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.