{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,23]],"date-time":"2026-03-23T14:46:47Z","timestamp":1774277207177,"version":"3.50.1"},"reference-count":35,"publisher":"MDPI AG","issue":"16","license":[{"start":{"date-parts":[[2021,8,5]],"date-time":"2021-08-05T00:00:00Z","timestamp":1628121600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>The specificity of inhibition by 6,6\u2032-dihydroxythiobinupharidine (DTBN) on cysteine proteases was demonstrated in this work. There were differences in the extent of inhibition, reflecting active site structural-steric and biochemical differences. Cathepsin S (IC50 = 3.2 \u03bcM) was most sensitive to inhibition by DTBN compared to Cathepsin B, L and papain (IC50 = 1359.4, 13.2 and 70.4 \u03bcM respectively). DTBN is inactive for the inhibition of Mpro of SARS-CoV-2. Docking simulations suggested a mechanism of interaction that was further supported by the biochemical results. In the docking results, it was shown that the cysteine sulphur of Cathepsin S, L and B was in close proximity to the DTBN thiaspirane ring, potentially forming the necessary conditions for a nucleophilic attack to form a disulfide bond. Covalent docking and molecular dynamic simulations were performed to validate disulfide bond formation and to determine the stability of Cathepsins-DTBN complexes, respectively. The lack of reactivity of DTBN against SARS-CoV-2 Mpro was attributed to a mismatch of the binding conformation of DTBN to the catalytic binding site of Mpro. Thus, gradations in reactivity among the tested Cathepsins may be conducive for a mechanism-based search for derivatives of nupharidine against COVID-19. This could be an alternative strategy to the large-scale screening of electrophilic inhibitors.<\/jats:p>","DOI":"10.3390\/molecules26164743","type":"journal-article","created":{"date-parts":[[2021,8,5]],"date-time":"2021-08-05T09:35:32Z","timestamp":1628156132000},"page":"4743","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Inhibition of Cysteine Proteases by 6,6\u2032-Dihydroxythiobinupharidine (DTBN) from Nuphar lutea"],"prefix":"10.3390","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0665-9370","authenticated-orcid":false,"given":"Kamran","family":"Waidha","sequence":"first","affiliation":[{"name":"DRDO-Defence Institute of High Altitude Research (DIHAR), Leh, Ladakh UT-194101, India"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Udi","family":"Zurgil","sequence":"additional","affiliation":[{"name":"The Jacob Blaustein Institutes for Desert Research (BIDR), French Associates Institute for Agriculture and Biotechnology of Drylands, Sede Boqer Campus, Ben-Gurion University of the Negev, Sede Boqer 8499000, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9954-3248","authenticated-orcid":false,"given":"Efrat","family":"Ben-Zeev","sequence":"additional","affiliation":[{"name":"Nancy and Stephen Grand Israel National Center for Personalized Medicine, The Department of Life Sciences Core Facilities, Weizmann Institute of Science, Rehovot 76100, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9289-8898","authenticated-orcid":false,"given":"Jacob","family":"Gopas","sequence":"additional","affiliation":[{"name":"The Shraga Segal Department of Microbiology, Immunology and Genetics Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer Sheva 8410501, Israel"},{"name":"Department of Oncology, Soroka University Medical Center, Beer Sheva 8410501, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7440-4038","authenticated-orcid":false,"given":"Saravanakumar","family":"Rajendran","sequence":"additional","affiliation":[{"name":"Chemistry Division, SAS, Vellore Institute of Technology Chennai Campus, Chennai 600127, India"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Avi","family":"Golan-Goldhirsh","sequence":"additional","affiliation":[{"name":"The Jacob Blaustein Institutes for Desert Research (BIDR), French Associates Institute for Agriculture and Biotechnology of Drylands, Sede Boqer Campus, Ben-Gurion University of the Negev, Sede Boqer 8499000, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2021,8,5]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1241","DOI":"10.1248\/bpb.19.1241","article-title":"Potent immunosuppressive principles, dimeric sesquiterpene thioalkaloids, isolated from nupharis rhizoma, the rhizoma of Nuphar pumilum (nymphaeaceae): Structure-requirement of nuphar-alkaloid for immunosuppressive activity","volume":"19","author":"Yamahara","year":"1996","journal-title":"Biol. Pharm. Bull."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"4445","DOI":"10.1016\/j.bmcl.2003.09.019","article-title":"Potent anti-metastatic activity of dimeric sesquiterpene thioalkaloids from the rhizome of Nuphar pumilum","volume":"13","author":"Matsuda","year":"2003","journal-title":"Bioorg. Med. Chem. Lett."