PROJECT SUMMARY Human coronaviruses generally cause the common cold, a mild upper respiratory illness, however, global outbreaks of new human coronavirus infections with severe respiratory disease have periodically emerged from animals. These include Severe Acute Respiratory coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and, most recently, SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19). Currently, there are no licensed vaccines or antiviral drugs against these viruses, underscoring an urgent need for the development of preventive and therapeutic measures against coronaviruses. Coronavirus genomes encode large polyproteins which are processed by a 3C-like protease (3CLpro) and a papain-like protease. Both proteases are essential for viral replication, making them attractive targets for drug development. Our foray in this area has resulted in the discovery of broad-spectrum inhibitors of multiple viruses, including coronaviruses and noroviruses that encode 3CLpro, as well as the first demonstration of clinical efficacy by a feline coronavirus 3CLpro inhibitor. Recently, we have demonstrated that a dipeptidyl series of compounds potently inhibit human coronaviruses, including MERS-CoV and SARS-CoV-2 in cell culture, and display in vivo efficacy in the DPP4-KI mouse model of MERS-CoV infection. The antiviral target of the compounds was validated by obtaining high resolution crystal structures 3CLpro-inhibitor complexes from SARS-CoV, SARS- CoV-2 and MERS-CoV. We hypothesize herein that the identified series can serve as a launching pad for the development of SARS-CoV-2-specific antivirals. The immediate and overarching goal of the proposed studies is to further optimize the pharmacological activity PK parameters of identified lead inhibitors of SARS-CoV-2 3CLpro and the demonstration of in vivo efficacy against SARS-CoV-2. The expected outcome of our studies is the selection of a preclinical candidate (and 1-2 backup compounds) that is well-suited to conducting further preclinical studies, ultimately leading to the development of a COVID-19-specific antiviral therapeutic.