{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,24]],"date-time":"2026-08-24T23:54:37Z","timestamp":1787615677412,"version":"build-2736575974"},"reference-count":38,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2025,6,4]],"date-time":"2025-06-04T00:00:00Z","timestamp":1748995200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Guangzhou National Laboratory","award":["GZNL202301004"],"award-info":[{"award-number":["GZNL202301004"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Norovirus, a major cause of acute gastroenteritis, possesses a single-stranded positive-sense RNA genome. The viral 3C-like cysteine protease (3CLpro) plays a critical role in processing the viral polyprotein into mature non-structural proteins, a step essential for viral replication. Targeting 3CLpro has emerged as a promising strategy for developing small-molecule inhibitors against Norovirus. In this study, we employed a combination of virtual screening and the FlipGFP assay to identify potential inhibitors targeting the 3CLpro of Norovirus genotype GII.4. A library of approximately 58,800 compounds was screened using AutoDock Vina tool, yielding 20 candidate compounds based on their Max Affinity scores. These compounds were subsequently evaluated using a cell-based FlipGFP assay. Among them, eight compounds demonstrated significant inhibitory activity against 3CLpro, with Gedatolisib showing the most potent effect (IC50 = 0.06 \u00b1 0.01 \u03bcM). Molecular docking and molecular dynamics simulations were conducted to explore the binding mechanisms and structural stability of the inhibitor\u20133CLpro complexes. Our findings provide valuable insights into the development of antiviral drugs targeting Norovirus 3CLpro, offering potential therapeutic strategies to combat Norovirus infections.<\/jats:p>","DOI":"10.3390\/v17060814","type":"journal-article","created":{"date-parts":[[2025,6,4]],"date-time":"2025-06-04T08:51:04Z","timestamp":1749027064000},"page":"814","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Discovery of Small-Molecule Inhibitors Against Norovirus 3CLpro Using Structure-Based Virtual Screening and FlipGFP Assay"],"prefix":"10.3390","volume":"17","author":[{"given":"Hao","family":"Shen","sequence":"first","affiliation":[{"name":"School of Basic Medical Sciences, Anhui Medical University, Hefei 230032, China"},{"name":"State Key Laboratory of Medical Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing 102206, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shiqi","family":"Liu","sequence":"additional","affiliation":[{"name":"School of Basic Medical Sciences, Anhui Medical University, Hefei 230032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Limin","family":"Shang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Medical Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing 102206, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuchen","family":"Liu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Medical Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing 102206, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yijin","family":"Sha","sequence":"additional","affiliation":[{"name":"School of Basic Medical Sciences, Anhui Medical University, Hefei 230032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dingwei","family":"Lei","sequence":"additional","affiliation":[{"name":"School of Pharmaceutical Sciences, Peking University, Beijing 100091, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuehui","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Medical Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing 102206, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chaozhi","family":"Jin","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Medical Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing 102206, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shanshan","family":"Wu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Medical Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing 102206, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3513-7766","authenticated-orcid":false,"given":"Mingyang","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Medical Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing 102206, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Han","family":"Wen","sequence":"additional","affiliation":[{"name":"AI for Science Institute, Beijing 100085, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chenxi","family":"Jia","sequence":"additional","affiliation":[{"name":"School of Basic Medical Sciences, Anhui Medical University, Hefei 230032, China"},{"name":"State Key Laboratory of Medical Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing 102206, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8116-7691","authenticated-orcid":false,"given":"Jian","family":"Wang","sequence":"additional","affiliation":[{"name":"School of Basic Medical Sciences, Anhui Medical University, Hefei 230032, China"},{"name":"State Key Laboratory of Medical Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing 102206, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,6,4]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"860","DOI":"10.1002\/med.21545","article-title":"Norovirus antivirals: Where are we now?","volume":"39","author":"Netzler","year":"2019","journal-title":"Med. Res. Rev."