PROJECT SUMMARY/ABSTRACT Human Noroviruses (hNoV), which belong to the Caliciviridae family, are the leading cause of viral gastroenteritis and food-borne disease worldwide. Each year in the United States alone, hNoV is responsible for greater than 21 million cases of acute gastroenteritis, leading to an estimated 71,000 hospitalizations per year. While most cases resolve within a week, immunocompromised patients, children, and the elderly have an elevated risk of long-term and even fatal infections. Currently, there are no antiviral drugs or vaccines approved for the prevention or treatment of chronic hNoV infections. The main goal of this project is to harness the medical benefits that are offered by the inhibition of viral enzymes, including the hNoV protease. The overall objective of this proposal is to design and develop a protease inhibitor for the treatment of hNoV infections. The hNoV NS6 protease has become an attractive drug target due to its essential role in the viral replication cycle. The central hypothesis is that small-molecule inhibitors targeting the viral protease will limit or eliminate hNoV infections. The rationale for the proposed research is based on our preliminary data, specifically compound 1 and its analogs. These compounds are unique, non-toxic, small peptidomimetic molecules that inhibit NoV replication selectively in culture at 20 nM EC50 by interacting with the hNoV protease. Pharmacological manipulation of the hNoV protease is expected to result in new and innovative approaches to the prevention and treatment of NoV infections. Guided by strong preliminary data, this hypothesis will be tested by pursuing three specific aims: Aim 1) To optimize and discover and develop broad-spectrum oral NoV PIs by multiparameter optimization and molecular modeling approaches of lead compounds from our proprietary library. Aim 2) To perform mechanism of action (MOA) and resistance studies of the lead NoV PIs. Aim 3) To evaluate the lead NoV PIs in an immunocompromised mouse model. Under the first aim, about 25 compounds will be designed, synthesized, and tested per year. Under the second aim, enzyme kinetic, crystallographic, host protease inhibition, inhibition of NoV protease production, and drug resistance studies will be performed with the lead compounds. Under the third aim, anti-NoV efficacy studies will be performed in an immunodeficient mouse model. This approach is innovative because we developed a research plan taking full advantage of five recent advances: (1) producing hNoV PR recombinant proteins in milligram amounts; (2) solving the crystal structure of the NoV GII.4 PR - the main drug target; (3) a cell-based hNoV replicon assay; (4) 3D human intestinal enteroid model, and (5) an immunocompromised mouse disease model to study human NoV infection. The proposed research is significant because in-depth pre-clinical studies and characterization of these new peptidomimetics could lead to the approval of the first safe an...