{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,9,30]],"date-time":"2025-09-30T00:08:09Z","timestamp":1759190889669,"version":"3.44.0"},"reference-count":57,"publisher":"MDPI AG","issue":"10","license":[{"start":{"date-parts":[[2025,9,28]],"date-time":"2025-09-28T00:00:00Z","timestamp":1759017600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Italian Ministry of University and Research","award":["FISR2020IP_00850"],"award-info":[{"award-number":["FISR2020IP_00850"]}]}],"content-domain":{"domain":["www.mdpi.com"],"crossmark-restriction":true},"short-container-title":["Pharmaceuticals"],"abstract":"<jats:p>Background\/Objectives: SARS-CoV-2 pandemic led to the identification of peptide-based main protease (Mpro) inhibitors. The overwhelming majority of them carry an electrophilic warhead and a \u03b3-lactam at the P1 position. During the selectivity assessment of an in-house Michael acceptors targeting SARS-CoV-2 Mpro, we unexpectedly observed a significant inhibition of human cathepsin S (hCatS). Methods: The biological investigation of three compounds (i.e., SPR38, SPR39, and SPR41) against hCatS was performed. The binding mode of SPRs was investigated by docking and molecular dynamics simulations. Results: Biological investigation has corroborated that hCatS is sensitive to peptide-based analogues harbouring \u03b3-lactam at the P1 position and a vinyl methyl ketone warhead. In silico studies revealed that despite being solvent exposed, the \u03b3-lactam at P1 might be involved in water-mediated H-bonds that could be optimized to gain inhibition potency and selectivity. Conclusions: The molecules repurposing of peptide-based SARS-CoV-2 Mpro inhibitors carrying the \u03b3-lactam at the P1 site could pave the way for the identification of novel potent and selective hCatS ligands.<\/jats:p>","DOI":"10.3390\/ph18101462","type":"journal-article","created":{"date-parts":[[2025,9,29]],"date-time":"2025-09-29T10:49:34Z","timestamp":1759142974000},"page":"1462","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A Second Opportunity for the Peptide-Based Analogues with \u03b3-Lactam at the P1 Position: Human Cathepsin S Inhibition"],"prefix":"10.3390","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8473-3321","authenticated-orcid":false,"given":"Santo","family":"Previti","sequence":"first","affiliation":[{"name":"Department of Chemical, Biological, Pharmaceutical, and Environmental Sciences, University of Messina, Viale Ferdinando Stagno d\u2019Alcontres 31, 98166 Messina, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1359-8684","authenticated-orcid":false,"given":"Nunzio","family":"Iraci","sequence":"additional","affiliation":[{"name":"Department of Chemical, Biological, Pharmaceutical, and Environmental Sciences, University of Messina, Viale Ferdinando Stagno d\u2019Alcontres 31, 98166 Messina, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9165-9270","authenticated-orcid":false,"given":"Elsa","family":"Calcaterra","sequence":"additional","affiliation":[{"name":"Department of Chemical, Biological, Pharmaceutical, and Environmental Sciences, University of Messina, Viale Ferdinando Stagno d\u2019Alcontres 31, 98166 Messina, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9020-2068","authenticated-orcid":false,"given":"Roberta","family":"Ettari","sequence":"additional","affiliation":[{"name":"Department of Chemical, Biological, Pharmaceutical, and Environmental Sciences, University of Messina, Viale Ferdinando Stagno d\u2019Alcontres 31, 98166 Messina, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8942-797X","authenticated-orcid":false,"given":"Maria","family":"Zappal\u00e0","sequence":"additional","affiliation":[{"name":"Department of Chemical, Biological, Pharmaceutical, and Environmental Sciences, University of Messina, Viale Ferdinando Stagno d\u2019Alcontres 31, 98166 Messina, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,9,28]]},"reference":[{"key":"ref_1","first-page":"21.2.1","article-title":"Papain-like cysteine proteases","volume":"21","author":"Bromme","year":"2001","journal-title":"Curr. Protoc. Protein Sci."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"526","DOI":"10.1016\/j.biopha.2018.05.148","article-title":"Cathepsins: Proteases that are vital for survival but can also be fatal","volume":"105","author":"Patel","year":"2018","journal-title":"Biomed. Pharmacother."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"62","DOI":"10.1016\/j.biochi.2024.04.010","article-title":"Compensational role between cathepsins","volume":"226","author":"Kos","year":"2024","journal-title":"Biochimie"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"68","DOI":"10.1016\/j.bbapap.2011.10.002","article-title":"Cysteine cathepsins: From structure, function and regulation to new frontiers","volume":"1824","author":"Turk","year":"2012","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Yadati, T., Houben, T., Bitorina, A., and Shiri-Sverdlov, R. (2020). The ins and outs of cathepsins: Physiological function and role in disease management. Cells, 9.","DOI":"10.3390\/cells9071679"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"4459","DOI":"10.1021\/cr0101656","article-title":"Human and parasitic papain-like cysteine proteases: Their role in physiology and pathology and recent developments in inhibitor design","volume":"102","author":"Lecaille","year":"2002","journal-title":"Chem. Rev."