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Bartsch, S.M., Lopman, B.A., Ozawa, S., Hall, A.J., and Lee, B.Y. (2016). Global Economic Burden of Norovirus Gastroenteritis. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0151219"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.jcv.2008.10.009","article-title":"Noroviruses: A comprehensive review","volume":"44","author":"Patel","year":"2009","journal-title":"J. Clin. Virol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"421","DOI":"10.1038\/nrmicro.2016.48","article-title":"Human norovirus transmission and evolution in a changing world","volume":"14","author":"Koopmans","year":"2016","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"233","DOI":"10.1016\/j.tim.2011.01.002","article-title":"Mechanisms of GII.4 norovirus evolution","volume":"19","author":"Bull","year":"2011","journal-title":"Trends Microbiol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"163","DOI":"10.3201\/eid3001.231003","article-title":"Emergence of Novel Norovirus GII.4 Variant","volume":"30","author":"Chhabra","year":"2024","journal-title":"Emerg. Infect. Dis."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1027","DOI":"10.1099\/jgv.0.001088","article-title":"Evolutionary dynamics of non-GII genotype 4 (GII.4) noroviruses reveal limited and independent diversification of variants","volume":"99","author":"Tohma","year":"2018","journal-title":"J. Gen. Virol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1900994","DOI":"10.1080\/19490976.2021.1900994","article-title":"Understanding the relationship between norovirus diversity and immunity","volume":"13","author":"Tohma","year":"2021","journal-title":"Gut Microbes"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"9438","DOI":"10.1021\/acs.jmedchem.5b00762","article-title":"Recent Advances in the Discovery of Norovirus Therapeutics","volume":"58","author":"Kim","year":"2015","journal-title":"J. Med. Chem."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"579","DOI":"10.1002\/prot.25682","article-title":"Putative structural rearrangements associated with the interaction of macrocyclic inhibitors with norovirus 3CL protease","volume":"87","author":"Weerawarna","year":"2019","journal-title":"Proteins"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Ebenezer, O., Jordaan, M.A., Damoyi, N., and Shapi, M. (2020). Discovery of Potential Inhibitors for RNA-Dependent RNA Polymerase of Norovirus: Virtual Screening, and Molecular Dynamics. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22010171"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"437","DOI":"10.1016\/j.virusres.2013.09.008","article-title":"Structural and inhibitor studies of norovirus 3C-like proteases","volume":"178","author":"Takahashi","year":"2013","journal-title":"Virus Res."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"11754","DOI":"10.1128\/JVI.01348-12","article-title":"Broad-spectrum antivirals against 3C or 3C-like proteases of picornaviruses, noroviruses, and coronaviruses","volume":"86","author":"Kim","year":"2012","journal-title":"J. Virol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"5315","DOI":"10.1016\/j.bmcl.2011.07.016","article-title":"Design, synthesis, and evaluation of inhibitors of Norwalk virus 3C protease","volume":"21","author":"Tiew","year":"2011","journal-title":"Bioorg Med. Chem. Lett."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"882","DOI":"10.1038\/nrd941","article-title":"Integration of virtual and high-throughput screening","volume":"1","author":"Bajorath","year":"2002","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"527","DOI":"10.1124\/pharmrev.120.000246","article-title":"Structure-Based Virtual Screening for Ligands of G Protein-Coupled Receptors: What Can Molecular Docking Do for You?","volume":"73","author":"Ballante","year":"2021","journal-title":"Pharmacol. Rev."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"269","DOI":"10.1016\/0022-2836(82)90153-X","article-title":"A geometric approach to macromolecule-ligand interactions","volume":"161","author":"Kuntz","year":"1982","journal-title":"J. Mol. Biol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"935","DOI":"10.1038\/nrd1549","article-title":"Docking and scoring in virtual screening for drug discovery: Methods and applications","volume":"3","author":"Kitchen","year":"2004","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"3676","DOI":"10.1093\/bioinformatics\/btw514","article-title":"PRODIGY: A web server for predicting the binding affinity of protein-protein complexes","volume":"32","author":"Xue","year":"2016","journal-title":"Bioinformatics"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1731","DOI":"10.1021\/ja026939x","article-title":"HADDOCK: A protein-protein docking approach based on biochemical or biophysical information","volume":"125","author":"Dominguez","year":"2003","journal-title":"J. Am. Chem. Soc."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1245","DOI":"10.1021\/acscentsci.1c00519","article-title":"Discovery of SARS-CoV-2 Papain-like Protease Inhibitors through a Combination of High-Throughput Screening and a FlipGFP-Based Reporter Assay","volume":"7","author":"Ma","year":"2021","journal-title":"ACS Cent. Sci."