp3-PLpro that will enter the pipeline in Aims 2 and 3, as well as multiple early hit candidates for development. Thus, Project 1 will have compounds at all stages from fundamental discovery to advanced leads with oral availability, animal testing and IND enabling pharmacokinetic studies at the outset and through the course of the project.