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1805","DOI":"10.1002\/prot.26845","article-title":"Novel insights into the dynamic conformational transitions and active site plasticity of human immunoregulatory cathepsin S","volume":"93","author":"Geetha","year":"2025","journal-title":"Proteins"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"573","DOI":"10.1080\/14728222.2020.1746765","article-title":"Cysteine cathepsins as therapeutic targets in inflammatory diseases","volume":"24","author":"Vidak","year":"2020","journal-title":"Expert Opin. Ther. Targets"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"e3117","DOI":"10.1002\/dmrr.3117","article-title":"Elevated circulating cathepsin S levels are associated with metabolic syndrome in overweight and obese individuals","volume":"35","author":"Chen","year":"2019","journal-title":"Diabetes Metab. Res. Rev."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"16","DOI":"10.4149\/neo_2015_003","article-title":"Cathepsin S as a cancer target","volume":"62","author":"Zhang","year":"2015","journal-title":"Neoplasma"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"88","DOI":"10.3389\/fcvm.2017.00088","article-title":"Cathepsin S as an inhibitor of cardiovascular inflammation and calcification in chronic kidney disease","volume":"4","author":"Sena","year":"2018","journal-title":"Front. Cardiovasc. Med."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"114","DOI":"10.3389\/fcell.2017.00114","article-title":"The multifaceted role of the lysosomal protease cathepsins in kidney disease","volume":"5","author":"Cocchiaro","year":"2017","journal-title":"Front. Cell Dev. Biol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"111","DOI":"10.1186\/s12931-020-01381-5","article-title":"Cathepsin S: Investigating an old player in lung disease pathogenesis, comorbidities, and potential therapeutics","volume":"21","author":"Brown","year":"2020","journal-title":"Respir. Res."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Senjor, E., Kos, J., and Nanut, M.P. (2023). Cysteine cathepsins as therapeutic targets in immune regulation and immune disorders. Biomedicines, 11.","DOI":"10.3390\/biomedicines11020476"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Sayed, S., Faruq, O., Preya, U.H., and Kim, J.T. (2023). Cathepsin S knockdown suppresses endothelial inflammation, angiogenesis, and complement protein activity under hyperglycemic conditions in vitro by inhibiting NF-\u03baB signaling. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms24065428"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"775","DOI":"10.1083\/jcb.147.4.775","article-title":"Cathepsin S controls the trafficking and maturation of MHC class II molecules in dendritic cells","volume":"147","author":"Driessen","year":"1999","journal-title":"J. Cell Biol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"197","DOI":"10.1016\/S1074-7613(00)80020-5","article-title":"Cathepsin S required for normal MHC class II peptide loading and germinal center development","volume":"10","author":"Shi","year":"1999","journal-title":"Immunity"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1205","DOI":"10.4049\/jimmunol.174.3.1205","article-title":"Cathepsin S controls MHC class II-mediated antigen presentation by epithelial cells in vivo","volume":"174","author":"Beers","year":"2005","journal-title":"J. Immunol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"867","DOI":"10.1515\/hsz-2015-0114","article-title":"Cathepsin S: Therapeutic, diagnostic, and prognostic potential","volume":"396","author":"Wilkinson","year":"2015","journal-title":"Biol. Chem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"101106","DOI":"10.1016\/j.mam.2022.101106","article-title":"Cathepsin S (CTSS) activity in health and disease\u2014A treasure trove of untapped clinical potential","volume":"88","author":"Smyth","year":"2022","journal-title":"Mol. Aspects Med."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"471","DOI":"10.3390\/pathophysiology31030035","article-title":"Advances in cathepsin S inhibition: Challenges and breakthroughs in drug development","volume":"31","author":"Ajani","year":"2024","journal-title":"Pathophysiology"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"11801","DOI":"10.1021\/acs.jmedchem.0c00949","article-title":"Highly selective sub-nanomolar cathepsin S inhibitors by merging fragment binders with nitrile inhibitors","volume":"63","author":"Schade","year":"2020","journal-title":"J. Med. Chem."