{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,27]],"date-time":"2026-05-27T13:11:40Z","timestamp":1779887500388,"version":"3.53.1"},"reference-count":35,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2025,6,12]],"date-time":"2025-06-12T00:00:00Z","timestamp":1749686400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Natural Science Foundation of China","award":["22407078"],"award-info":[{"award-number":["22407078"]}]},{"name":"National Natural Science Foundation of China","award":["SYS202205"],"award-info":[{"award-number":["SYS202205"]}]},{"name":"National Natural Science Foundation of China","award":["ZR2022QH312"],"award-info":[{"award-number":["ZR2022QH312"]}]},{"name":"Shandong Laboratory Program","award":["22407078"],"award-info":[{"award-number":["22407078"]}]},{"name":"Shandong Laboratory Program","award":["SYS202205"],"award-info":[{"award-number":["SYS202205"]}]},{"name":"Shandong Laboratory Program","award":["ZR2022QH312"],"award-info":[{"award-number":["ZR2022QH312"]}]},{"name":"Natural Science Foundation of Shandong Province","award":["22407078"],"award-info":[{"award-number":["22407078"]}]},{"name":"Natural Science Foundation of Shandong Province","award":["SYS202205"],"award-info":[{"award-number":["SYS202205"]}]},{"name":"Natural Science Foundation of Shandong Province","award":["ZR2022QH312"],"award-info":[{"award-number":["ZR2022QH312"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Human noroviruses (HuNoVs) are the primary cause of acute viral gastroenteritis. There are no antivirals or vaccines available to treat and\/or prevent HuNoV. Norovirus 3C-like protease (3CLpro) is essential for viral replication; consequently, the inhibition of this enzyme is a fruitful avenue for antinorovirus therapeutics. To discover novel 3CLpro inhibitors with diverse scaffolds, a multi-stage virtual screening approach was performed by docking &gt;10 million compounds into the 3CLpro catalytic site. An initial subset of 18 compounds was selected, and compounds YY-1029 and YY-4204 were identified as the best two molecules. Molecular dynamics (MD) simulations and binding free energy calculations (MM\/GBSA) of YY-1029 and YY-4204 were performed to elucidate the binding mechanisms. The ADMET properties were also estimated to predict the potential druggability of representative molecules. All 18 compounds were evaluated for their antinorovirus activity and cytotoxicity in a cell-based replicon system. This work could provide information for the development of 3CL pro inhibitors.<\/jats:p>","DOI":"10.3390\/ijms26125625","type":"journal-article","created":{"date-parts":[[2025,6,12]],"date-time":"2025-06-12T03:59:17Z","timestamp":1749700757000},"page":"5625","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Discovery of Small Molecules Targeting Norovirus 3CL Protease by Multi-Stage Virtual Screening"],"prefix":"10.3390","volume":"26","author":[{"given":"Zhongling","family":"Shi","sequence":"first","affiliation":[{"name":"Department of Medicinal Chemistry, Shandong Key Laboratory of Druggability Optimization and Evaluation for Lead Compounds, School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, 44 West Culture Road, Jinan 250012, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Na","family":"Liu","sequence":"additional","affiliation":[{"name":"Department of Medicinal Chemistry, Shandong Key Laboratory of Druggability Optimization and Evaluation for Lead Compounds, School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, 44 West Culture Road, Jinan 250012, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fabao","family":"Zhao","sequence":"additional","affiliation":[{"name":"Department of Medicinal Chemistry, Shandong Key Laboratory of Druggability Optimization and Evaluation for Lead Compounds, School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, 44 West Culture Road, Jinan 250012, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9232-953X","authenticated-orcid":false,"given":"Dongwei","family":"Kang","sequence":"additional","affiliation":[{"name":"Department of Medicinal Chemistry, Shandong Key Laboratory of Druggability Optimization and Evaluation for Lead Compounds, School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, 44 West Culture Road, Jinan 250012, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3872-6558","authenticated-orcid":false,"given":"Steven","family":"De Jonghe","sequence":"additional","affiliation":[{"name":"Molecular, Structural and Translational Virology Research Group, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, KU Leuven, Herestraat 49, B-3000 Leuven, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0033-7514","authenticated-orcid":false,"given":"Johan","family":"Neyts","sequence":"additional","affiliation":[{"name":"Antiviral Drug & Vaccine Research Group, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, KU Leuven, Herestraat 49, B-3000 Leuven, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ni","family":"Gao","sequence":"additional","affiliation":[{"name":"Department of Medicinal Chemistry, Shandong Key Laboratory of Druggability Optimization and Evaluation for Lead Compounds, School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, 44 West Culture Road, Jinan 250012, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7302-2214","authenticated-orcid":false,"given":"Xinyong","family":"Liu","sequence":"additional","affiliation":[{"name":"Department of Medicinal Chemistry, Shandong Key Laboratory of Druggability Optimization and Evaluation for Lead Compounds, School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, 44 West Culture Road, Jinan 250012, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,6,12]]},"reference":[{"key":"ref_1","unstructured":"WHO (2022). Immunization, Vaccines and Biologicals."