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"4526","DOI":"10.1021\/jacs.8b13042","article-title":"Designing a Green Fluorogenic Protease Reporter by Flipping a Beta Strand of GFP for Imaging Apoptosis in Animals","volume":"141","author":"Zhang","year":"2019","journal-title":"J. Am. Chem. Soc."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"529","DOI":"10.1080\/17460441.2016.1178231","article-title":"Current tools for norovirus drug discovery","volume":"11","author":"Weerasekara","year":"2016","journal-title":"Expert. Opin. Drug Discov."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"5050","DOI":"10.1128\/JVI.80.10.5050-5058.2006","article-title":"X-ray crystallographic structure of the Norwalk virus protease at 1.5-A resolution","volume":"80","author":"Zeitler","year":"2006","journal-title":"J. Virol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"4281","DOI":"10.1128\/JVI.02869-12","article-title":"Structural basis of substrate specificity and protease inhibition in Norwalk virus","volume":"87","author":"Muhaxhiri","year":"2013","journal-title":"J. Virol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"102323","DOI":"10.1016\/j.xpro.2023.102323","article-title":"FlipGFP protease assay for evaluating in vitro inhibitory activity against SARS-CoV-2 M(pro) and PL(pro)","volume":"4","author":"Tan","year":"2023","journal-title":"STAR Protoc."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"493","DOI":"10.1038\/s41586-024-07487-w","article-title":"Accurate structure prediction of biomolecular interactions with AlphaFold 3","volume":"630","author":"Abramson","year":"2024","journal-title":"Nature"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1186\/1758-2946-3-33","article-title":"Open Babel: An open chemical toolbox","volume":"3","author":"Banck","year":"2011","journal-title":"J. Cheminform"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"417","DOI":"10.1007\/s10822-010-9352-6","article-title":"Ligand docking and binding site analysis with PyMOL and Autodock\/Vina","volume":"24","author":"Seeliger","year":"2010","journal-title":"J. Comput. Aided Mol. Des."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"455","DOI":"10.1002\/jcc.21334","article-title":"AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading","volume":"31","author":"Trott","year":"2010","journal-title":"J. Comput. Chem."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"955","DOI":"10.1038\/nrd1907","article-title":"Tipranavir","volume":"4","author":"Flexner","year":"2005","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"35","DOI":"10.1038\/s41392-024-02075-w","article-title":"Hepatocellular carcinoma: Signaling pathways and therapeutic advances","volume":"10","author":"Zheng","year":"2025","journal-title":"Signal Transduct. Target. Ther."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"177","DOI":"10.1016\/j.jtho.2024.10.004","article-title":"Clinical Significance of a Prospective Large Genomic Screening for SCLC: The Genetic Classification and a Biomarker-Driven Phase 2 Trial of Gedatolisib","volume":"20","author":"Umemura","year":"2025","journal-title":"J. Thorac. Oncol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1856","DOI":"10.1093\/cvr\/cvad089","article-title":"Vascular smooth muscle cells specific deletion of angiopoietin-like protein 8 prevents angiotensin II-promoted hypertension and cardiovascular hypertrophy","volume":"119","author":"Jiao","year":"2023","journal-title":"Cardiovasc. Res."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"2140","DOI":"10.1200\/JCO.19.02654","article-title":"Phase I, Open-Label, Dose-Escalation\/Dose-Expansion Study of Lifirafenib (BGB-283), an RAF Family Kinase Inhibitor, in Patients With Solid Tumors","volume":"38","author":"Desai","year":"2020","journal-title":"J. Clin. Oncol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"111607","DOI":"10.1016\/j.ejmech.2019.111607","article-title":"1,2,4-Oxadiazole derivatives targeting EGFR and c-Met degradation in TKI resistant NSCLC","volume":"182","author":"Dokla","year":"2019","journal-title":"Eur. J. Med. Chem."},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"(2022). PROTAC Shrinks Mutated Prostate Tumors. Cancer Discov., 12, Of2.","DOI":"10.1158\/2159-8290.CD-NB2022-0020"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"563","DOI":"10.1038\/s41419-021-03843-0","article-title":"Epithelial PBLD attenuates intestinal inflammatory response and improves intestinal barrier function by inhibiting NF-\u03baB signaling","volume":"12","author":"Chen","year":"2021","journal-title":"Cell Death Dis."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/17\/6\/814\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T17:46:14Z","timestamp":1760031974000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/17\/6\/814"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,6,4]]},"references-count":38,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2025,6]]}},"alternative-id":["v17060814"],"URL":"https:\/\/doi.org\/10.3390\/v17060814","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,6,4]]}}}