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1567","DOI":"10.1016\/j.bmcl.2005.12.032","article-title":"Nuphar alkaloids with immediately apoptosis-inducing activity from Nuphar pumilum and their structural requirements for the activity","volume":"16","author":"Matsuda","year":"2006","journal-title":"Bioorg. Med. Chem. Lett."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"477","DOI":"10.2174\/1568011054866973","article-title":"Sesquiterpenes: Natural products that decrease cancer growth","volume":"5","author":"Modzelewska","year":"2005","journal-title":"Curr. Med. Chem. Anticancer Agents"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1860","DOI":"10.4161\/cbt.8.19.9567","article-title":"Nuphar lutea thioalkaloids inhibit the nuclear factor kappaB pathway, potentiate apoptosis and are synergistic with cisplatin and etoposide","volume":"8","author":"Ozer","year":"2009","journal-title":"Cancer Biol. Ther."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"86","DOI":"10.1016\/j.jep.2014.11.048","article-title":"Anti-inflammatory effect of a Nuphar lutea partially purified leaf extract in murine models of septic shock","volume":"161","author":"Ozer","year":"2015","journal-title":"J. Ethnopharmacol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"616","DOI":"10.1016\/S1995-7645(13)60106-6","article-title":"In vitro antibacterial and antitumor activities of some medicinal plant extracts, growing in Turkey","volume":"6","author":"Yildirim","year":"2013","journal-title":"Asian Pac. J. Trop. Med."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1245","DOI":"10.1016\/j.bbagen.2015.02.012","article-title":"Action mechanism of 6, 6\u2032-dihydroxythiobinupharidine from Nuphar japonicum, which showed anti-MRSA and anti-VRE activities","volume":"1850","author":"Okamura","year":"2015","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"826","DOI":"10.1002\/jps.2600620530","article-title":"Isolation and in vitro antifungal activity of 6,6\u2032-dihydroxythiobinupharidine","volume":"62","author":"Cullen","year":"1973","journal-title":"J. Pharm. Sci."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"788","DOI":"10.1016\/j.phymed.2009.01.011","article-title":"Nuphar lutea: In vitro anti-leishmanial activity against Leishmania major promastigotes and amastigotes","volume":"16","author":"Ozer","year":"2009","journal-title":"Phytomedicine"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"510","DOI":"10.1016\/j.exppara.2010.05.025","article-title":"Leishmania major: Anti-leishmanial activity of Nuphar lutea extract mediated by the activation of transcription factor NF-kappaB","volume":"126","author":"Ozer","year":"2010","journal-title":"Exp. Parasitol."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Jain, S., Jacob, M., Walker, L., and Tekwani, B. (2016). Screening North American plant extracts in vitro against Trypanosoma brucei for discovery of new antitrypanosomal drug leads. BMC Complement. Altern. Med., 16.","DOI":"10.1186\/s12906-016-1122-0"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1433","DOI":"10.7150\/jca.18435","article-title":"Anti-Metastatic Effect of Semi-Purified Nuphar Lutea Leaf Extracts","volume":"8","author":"Ozer","year":"2017","journal-title":"J. Cancer"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Winer, H., Ozer, J., Shemer, Y., Reichenstein, I., Eilam-Frenkel, B., Benharroch, D., Golan-Goldhirsh, A., and Gopas, J. (2020). Nuphar lutea Extracts Exhibit Anti-Viral Activity against the Measles Virus. Molecules, 25.","DOI":"10.3390\/molecules25071657"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Waidha, K., Anto, N.P., Jayaram, D.R., Golan-Goldhirsh, A., Rajendran, S., Livneh, E., and Gopas, J. (2021). 6,6\u2032-Dihydroxythiobinupharidine (DTBN) Purified from Nuphar lutea Leaves Is an Inhibitor of Protein Kinase C Catalytic Activity. Molecules, 26.","DOI":"10.3390\/molecules26092785"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"De Pasquale, V., Moles, A., and Pavone, L.M. (2020). Cathepsins in the Pathophysiology of Mucopolysaccharidoses: New Perspectives for Therapy. Cells, 9.","DOI":"10.3390\/cells9040979"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"767","DOI":"10.2174\/138920312804871102","article-title":"The role of cysteine proteinases and their inhibitors in the host-pathogen cross talk","volume":"13","year":"2012","journal-title":"Curr. Protein Pept. Sci."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Dana, D., and Pathak, S.K. (2020). A Review of Small Molecule Inhibitors and Functional Probes of Human Cathepsin L.. Molecules, 25.","DOI":"10.3390\/molecules25030698"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"401","DOI":"10.1021\/acscentsci.6b00113","article-title":"Synthesis and Sulfur Electrophilicity of the Nuphar Thiaspirane Pharmacophore","volume":"2","author":"Tada","year":"2016","journal-title":"ACS Cent. Sci."