Abstract Title
Broad-Spectrum Inhibition of Norovirus Protease Across Diverse Genotypes by Substrate-Mimetic Compounds
Presenter
Ladislau Kovari, Wayne State University
Co-Author(s)
Franck Amblard2, Dharmeshkumar Patel2, Shaoman Zhou2, Hongwang Zhang2, Chengwei Li2, Niloufar Azadi2, Tamara R. McBrayer2, Michael Muczynski1, Abdullah Al-Homoudi1, Joseph Engel1, Raymond F. Schinazi2, Christiane E. Wobus3
1Department of Biochemistry, Microbiology, and Immunology, Wayne State University School of Medicine, Detroit, MI 48201, USA
2Center for ViroScience and Cure, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, and Children’s Healthcare of Atlanta, GA 30322, USA
3Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109, USA
Abstract Category
Prevention & Control (antivirals)
Abstract
Background and Significance: Norovirus is a major global cause of acute viral gastroenteritis. Despite its health burden and genetic diversity, no approved antiviral therapies exist.
Objective: We evaluated substrate-mimetic small-molecule inhibitors targeting the norovirus NS6 protease (PR) across diverse human norovirus genotypes.
Methods: NIP-22c and CIP-1, previously reported as low-nanomolar inhibitors, covalently bind the NS6 PR active site Cys139. Molecular docking and molecular dynamics simulations utilized our 2.4 Å crystal structure (PDB: 6B6I). Protease variants from genotypes GI.1, GII.4, GII.17, and GIV.1 were expressed using a His-SUMO-NS6 system. Protease inhibitory activity was evaluated with a FRET-based assay, and antiviral efficacy was assessed in the HNoV GI.1 replicon system. Cytotoxicity was measured in primary human lymphocytes, CEM, Huh-7, Vero, Caco-2, and Calu-3 cells. Toxicity and antiviral efficacy against GII.4 were further evaluated in 3D human intestinal enteroids (HIE), a physiologically relevant model of the intestinal epithelium.
Results: NIP-22c and CopSi inhibited multiple NoV genotypes by blocking polyprotein processing via NS6 PR. Despite sequence and structural variation among genogroups, the catalytic triad (His30, Glu54, Cys139) is conserved, supporting the feasibility of broad-spectrum inhibitor design. These compounds are potent and selective, showing promising antiviral activity in both cell-based and HIE systems.
Conclusions: These findings support the development of low-nanomolar, broad-spectrum NS6 PR inhibitors as candidate therapeutics.
Acknowledgments: Supported by NIAID/NIH R01-AI-173229 (RFS, LCK, CEW).
Objective: We evaluated substrate-mimetic small-molecule inhibitors targeting the norovirus NS6 protease (PR) across diverse human norovirus genotypes.
Methods: NIP-22c and CIP-1, previously reported as low-nanomolar inhibitors, covalently bind the NS6 PR active site Cys139. Molecular docking and molecular dynamics simulations utilized our 2.4 Å crystal structure (PDB: 6B6I). Protease variants from genotypes GI.1, GII.4, GII.17, and GIV.1 were expressed using a His-SUMO-NS6 system. Protease inhibitory activity was evaluated with a FRET-based assay, and antiviral efficacy was assessed in the HNoV GI.1 replicon system. Cytotoxicity was measured in primary human lymphocytes, CEM, Huh-7, Vero, Caco-2, and Calu-3 cells. Toxicity and antiviral efficacy against GII.4 were further evaluated in 3D human intestinal enteroids (HIE), a physiologically relevant model of the intestinal epithelium.
Results: NIP-22c and CopSi inhibited multiple NoV genotypes by blocking polyprotein processing via NS6 PR. Despite sequence and structural variation among genogroups, the catalytic triad (His30, Glu54, Cys139) is conserved, supporting the feasibility of broad-spectrum inhibitor design. These compounds are potent and selective, showing promising antiviral activity in both cell-based and HIE systems.
Conclusions: These findings support the development of low-nanomolar, broad-spectrum NS6 PR inhibitors as candidate therapeutics.
Acknowledgments: Supported by NIAID/NIH R01-AI-173229 (RFS, LCK, CEW).