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"539","DOI":"10.1080\/14760584.2020.1777860","article-title":"Recent advances in human norovirus research and implications for candidate vaccines","volume":"19","author":"Cates","year":"2020","journal-title":"Expert Rev. Vaccines"},{"key":"ref_3","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_4","doi-asserted-by":"crossref","unstructured":"Lin, S.-C., Bai, G.-H., Lin, P.-C., Chen, C.-Y., Hsu, Y.-H., Lee, Y.-C., and Chen, S.-Y. (2023). Molecular and Genetics-Based Systems for Tracing the Evolution and Exploring the Mechanisms of Human Norovirus Infections. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms24109093"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Ushijima, H., Hoque, S.A., Akari, Y., Pham, N.T.K., Phan, T., Nishimura, S., Kobayashi, M., Sugita, K., Okitsu, S., and Komoto, S. (2024). Molecular Evolution of GII.P31\/GII.4_Sydney_2012 Norovirus over a Decade in a Clinic in Japan. Int. J. Mol. Sci., 25.","DOI":"10.3390\/ijms25073619"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Campillay-V\u00e9liz, C.P., Carvajal, J.J., Avellaneda, A.M., Escobar, D., Covi\u00e1n, C., Kalergis, A.M., and Lay, M.K. (2020). Human Norovirus Proteins: Implications in the Replicative Cycle, Pathogenesis, and the Host Immune Response. Front. Immunol., 11.","DOI":"10.3389\/fimmu.2020.00961"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1393","DOI":"10.1099\/jgv.0.001318","article-title":"Updated classification of norovirus genogroups and genotypes","volume":"100","author":"Chhabra","year":"2019","journal-title":"J. Gen. Virol."},{"key":"ref_8","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_9","doi-asserted-by":"crossref","first-page":"13685","DOI":"10.1128\/JVI.79.21.13685-13693.2005","article-title":"A norovirus protease structure provides insights into active and substrate binding site integrity","volume":"79","author":"Nakamura","year":"2005","journal-title":"J. Virol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"509","DOI":"10.1093\/jb\/mvp094","article-title":"Insights into the enzyme-substrate interaction in the norovirus 3C-like protease","volume":"146","author":"Someya","year":"2009","journal-title":"J. Biochem."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Chang, K.O., Kim, Y., Lovell, S., Rathnayake, A.D., and Groutas, W.C. (2019). Antiviral Drug Discovery: Norovirus Proteases and Development of Inhibitors. Viruses, 11.","DOI":"10.3390\/v11020197"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Van Dycke, J., Dai, W., Stylianidou, Z., Li, J., Cuvry, A., Roux, E., Li, B., Rymenants, J., Bervoets, L., and de Witte, P. (2021). A Novel Class of Norovirus Inhibitors Targeting the Viral Protease with Potent Antiviral Activity In Vitro and In Vivo. Viruses, 13.","DOI":"10.3390\/v13091852"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"11945","DOI":"10.1021\/acs.jmedchem.0c01252","article-title":"Structure-Guided Optimization of Dipeptidyl Inhibitors of Norovirus 3CL Protease","volume":"63","author":"Rathnayake","year":"2020","journal-title":"J. Med. Chem."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"6239","DOI":"10.1021\/acs.jmedchem.7b00497","article-title":"Design, Synthesis, and Evaluation of Novel Prodrugs of Transition State Inhibitors of Norovirus 3CL Protease","volume":"60","author":"Kim","year":"2017","journal-title":"J. Med. Chem."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"3144","DOI":"10.1021\/jm5019934","article-title":"Structure-guided design and optimization of dipeptidyl inhibitors of norovirus 3CL protease. Structure-activity relationships and biochemical, X-ray crystallographic, cell-based, and in vivo studies","volume":"58","author":"Kim","year":"2015","journal-title":"J. Med. Chem."},{"key":"ref_16","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_17","doi-asserted-by":"crossref","unstructured":"Wang, Y., Xu, B., Ma, S., Wang, H., Shang, L., Zhu, C., and Ye, S. (2022). Discovery of SARS-CoV-2 3CL(Pro) Peptidomimetic Inhibitors through the Catalytic Dyad Histidine-Specific Protein-Ligand Interactions. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms23042392"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"452","DOI":"10.1038\/s41586-021-04220-9","article-title":"Synthon-based ligand discovery in virtual libraries of over 11 billion compounds","volume":"601","author":"Sadybekov","year":"2022","journal-title":"Nature"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Gorgulla, C., Jayaraj, A., Fackeldey, K., and Arthanari, H. (2022). Emerging frontiers in virtual drug discovery: From quantum mechanical methods to deep learning approaches. Curr. Opin. Chem. Biol., 69.","DOI":"10.1016\/j.cbpa.2022.102156"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Obaid, R.J., Shafie, A., Malik, M.S., Al-Rooqi, M.M., Moussa, Z., Abdulaziz, O., Aljuaid, A., Allahyani, M., Almehmadi, M., and Anjum, F. (2023). In Silico Screening and Molecular Dynamics Simulation Studies in the Identification of Natural Compound Inhibitors Targeting the Human Norovirus RdRp Protein to Fight Gastroenteritis. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms24055003"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Ebenezer, O., Jordaan, M.A., Damoyi, N., and Shapi, M. (2021). 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_22","doi-asserted-by":"crossref","unstructured":"Pitasi, G., Brancale, A., Floris, S., Fais, A., Gitto, R., and De Luca, L. (2024). Computational Approach to Identifying New Chemical Entities as Elastase Inhibitors with Potential Antiaging Effects. Int. J. Mol. Sci., 25.","DOI":"10.3390\/ijms252011174"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Suenaga, A., Okimoto, N., Hirano, Y., and Fukui, K. (2012). An efficient computational method for calculating ligand binding affinities. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0042846"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"6245","DOI":"10.1080\/07391102.2023.2242950","article-title":"Computational screening and MM\/GBSA-based MD simulation studies reveal the high binding potential of FDA-approved drugs against Cutibacterium acnes sialidase","volume":"42","author":"Singh","year":"2024","journal-title":"J. Biomol. Struct. Dyn."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"2489","DOI":"10.1021\/acs.jmedchem.0c02227","article-title":"Decision Making in Structure-Based Drug Discovery: Visual Inspection of Docking Results","volume":"64","author":"Fischer","year":"2021","journal-title":"J. Med. Chem."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1021\/jp992433y","article-title":"Understanding Modern Molecular Dynamics:\u2009 Techniques and Applications","volume":"104","author":"Tuckerman","year":"2000","journal-title":"J. Phys. Chem. B"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"7986","DOI":"10.1080\/07391102.2022.2127907","article-title":"In silico, in vitro VEGFR-2 inhibition, and anticancer activity of a 3-(hydrazonomethyl)naphthalene-2-ol derivative","volume":"41","author":"Elkaeed","year":"2023","journal-title":"J. Biomol. Struct. Dyn."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"108285","DOI":"10.1016\/j.isci.2023.108285","article-title":"ADMET property prediction via multi-task graph learning under adaptive auxiliary task selection","volume":"26","author":"Du","year":"2023","journal-title":"iScience"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1473","DOI":"10.1080\/17460441.2020.1798926","article-title":"Open access in silico tools to predict the ADMET profiling of drug candidates","volume":"15","author":"Kar","year":"2020","journal-title":"Expert Opin. Drug Discov."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"De Carlo, A., Ronchi, D., Piastra, M., Tosca, E.M., and Magni, P. (2024). Predicting ADMET Properties from Molecule SMILE: A Bottom-Up Approach Using Attention-Based Graph Neural Networks. Pharmaceutics, 16.","DOI":"10.3390\/pharmaceutics16060776"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"W422","DOI":"10.1093\/nar\/gkae236","article-title":"ADMETlab 3.0: An updated comprehensive online ADMET prediction platform enhanced with broader coverage, improved performance, API functionality and decision support","volume":"52","author":"Fu","year":"2024","journal-title":"Nucleic. Acids Res."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"W257","DOI":"10.1093\/nar\/gky318","article-title":"ProTox-II: A webserver for the prediction of toxicity of chemicals","volume":"46","author":"Banerjee","year":"2018","journal-title":"Nucleic Acids Res."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"777","DOI":"10.1016\/j.bbrc.2012.07.034","article-title":"Favipiravir (T-705) inhibits in vitro norovirus replication","volume":"424","author":"Jochmans","year":"2012","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"11798","DOI":"10.1128\/JVI.02064-13","article-title":"The viral polymerase inhibitor 2\u2032-C-methylcytidine inhibits Norwalk virus replication and protects against norovirus-induced diarrhea and mortality in a mouse model","volume":"87","author":"Jochmans","year":"2013","journal-title":"J. Virol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"20402066211026852","DOI":"10.1177\/20402066211026852","article-title":"Assessment of the anti-norovirus activity in cell culture using the mouse norovirus: Identification of active compounds","volume":"29","author":"Rymenants","year":"2021","journal-title":"Antivir. Chem. Chemother."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/12\/5625\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T17:50:32Z","timestamp":1760032232000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/12\/5625"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,6,12]]},"references-count":35,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2025,6]]}},"alternative-id":["ijms26125625"],"URL":"https:\/\/doi.org\/10.3390\/ijms26125625","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,6,12]]}}}