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"3202","DOI":"10.1038\/s41467-020-16954-7","article-title":"Structural plasticity of SARS-CoV-2 3CL M(pro) active site cavity revealed by room temperature X-ray crystallography","volume":"11","author":"Kneller","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1825","DOI":"10.1093\/nar\/18.7.1825","article-title":"The primary structure and expression of the second open reading frame of the polymerase gene of the coronavirus MHV-A59; a highly conserved polymerase is expressed by an efficient ribosomal frameshifting mechanism","volume":"18","author":"Bredenbeek","year":"1990","journal-title":"Nucleic Acids Res."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"2305","DOI":"10.1099\/vir.0.19424-0","article-title":"Mechanisms and enzymes involved in SARS coronavirus genome expression","volume":"84","author":"Thiel","year":"2003","journal-title":"J. Gen. Virol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"581","DOI":"10.1038\/s41557-020-0496-2","article-title":"Crowdsourcing drug discovery for pandemics","volume":"12","author":"Chodera","year":"2020","journal-title":"Nat. Chem."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"13272","DOI":"10.1021\/jacs.7b07685","article-title":"Total Synthesis of Unsymmetrically Oxidized Nuphar Thioalkaloids via Copper-Catalyzed Thiolane Assembly","volume":"139","author":"Lacharity","year":"2017","journal-title":"J. Am. Chem. Soc."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1881","DOI":"10.1016\/j.bmcl.2019.06.003","article-title":"6,6\u2032-Dihydroxythiobinupharidine as a poison of human type II topoisomerases","volume":"29","author":"Dalvie","year":"2019","journal-title":"Bioorg. Med. Chem. Lett."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"4524","DOI":"10.1021\/jm010206q","article-title":"Identification of dipeptidyl nitriles as potent and selective inhibitors of cathepsin B through structure-based drug design","volume":"44","author":"Greenspan","year":"2001","journal-title":"J. Med. Chem."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"3350","DOI":"10.1021\/acs.jmedchem.7b01869","article-title":"Repurposing a Library of Human Cathepsin L Ligands: Identification of Macrocyclic Lactams as Potent Rhodesain and Trypanosoma brucei Inhibitors","volume":"61","author":"Giroud","year":"2018","journal-title":"J. Med. Chem."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"4507","DOI":"10.1016\/j.bmcl.2010.06.043","article-title":"4-(3-Trifluoromethylphenyl)-pyrimidine-2-carbonitrile as cathepsin S inhibitors: N3, not N1 is critically important","volume":"20","author":"Cai","year":"2010","journal-title":"Bioorg. Med. Chem. Lett."},{"key":"ref_29","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 alpha-ketoamide inhibitors","volume":"368","author":"Zhang","year":"2020","journal-title":"Science"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1331","DOI":"10.1126\/science.abb4489","article-title":"Structure-based design of antiviral drug candidates targeting the SARS-CoV-2 main protease","volume":"368","author":"Dai","year":"2020","journal-title":"Science"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"289","DOI":"10.1038\/s41586-020-2223-y","article-title":"Structure of M(pro) from SARS-CoV-2 and discovery of its inhibitors","volume":"582","author":"Jin","year":"2020","journal-title":"Nature"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"529","DOI":"10.1038\/s41594-020-0440-6","article-title":"Structural basis for the inhibition of SARS-CoV-2 main protease by antineoplastic drug carmofur","volume":"27","author":"Jin","year":"2020","journal-title":"Nat. Struct. Mol. Biol."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Arun, K.G., Sharanya, C.S., Abhithaj, J., Francis, D., and Sadasivan, C. (2020). Drug repurposing against SARS-CoV-2 using E-pharmacophore based virtual screening, molecular docking and molecular dynamics with main protease as the target. J. Biomol. Struct. Dyn., 1\u201312.","DOI":"10.1080\/07391102.2020.1779819"},{"key":"ref_34","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_35","doi-asserted-by":"crossref","first-page":"5047","DOI":"10.1038\/s41467-020-18709-w","article-title":"Crystallographic and electrophilic fragment screening of the SARS-CoV-2 main protease","volume":"11","author":"Douangamath","year":"2020","journal-title":"Nat. Commun."}],"container-title":["Molecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1420-3049\/26\/16\/4743\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T06:41:06Z","timestamp":1760164866000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1420-3049\/26\/16\/4743"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,8,5]]},"references-count":35,"journal-issue":{"issue":"16","published-online":{"date-parts":[[2021,8]]}},"alternative-id":["molecules26164743"],"URL":"https:\/\/doi.org\/10.3390\/molecules26164743","relation":{},"ISSN":["1420-3049"],"issn-type":[{"value":"1420-3049","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,8,5]]}}}