{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,28]],"date-time":"2026-09-28T09:49:40Z","timestamp":1790588980088,"version":"4.1.0"},"reference-count":42,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2024,5,27]],"date-time":"2024-05-27T00:00:00Z","timestamp":1716768000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"French cancer league (Comit\u00e9 17 de la Ligue Nationale contre le Cancer)","award":["ANR-22-CE18-0028"],"award-info":[{"award-number":["ANR-22-CE18-0028"]}]},{"name":"Project NANOLIGO","award":["ANR-22-CE18-0028"],"award-info":[{"award-number":["ANR-22-CE18-0028"]}]},{"name":"French Ministry of Research and Higher Education","award":["ANR-22-CE18-0028"],"award-info":[{"award-number":["ANR-22-CE18-0028"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Marine Drugs"],"abstract":"<jats:p>Brown seaweeds of the Fucus genus represent a rich source of natural antiviral products. In this study, a Fucus ceranoides hydroalcoholic extract (FCHE) was found to inhibit 74.2 \u00b1 1.3% of the proteolytic activity of the free SARS-CoV-2 3CL protease (3CLpro), an enzyme that plays a pivotal role in polyprotein processing during coronavirus replication and has been identified as a relevant drug discovery target for SARS- and MERS-CoVs infections. To purify and identify 3CLpro ligands with potential inhibitory activity using a one-step approach, we immobilized the enzyme onto magnetic microbeads (3CLpro-MPs), checked that the enzymatic activity was maintained after grafting, and used this bait for a ligand-fishing strategy followed by a high-resolution mass spectrometry analysis of the fished-out molecules. Proof of concept for the ligand-fishing capacity of the 3CLpro-MPs was demonstrated by doping the FCHE extract with the substrate peptide TSAVLQ-pNA, resulting in the preferential capture of this high-affinity peptide within the macroalgal complex matrix. Ligand fishing in the FCHE alone led to the purification and identification via high-resolution mass spectrometry (HRMS) of seven hepta-, octa-, and decapeptides in an eluate mix that significantly inhibited the free 3CLpro more than the starting FCHE (82.7 \u00b1 2.2% inhibition). Molecular docking simulations of the interaction between each of the seven peptides and the 3CLpro demonstrated a high affinity for the enzyme\u2019s proteolytic active site surpassing that of the most affine peptide ligand identified so far (a co-crystallographic peptide). Testing of the corresponding synthetic peptides demonstrated that four out of seven significantly inhibited the free 3CLpro (from 46.9 \u00b1 6.4 to 76.8 \u00b1 3.6% inhibition at 10 \u00b5M). This study is the first report identifying peptides from Fucus ceranoides with high inhibitory activity against the SARS-CoV-2 3CLprotease which bind with high affinity to the protease\u2019s active site. It also confirms the effectiveness of the ligand-fishing strategy for the single-step purification of enzyme inhibitors from complex seaweed matrices.<\/jats:p>","DOI":"10.3390\/md22060244","type":"journal-article","created":{"date-parts":[[2024,5,27]],"date-time":"2024-05-27T08:36:22Z","timestamp":1716798982000},"page":"244","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["The Identification of Peptide Inhibitors of the Coronavirus 3CL Protease from a Fucus ceranoides L. Hydroalcoholic Extract Using a Ligand-Fishing Strategy"],"prefix":"10.3390","volume":"22","author":[{"given":"Luiz Antonio Miranda de Souza","family":"Duarte Filho","sequence":"first","affiliation":[{"name":"Littoral Environnement et Societ\u00e9s (LIENSs), UMRi CNRS 7266, La Rochelle Universit\u00e9, 17042 La Rochelle, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cintia Emi","family":"Yanaguibashi Leal","sequence":"additional","affiliation":[{"name":"Unit\u00e9 de G\u00e9nie Enzymatique et Cellulaire, UMR CNRS 7025, Universit\u00e9 de Picardie Jules Verne, 80039 Amiens, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1904-4823","authenticated-orcid":false,"given":"Pierre-Edouard","family":"Bodet","sequence":"additional","affiliation":[{"name":"Plateforme D\u2019analyse Haute R\u00e9solution des Biomol\u00e9cules, UMR CNRS 7266 LIENSs, La Rochelle Universit\u00e9, 17042 La Rochelle, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1000-0114","authenticated-orcid":false,"given":"Edilson","family":"Beserra de Alencar Filho","sequence":"additional","affiliation":[{"name":"Department of Pharmacy, Universidade Federal do Vale do S\u00e3o Francisco, Petrolina 56304-205, PE, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0867-1357","authenticated-orcid":false,"given":"Jackson