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"837","DOI":"10.1021\/acsmedchemlett.4c00050","article-title":"Structural modifications of covalent cathepsin S inhibitors: Impact on affinity, selectivity, and permeability","volume":"15","author":"Meta","year":"2024","journal-title":"ACS Med. Chem. Lett."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"9789","DOI":"10.1021\/jm401528k","article-title":"Identification of potent and selective cathepsin S inhibitors containing different central cyclic scaffolds","volume":"56","author":"Hilpert","year":"2013","journal-title":"J. Med. Chem."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"717","DOI":"10.2174\/156802610791113432","article-title":"Recent advances in the design of cathepsin S inhibitors","volume":"10","author":"Sun","year":"2010","journal-title":"Curr. Top. Med. Chem."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"602","DOI":"10.1021\/acsmedchemlett.3c00562","article-title":"Identification of dual inhibitors targeting main protease (Mpro) and cathepsin L as potential anti-SARS-CoV-2 agents","volume":"15","author":"Previti","year":"2024","journal-title":"ACS Med. Chem. Lett."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"12500","DOI":"10.1021\/acs.jmedchem.2c01005","article-title":"Targeting SARS-CoV-2 main protease for treatment of COVID-19: Covalent inhibitors structure-activity relationship insights and evolution perspectives","volume":"65","author":"Bono","year":"2022","journal-title":"J. Med. Chem."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1433","DOI":"10.1039\/D0SC06195F","article-title":"Mechanism of inhibition of SARS-CoV-2 M(pro) by N3 peptidyl Michael acceptor explained by QM\/MM simulations and design of new derivatives with tunable chemical reactivity","volume":"12","author":"Arafet","year":"2020","journal-title":"Chem. Sci."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"115491","DOI":"10.1016\/j.ejmech.2023.115491","article-title":"The research progress of SARS-CoV-2 main protease inhibitors from 2020 to 2022","volume":"257","author":"Pang","year":"2023","journal-title":"Eur. J. Med. Chem."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"115021","DOI":"10.1016\/j.ejmech.2022.115021","article-title":"Structure-based lead optimization of peptide-based vinyl methyl ketones as SARS-CoV-2 main protease inhibitors","volume":"247","author":"Previti","year":"2023","journal-title":"Eur. J. Med. Chem."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"3193","DOI":"10.1021\/jm00017a002","article-title":"Vinyl sulfones as mechanism-based cysteine protease inhibitors","volume":"38","author":"Palmer","year":"1995","journal-title":"J. Med. Chem."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Muller, P., Meta, M., Meidner, J.L., Schwickert, M., Meyr, J., Schwickert, K., Kersten, C., Zimmer, C., Hammerschmidt, S.J., and Frey, A. (2023). Investigation of the compatibility between warheads and peptidomimetic sequences of protease inhibitors-a comprehensive reactivity and selectivity study. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms24087226"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"e202300160","DOI":"10.1002\/cmdc.202300160","article-title":"Subnanomolar cathepsin S inhibitors with high selectivity: Optimizing covalent reversible alpha-fluorovinylsulfones and alpha-sulfonates as potential immunomodulators in cancer","volume":"18","author":"Fuchs","year":"2023","journal-title":"ChemMedChem"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1479","DOI":"10.1007\/s43440-020-00155-6","article-title":"Drug repurposing approach to fight COVID-19","volume":"72","author":"Singh","year":"2020","journal-title":"Pharmacol. Rep."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"588654","DOI":"10.3389\/fphar.2020.588654","article-title":"Challenges for drug repurposing in the COVID-19 pandemic era","volume":"11","author":"Sultana","year":"2020","journal-title":"Front. Pharmacol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"34196","DOI":"10.1021\/acsomega.4c03023","article-title":"Progress in research on inhibitors targeting SARS-CoV-2 main protease (Mpro)","volume":"9","author":"Yang","year":"2024","journal-title":"ACS Omega"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"115667","DOI":"10.1016\/j.ejmech.2023.115667","article-title":"Exploring diverse reactive warheads for the design of SARS-CoV-2 main protease inhibitors","volume":"259","author":"Tan","year":"2023","journal-title":"Eur. J. Med. Chem."},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Agost-Beltr\u00e1n, L., de la Hoz-Rodr\u00edguez, S., Bou-Iserte, L., Rodr\u00edguez, S., Fern\u00e1ndez-de-la-Pradilla, A., and Gonz\u00e1lez, F.V. (2022). Advances in the development of SARS-CoV-2 Mpro inhibitors. Molecules, 27.","DOI":"10.20944\/preprints202204.0065.v1"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1345","DOI":"10.1021\/acsmedchemlett.2c00260","article-title":"A warhead substitution study on the coronavirus main protease inhibitor nirmatrelvir","volume":"13","author":"Vankadara","year":"2022","journal-title":"ACS Med. Chem. Lett."