Discovery of Small-Molecule Inhibitors Against Norovirus 3CLpro Using Structure-Based Virtual Screening and FlipGFP Assay
Abstract
1. Introduction
2. Materials and Methods
2.1. Plasmid Construction
2.2. Cell Culture and Transfection
2.3. Fluorescence Imaging and Fluorescence Intensity Measurement
2.4. FlipGFP Assay
2.5. Docking Library Preparation and Molecular Docking
2.6. Molecular Dynamics Simulations
2.7. Western Blot
3. Results
3.1. Development of a Flipgfp Assay for Detecting Norovirus 3CLpro Inhibitors
3.2. Screening of 3CLpro Protease Inhibitors by Virtual Screening
3.3. Identifying Candidate Inhibitors Using a Cell-Based FlipGFP Assay
3.4. Crystal Structures of Norovirus 3CLpro in Complex with Hit Inhibitors
3.5. Molecular Dynamics Simulation of Hit Compound–3CLpro Complexes
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
3CLpro | 3C-like cysteine protease |
GFP | Green fluorescent protein |
WT | Wild-type |
HTS | High-throughput screening |
HAS | High-accuracy screening |
MD | Molecular dynamics |
RMSD | Root mean square deviation |
Rg | Radius of gyration |
RMSF | Root mean square fluctuation |
HIV | Human immunodeficiency virus |
HCV | Hepatitis C virus |
PROTAC | Proteolysis-targeting chimera |
EGFR | Epidermal growth factor receptor |
AR | Androgen receptor |
IKK | IκB kinase |
SASA | Solvent-accessible surface area |
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Library | Number | Description |
---|---|---|
Golden Scaffold Library | 5000 | Representative drug-like compounds selected from the ChemDiv core library, covering 5000 skeletal structures |
Selleck Compound Library | 20,550 | Representative bioactive molecules from the ChEMBL database |
Inhibitor Library | 7315 | Representative known inhibitors |
Immunology or Inflammation Compound Library | 14,036 | Multiple compounds related to immune inflammation from MCE Company |
Approved FDA Drugs Database | 11,935 | Preclinical, clinical, and FDA-approved compounds |
Total number of compounds | 58,836 |
PubChem ID | Compound Name | Max Affinity (kcal/mol) | H-Bonded Residues |
---|---|---|---|
2222112-77-6 | Bavdegalutamide | −12.41 | Thr81, Val82, Asn165 and Thr166 |
44516953 | Gedatolisib | −11.41 | Val82 |
1446090-79-4 | Lifirafenib | −11.15 | Val82, Arg100, Ser125 and Met130 |
2135642-56-5 | TAM-IN-2 | −11.13 | Thr84, Arg100, Ser125 and Met130 |
9549298 | IKK 16 | −11.12 | Thr103 |
1089283-49-7 | GSK1904529A | −11.08 | Arg100, Gly124, Ser125, Asn126 and Met130 |
139035057 | EGFR-IN-8 | −11.07 | Glu79 and Asn165 |
135398501 | Akt inhibitor VIII | −11.06 | Gly119 and Thr166 |
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Shen, H.; Liu, S.; Shang, L.; Liu, Y.; Sha, Y.; Lei, D.; Zhang, Y.; Jin, C.; Wu, S.; Zhang, M.; et al. Discovery of Small-Molecule Inhibitors Against Norovirus 3CLpro Using Structure-Based Virtual Screening and FlipGFP Assay. Viruses 2025, 17, 814. https://doi.org/10.3390/v17060814
Shen H, Liu S, Shang L, Liu Y, Sha Y, Lei D, Zhang Y, Jin C, Wu S, Zhang M, et al. Discovery of Small-Molecule Inhibitors Against Norovirus 3CLpro Using Structure-Based Virtual Screening and FlipGFP Assay. Viruses. 2025; 17(6):814. https://doi.org/10.3390/v17060814
Chicago/Turabian StyleShen, Hao, Shiqi Liu, Limin Shang, Yuchen Liu, Yijin Sha, Dingwei Lei, Yuehui Zhang, Chaozhi Jin, Shanshan Wu, Mingyang Zhang, and et al. 2025. "Discovery of Small-Molecule Inhibitors Against Norovirus 3CLpro Using Structure-Based Virtual Screening and FlipGFP Assay" Viruses 17, no. 6: 814. https://doi.org/10.3390/v17060814
APA StyleShen, H., Liu, S., Shang, L., Liu, Y., Sha, Y., Lei, D., Zhang, Y., Jin, C., Wu, S., Zhang, M., Wen, H., Jia, C., & Wang, J. (2025). Discovery of Small-Molecule Inhibitors Against Norovirus 3CLpro Using Structure-Based Virtual Screening and FlipGFP Assay. Viruses, 17(6), 814. https://doi.org/10.3390/v17060814