PROJECT SUMMARY/ABSTRACT COVID-19 was first identified in December 2019 in Wuhan, Hubei province, China, resulting in the ongoing 2019- 2020 pandemic. COVID-19 is caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV- 2). Common symptoms of the disease include fever, dry cough, shortness of breath, diarrhea, and loss of smell. Complications may include pneumonia, viral sepsis, and acute respiratory distress syndrome. As of today, other than remdesivir, there is no approved small molecule drug for the treatment of COVID-19 and the discovery of an effective vaccine remains uncertain. Our long-term goal is to develop antiviral drugs for the treatment of COVID-19 and human coronavirus infections in general. Our central hypothesis is that inhibition of SARS-CoV- 2 polyprotein cleavage results in the prevention and early treatment of COVID-19 before it progresses to its more severe form. We will identify nanomolar inhibitors of the CoV 3C-like protease (3CLpro) suitable to be developed as antiviral agents for the treatment of COVID-19 and other coronavirus infections. The proposal targets the 3CLpro, a key enzyme for SARS-CoV-2 polyprotein cleavage and viral replication. Our overall premise is that small molecule inhibitors targeting this essential viral enzyme will inhibit replication, and therefore have the potential to be of both preventive and therapeutic value. Thus, our primary objective is to design and develop structure-based small-molecule inhibitors targeting coronavirus 3CLpro using our established and proven drug discovery expertise. Guided by strong preliminary data, the inhibition of polyprotein cleavage hypothesis will be tested by pursuing three specific aims: Aim 1) To inhibit SARS-CoV-2 polyprotein cleavage by developing covalent peptidic inhibitors of 3CLpro (nsp5).; Aim 2) To inhibit SARS-CoV-2 polyprotein cleavage by developing noncovalent nonpeptidic inhibitors of 3CLpro (nsp5).; and Aim 3) To determine the efficacy of covalent and noncovalent SARS-CoV-2 3CLpro inhibitors in a golden hamster model. Under the first aim, lead compound 3150 and its analogs will be tested in viral and enzyme assays for inhibitory activity of SARS-CoV-2 3CLpro. An aqueous soluble form of 3150 will be evaluated in the animal model. Structure-based drug design approaches will be employed to optimize 3150 for binding to the crystal structure of SARS-CoV-2 3CLpro. Under aim 2, Structure-based virtual screening and hybrid ligand screening approaches along with medicinal chemistry will be used to prepare and evaluate noncovalent nonpeptidic inhibitors of 3CLpro. Under the third aim, top-ranked SARS-CoV-2 3CLpro covalent and noncovalent inhibitors will be tested for pharmacokinetics and efficacy in a golden hamster COVID-19 model. The ultimate goal of the proposed studies is to advance an anti-COVID-19 drug candidate to the stage of filing an investigational new drug (IND) application. Overall, the results of this project will have a signifi...