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（SARS-CoV-2），COVID-19的病原体， 它深刻地影响了全球人类健康，并表明必须开发用于预防的抗病毒药物， 治疗靶向关键所需CoV复制功能并可口服的CoV。高度- 范德比尔特大学医学中心（VUMC）丹尼森实验室的CoV合作研究项目， 位于查佩尔山的Sheahan和Baric实验室30多年来一直是CoV复制的世界领导者， 演变、发病机制及对策。我们的项目领导了IND临床前研究， 核苷类似物抗病毒剂Remdesivir（RDV）和Molnupiravir（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）将并行进行，但高度 集成的抗病毒发现和开发项目集中在不同的复制酶功能上。项目1（本 项目）将集中在两个基本的病毒编码蛋白酶nsp 3-木瓜蛋白酶样蛋白酶（nsp 3-PLPro）和 nsp 5 - 3C样蛋白酶或主要蛋白酶（nsp 5 -3CLPro / Mpro），而项目2将针对抗病毒开发 nsp 12-RNA依赖性RNA聚合酶和nsp 13-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，药物开发， 冠状病毒蛋白酶生物学，以及来自学术界、工业界和 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， 靶向CoV蛋白酶的有效和广谱抗病毒药物，并设计增强活性和 防止出现对SARS-CoV-2和其他新出现的冠状病毒的耐药性。研究目的1 将发现和验证命中，并优先考虑和优化先导化合物。对于nsp 5 -3CLPro，我们将启动 使用来自Pardes Biosciences的已确定的先导化合物进行研究。对于nsp 3-PLPro，我们使用经验证的命中， 正在进行的基于片段结构的筛选方法的发现。我们将确定活动，广度， 在高通量病毒学测定中测定化合物的毒性，并优化铅的摄取和代谢特征。 目标2将使用先导化合物来确定病毒抗性的遗传基础，抗性突变体的病毒适应性， 和作用机制。我们将测试项目1的铅与其他蛋白酶抑制剂的组合， 核苷类似物（RDV、MPV）和项目2的先导化合物。在目标3中，我们将优化以下化合物的体内PK/PD： 化学先导物，确定优化的先导物在体内抗SARS-CoV-2和其他CoV的功效，确定 抗性对体内功效的影响，并测试组合的功效。我们已经有一个小组， 先导化合物和来自合作伙伴的针对nsp 5 -3CLPro和nsp 3-PLpro的经验证的命中， 目标2和3中的管道，以及多个早期开发候选项目。因此，项目1将具有 从基础发现到高级先导化合物的所有阶段，包括口服可用性、动物试验和 IND在项目开始时和整个项目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.