Roberto Guedes da Silva","family":"Almeida","sequence":"additional","affiliation":[{"name":"N\u00facleo de Estudos e Pesquisas de Plantas Medicinais (NEPLAME), Universidade Federal do Vale do S\u00e3o Francisco, Petrolina 56304-205, PE, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Manon","family":"Porta Zapata","sequence":"additional","affiliation":[{"name":"Littoral Environnement et Societ\u00e9s (LIENSs), UMRi CNRS 7266, La Rochelle Universit\u00e9, 17042 La Rochelle, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Oussama","family":"Achour","sequence":"additional","affiliation":[{"name":"Littoral Environnement et Societ\u00e9s (LIENSs), UMRi CNRS 7266, La Rochelle Universit\u00e9, 17042 La Rochelle, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1571-9400","authenticated-orcid":false,"given":"Hugo","family":"Groult","sequence":"additional","affiliation":[{"name":"Littoral Environnement et Societ\u00e9s (LIENSs), UMRi CNRS 7266, La Rochelle Universit\u00e9, 17042 La Rochelle, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carlos Arthur","family":"Gouveia Veloso","sequence":"additional","affiliation":[{"name":"Littoral Environnement et Societ\u00e9s (LIENSs), UMRi CNRS 7266, La Rochelle Universit\u00e9, 17042 La Rochelle, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7799-992X","authenticated-orcid":false,"given":"Claudio","family":"Viegas J\u00fanior","sequence":"additional","affiliation":[{"name":"Institute of Chemistry, Federal University of Alfenas, Alfenas 37130-000, MG, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7755-3357","authenticated-orcid":false,"given":"Nathalie","family":"Bourgougnon","sequence":"additional","affiliation":[{"name":"Laboratoire de Biotechnologie et Chimie Marines, LBCM, Universit\u00e9 Bretagne Sud, EMR CNRS 6076, IUEM, 56000 Vannes, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4656-3557","authenticated-orcid":false,"given":"Laurent","family":"Picot","sequence":"additional","affiliation":[{"name":"Littoral Environnement et Societ\u00e9s (LIENSs), UMRi CNRS 7266, La Rochelle Universit\u00e9, 17042 La Rochelle, France"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,5,27]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"e13952","DOI":"10.1016\/j.heliyon.2023.e13952","article-title":"An Update on COVID-19: SARS-CoV-2 Variants, Antiviral Drugs, and Vaccines","volume":"9","author":"Hillary","year":"2023","journal-title":"Heliyon"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1146\/annurev-med-042921-020956","article-title":"COVID-19: Challenges of Viral Variants","volume":"74","author":"Jacobs","year":"2023","journal-title":"Annu. Rev. Med."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Taglialatela-Scafati, O. (2021). New Hopes for Drugs against COVID-19 Come from the Sea. Mar. Drugs, 19.","DOI":"10.3390\/md19020104"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"409","DOI":"10.1126\/science.abb3405","article-title":"Crystal Structure of SARS-CoV-2 Main Protease Provides a Basis for Design of Improved \u03b1-Ketoamide Inhibitors","volume":"368","author":"Zhang","year":"2020","journal-title":"Science"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"583387","DOI":"10.3389\/fphar.2021.583387","article-title":"Plant Products as Inhibitors of Coronavirus 3CL Protease","volume":"12","author":"Mandal","year":"2021","journal-title":"Front. Pharmacol."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Zhuo, R., Liu, H., Liu, N., and Wang, Y. (2016). Ligand Fishing: A Remarkable Strategy for Discovering Bioactive Compounds from Complex Mixture of Natural Products. Molecules, 21.","DOI":"10.3390\/molecules21111516"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Catarino, M.D., Silva, A.M.S., and Cardoso, S.M. (2018). Phycochemical Constituents and Biological Activities of Fucus spp.. Mar. Drugs, 16.","DOI":"10.3390\/md16080249"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"685","DOI":"10.1080\/07391102.2020.1817786","article-title":"Virtual Screening of Approved Clinic Drugs with Main Protease (3CLpro) Reveals Potential Inhibitory Effects on SARS-CoV-2","volume":"40","author":"Wang","year":"2022","journal-title":"J. Biomol. Struct. Dyn."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"12997","DOI":"10.1073\/pnas.1601327113","article-title":"SARS-CoV 3CL Protease Cleaves Its C-Terminal Autoprocessing Site by Novel Subsite Cooperativity","volume":"113","author":"Muramatsu","year":"2016","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"8","DOI":"10.1039\/C7NP00052A","article-title":"Marine Natural Products","volume":"35","author":"Blunt","year":"2018","journal-title":"Nat. Prod. Rep."