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"534","DOI":"10.1021\/jm050540c","article-title":"Novel procedure for modeling ligand\/receptor induced fit effects","volume":"49","author":"Sherman","year":"2006","journal-title":"J. Med. Chem."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"3203","DOI":"10.1021\/bi027308i","article-title":"Specificity determinants of human cathepsin S revealed by crystal structures of complexes","volume":"42","author":"Pauly","year":"2003","journal-title":"Biochemistry"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"13793","DOI":"10.1021\/acs.jmedchem.1c01178","article-title":"An activity-based probe for cathepsin K imaging with excellent potency and selectivity","volume":"64","author":"Lemke","year":"2021","journal-title":"J. Med. Chem."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"955","DOI":"10.1002\/cbic.201300806","article-title":"A coumarin-labeled vinyl sulfone as tripeptidomimetic activity-based probe for cysteine cathepsins","volume":"15","author":"Mertens","year":"2014","journal-title":"Chembiochem"},{"key":"ref_44","unstructured":"Leatherbarrow, R.J. (2007). GraFit 6, Vol. Erithacus Software Limited."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"368","DOI":"10.2174\/156802610790725498","article-title":"Enzyme kinetics and hit validation in fluorimetric protease assays","volume":"10","author":"Ludewig","year":"2010","journal-title":"Curr. Top. Med. Chem."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1093\/nar\/28.1.235","article-title":"The Protein Data Bank","volume":"28","author":"Berman","year":"2000","journal-title":"Nucleic Acids Res."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"221","DOI":"10.1007\/s10822-013-9644-8","article-title":"Protein and ligand preparation: Parameters, protocols, and influence on virtual screening enrichments","volume":"27","author":"Sastry","year":"2013","journal-title":"J. Comput. Aided. Mol. Des."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"1941","DOI":"10.1002\/cmdc.201300287","article-title":"Computer-aided design, synthesis and validation of 2-phenylquinazolinone fragments as CDK9 inhibitors with anti-HIV-1 Tat-mediated transcription activity","volume":"8","author":"Sancineto","year":"2013","journal-title":"ChemMedChem"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"11225","DOI":"10.1021\/ja9621760","article-title":"Development and testing of the OPLS All-atom force field on conformational energetics and properties of organic liquids","volume":"118","author":"Jorgensen","year":"1996","journal-title":"J. Am. Chem. Soc."},{"key":"ref_50","unstructured":"(Schr\u00f6dinger Release 2025-2: Maestro, 2025). Schr\u00f6dinger Release 2025-2: Maestro."},{"key":"ref_51","unstructured":"(Schr\u00f6dinger Release 2025-2: MacroModel, 2025). Schr\u00f6dinger Release 2025-2: MacroModel."},{"key":"ref_52","unstructured":"(Schr\u00f6dinger Release 2025-2: Prime, 2025). Schr\u00f6dinger Release 2025-2: Prime."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"1739","DOI":"10.1021\/jm0306430","article-title":"Glide: A new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy","volume":"47","author":"Friesner","year":"2004","journal-title":"J. Med. Chem."},{"key":"ref_54","doi-asserted-by":"crossref","unstructured":"Bowers, K.J., Chow, E., Xu, H., Dror, R.O., Eastwood, M.P., Gregersen, B.A., Klepeis, J.L., Kolossvary, I., Moraes, M.A., and Sacerdoti, F.D. (2006, January 11\u201317). Scalable algorithms for molecular dynamics simulations on commodity clusters. Proceedings of the ACM\/IEEE Conference on Supercomputing, Tampa, FL, USA.","DOI":"10.1109\/SC.2006.54"},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"926","DOI":"10.1063\/1.445869","article-title":"Comparison of simple potential functions for simulating liquid water","volume":"79","author":"Jorgensen","year":"1983","journal-title":"J. Chem. Phys."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1016\/0263-7855(96)00018-5","article-title":"VMD: Visual molecular dynamics","volume":"14","author":"Humphrey","year":"1996","journal-title":"J. Mol. Graph."},{"key":"ref_57","unstructured":"(Schr\u00f6dinger Suite Release 2025-2, 2025). Schr\u00f6dinger Suite Release 2025-2."}],"container-title":["Pharmaceuticals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1424-8247\/18\/10\/1462\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,9,29]],"date-time":"2025-09-29T11:02:09Z","timestamp":1759143729000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1424-8247\/18\/10\/1462"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,9,28]]},"references-count":57,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2025,10]]}},"alternative-id":["ph18101462"],"URL":"https:\/\/doi.org\/10.3390\/ph18101462","relation":{},"ISSN":["1424-8247"],"issn-type":[{"value":"1424-8247","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,9,28]]}}}