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"128","DOI":"10.1016\/j.algal.2018.01.002","article-title":"Potential of Intensification Techniques for the Extraction and Depolymerization of Fucoidan","volume":"30","author":"Torres","year":"2018","journal-title":"Algal Res."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"119551","DOI":"10.1016\/j.carbpol.2022.119551","article-title":"A State-of-the-Art Review on Fucoidan as an Antiviral Agent to Combat Viral Infections","volume":"291","author":"Pradhan","year":"2022","journal-title":"Carbohydr. Polym."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"3525","DOI":"10.1007\/s00018-019-03138-w","article-title":"Antiviral Peptides as Promising Therapeutic Drugs","volume":"76","author":"Campos","year":"2019","journal-title":"Cell. Mol. Life Sci."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Andr\u00e9, R., Guedes, L., Melo, R., Ascens\u00e3o, L., Pacheco, R., Vaz, P.D., and Serralheiro, M.L. (2020). Effect of Food Preparations on In Vitro Bioactivities and Chemical Components of Fucus Vesiculosus. Foods, 9.","DOI":"10.3390\/foods9070955"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"2885","DOI":"10.1002\/adsc.201100163","article-title":"Potential of Different Enzyme Immobilization Strategies to Improve Enzyme Performance","volume":"353","author":"Rodrigues","year":"2011","journal-title":"Adv. Synth. Catal."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"4503","DOI":"10.1021\/acscatal.5b00996","article-title":"Enhanced Activity of Immobilized or Chemically Modified Enzymes","volume":"5","author":"Zhang","year":"2015","journal-title":"ACS Catal."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"2295","DOI":"10.1039\/C4TB01697A","article-title":"An Enzyme\u2013Inorganic Hybrid Nanoflower Based Immobilized Enzyme Reactor with Enhanced Enzymatic Activity","volume":"3","author":"Yin","year":"2015","journal-title":"J. Mater. Chem. B"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"295","DOI":"10.1007\/s12010-012-9772-y","article-title":"Magnetic Bead Cellulose as a Suitable Support for Immobilization of \u03b1-Chymotrypsin","volume":"168","author":"Prikryl","year":"2012","journal-title":"Appl. Biochem. Biotechnol."},{"key":"ref_19","unstructured":"Babick, F. (2020). Characterization of Nanoparticles, Elsevier."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Trindade Ximenes, I.A., de Oliveira, P.C.O., Wegermann, C.A., and de Moraes, M.C. (2021). Magnetic Particles for Enzyme Immobilization: A Versatile Support for Ligand Screening. J. Pharm. Biomed. Anal., 204.","DOI":"10.1016\/j.jpba.2021.114286"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1016\/j.molcatb.2011.01.001","article-title":"A Novel Method for the Immobilization of Urease on Phosphonate Grafted Iron Oxide Nanoparticle","volume":"69","author":"Sahoo","year":"2011","journal-title":"J. Mol. Catal. B Enzym."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"92","DOI":"10.1016\/j.procbio.2018.05.012","article-title":"Synthesis of Magnetic Gold Mesoporous Silica Nanoparticles Core Shell for Cellulase Enzyme Immobilization: Improvement of Enzymatic Activity and Thermal Stability","volume":"71","author":"Poorakbar","year":"2018","journal-title":"Process Biochem."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"127059","DOI":"10.1016\/j.colsurfa.2021.127059","article-title":"One-Step Elaboration of Janus Polymeric Nanoparticles: A Comparative Study of Different Emulsification Processes","volume":"626","author":"Vauthier","year":"2021","journal-title":"Colloids Surf. A Physicochem. Eng. Asp."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"170526","DOI":"10.1016\/j.peptides.2021.170526","article-title":"Antiviral Peptides against Coronaviridae Family: A Review","volume":"139","author":"Heydari","year":"2021","journal-title":"Peptides"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"622","DOI":"10.1016\/j.peptides.2005.09.006","article-title":"Synthesis and Activity of an Octapeptide Inhibitor Designed for SARS Coronavirus Main Proteinase","volume":"27","author":"Gan","year":"2006","journal-title":"Peptides"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"7962","DOI":"10.1021\/jm200870n","article-title":"Structure-Based Design, Synthesis, and Evaluation of Peptide-Mimetic SARS 3CL Protease Inhibitors","volume":"54","author":"Akaji","year":"2011","journal-title":"J. Med. Chem."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"e151","DOI":"10.1002\/mco2.151","article-title":"The SARS-CoV-2 Main Protease (Mpro): Structure, Function, and Emerging Therapies for COVID-19","volume":"3","author":"Hu","year":"2022","journal-title":"MedComm"},{"key":"ref_28","unstructured":"Hubbard, R.E., and Kamran Haider, M. (2010). Encyclopedia of Life Sciences, Wiley."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Amin, S.A., Banerjee, S., Ghosh, K., Gayen, S., and Jha, T. (2021). Protease Targeted COVID-19 Drug Discovery and Its Challenges: Insight into Viral Main Protease (Mpro) and Papain-like Protease (PLpro) Inhibitors. Bioorganic Med. Chem., 29.","DOI":"10.1016\/j.bmc.2020.115860"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"7017","DOI":"10.1021\/ac501200h","article-title":"Discrimination of Leucine and Isoleucine in Peptides Sequencing with Orbitrap Fusion Mass Spectrometer","volume":"86","author":"Lebedev","year":"2014","journal-title":"Anal. Chem."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1211","DOI":"10.1002\/jms.1598","article-title":"Metastable Atom-activated Dissociation Mass Spectrometry: Leucine\/Isoleucine Differentiation and Ring Cleavage of Proline Residues","volume":"44","author":"Cook","year":"2009","journal-title":"J. Mass Spectrom."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"66","DOI":"10.1016\/j.procbio.2019.11.026","article-title":"Parameters Necessary to Define an Immobilized Enzyme Preparation","volume":"90","author":"Boudrant","year":"2020","journal-title":"Process Biochem."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1016\/S0378-5173(00)00470-1","article-title":"Immobilization of Antibodies on Alginate-Chitosan Beads","volume":"206","author":"Albarghouthi","year":"2000","journal-title":"Int. J. Pharm."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Nguyen, T.T.H., Jung, J.-H., Kim, M.-K., Lim, S., Choi, J.-M., Chung, B., Kim, D.-W., and Kim, D. (2021). The Inhibitory Effects of Plant Derivate Polyphenols on the Main Protease of SARS Coronavirus 2 and Their Structure\u2013Activity Relationship. Molecules, 26.","DOI":"10.3390\/molecules26071924"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"2200964","DOI":"10.1002\/jssc.202200964","article-title":"Ligand Fishing as a Tool to Screen Natural Products with Anticancer Potential","volume":"46","author":"Picot","year":"2023","journal-title":"J. Sep. Sci."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Roepstorff, P., and Fohlman, J. (1984). Proposal for a Common Nomenclature for Sequence Ions in Mass Spectra of Peptides. Biomed. Mass Spectrom., 11.","DOI":"10.1002\/bms.1200111109"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"727","DOI":"10.1006\/jmbi.1996.0897","article-title":"Development and Validation of a Genetic Algorithm for Flexible docking 1","volume":"267","author":"Jones","year":"1997","journal-title":"J. Mol. Biol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"737","DOI":"10.1007\/s10822-012-9551-4","article-title":"Pose Prediction and Virtual Screening Performance of GOLD Scoring Functions in a Standardized Test","volume":"26","author":"Liebeschuetz","year":"2012","journal-title":"J. Comput. Aided Mol. Des."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1605","DOI":"10.1002\/jcc.20084","article-title":"UCSF Chimera\u2014A Visualization System for Exploratory Research and Analysis","volume":"25","author":"Pettersen","year":"2004","journal-title":"J. Comput. Chem."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"4745","DOI":"10.1021\/ct500592m","article-title":"Improved PEP-FOLD Approach for Peptide and Miniprotein Structure Prediction","volume":"10","author":"Shen","year":"2014","journal-title":"J. Chem. Theory Comput."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"112","DOI":"10.1002\/pro.3280","article-title":"Improvements to the APBS Biomolecular Solvation Software Suite","volume":"27","author":"Jurrus","year":"2018","journal-title":"Protein Sci."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"3106","DOI":"10.1002\/cmdc.202100397","article-title":"Amino Acid and Peptide-Based Antiviral Agents","volume":"16","author":"Skwarecki","year":"2021","journal-title":"ChemMedChem"}],"container-title":["Marine Drugs"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1660-3397\/22\/6\/244\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T14:48:52Z","timestamp":1760107732000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1660-3397\/22\/6\/244"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,5,27]]},"references-count":42,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2024,6]]}},"alternative-id":["md22060244"],"URL":"https:\/\/doi.org\/10.3390\/md22060244","relation":{},"ISSN":["1660-3397"],"issn-type":[{"value":"1660-3397","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,5,27]]}}}