{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T02:07:34Z","timestamp":1760148454077,"version":"build-2065373602"},"reference-count":38,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2023,5,5]],"date-time":"2023-05-05T00:00:00Z","timestamp":1683244800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100006544","name":"Israel Ministry of Health","doi-asserted-by":"publisher","award":["3-0-7374"],"award-info":[{"award-number":["3-0-7374"]}],"id":[{"id":"10.13039\/501100006544","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>We have previously published research on the anti-viral properties of an alkaloid mixture extracted from Nuphar lutea, the major components of the partially purified mixture found by NMR analysis. These are mostly dimeric sesquiterpene thioalkaloids called thiobinupharidines and thiobinuphlutidines against the negative strand RNA measles virus (MV). We have previously reported that this extract inhibits the MV as well as its ability to downregulate several MV proteins in persistently MV-infected cells, especially the P (phospho)-protein. Based on our observation that the Nuphar extract is effective in vitro against the MV, and the immediate need that the coronavirus disease 2019 (COVID-19) pandemic created, we tested here the ability of 6,6\u2032-dihydroxythiobinupharidine DTBN, an active small molecule, isolated from the Nuphar lutea extract, on COVID-19. As shown here, DTBN effectively inhibits SARS-CoV-2 production in Vero E6 cells at non-cytotoxic concentrations. The short-term daily administration of DTBN to infected mice delayed the occurrence of severe clinical outcomes, lowered virus levels in the lungs and improved survival with minimal changes in lung histology. The viral load on lungs was significantly reduced in the treated mice. DTBN is a pleiotropic small molecule with multiple targets. Its anti-inflammatory properties affect a variety of pathogens including SARS-CoV-2 as shown here. Its activity appears to target both pathogen specific (as suggested by docking analysis) as well as cellular proteins, such as NF-\u03baB, PKCs, cathepsins and topoisomerase 2, that we have previously identified in our work. Thus, this combined double action of virus inhibition and anti-inflammatory activity may enhance the overall effectivity of DTBN. The promising results from this proof-of-concept in vitro and in vivo preclinical study should encourage future studies to optimize the use of DTBN and\/or its molecular derivatives against this and other related viruses.<\/jats:p>","DOI":"10.3390\/ijms24098327","type":"journal-article","created":{"date-parts":[[2023,5,8]],"date-time":"2023-05-08T03:34:53Z","timestamp":1683516893000},"page":"8327","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["In Vitro and In Vivo Therapeutic Potential of 6,6\u2032-Dihydroxythiobinupharidine (DTBN) from Nuphar lutea on Cells and K18-hACE2 Mice Infected with SARS-CoV-2"],"prefix":"10.3390","volume":"24","author":[{"given":"Shay","family":"Weiss","sequence":"first","affiliation":[{"name":"Department of Infectious Diseases, Israel Institute for Biological Research, Ness Ziona 7410001, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0665-9370","authenticated-orcid":false,"given":"Kamran","family":"Waidha","sequence":"additional","affiliation":[{"name":"The Shraga Segal Department of Microbiology, Immunology & Genetics, Faculty of Health Sciences, Ben-Gurion University of the Negev, 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"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5178-5851","authenticated-orcid":false,"given":"Daniel","family":"Benharroch","sequence":"additional","affiliation":[{"name":"Department of Pathology, Soroka University Medical Center and Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer Sheva 8410501, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jannat","family":"Khalilia","sequence":"additional","affiliation":[{"name":"The Shraga Segal Department of Microbiology, Immunology & Genetics, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer Sheva 8410501, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1902-8535","authenticated-orcid":false,"given":"Haim","family":"Levy","sequence":"additional","affiliation":[{"name":"Department of Infectious Diseases, Israel Institute for Biological Research, Ness Ziona 7410001, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Elad","family":"Bar-David","sequence":"additional","affiliation":[{"name":"Department of Infectious Diseases, Israel Institute for Biological Research, Ness Ziona 7410001, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Avi","family":"Golan-Goldhirsh","sequence":"additional","affiliation":[{"name":"The Jacob Blaustein Institutes for Desert Research (BIDR), Ben-Gurion University of the Negev, Sede Boqer Campus, Sde Boker 8410501, 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 & Genetics, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer Sheva 8410501, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Amir","family":"Ben-Shmuel","sequence":"additional","affiliation":[{"name":"Department of Infectious Diseases, Israel Institute for Biological Research, Ness Ziona 7410001, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,5,5]]},"reference":[{"key":"ref_1","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_2","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_3","doi-asserted-by":"crossref","first-page":"65","DOI":"10.1186\/s12929-022-00847-6","article-title":"Beyond the vaccines: A glance at the small molecule and peptide-based anti-COVID19 arsenal","volume":"29","author":"Nepali","year":"2022","journal-title":"J. Biomed. Sci."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"118","DOI":"10.1055\/s-0042-1758837","article-title":"Use of Antiviral Agents and other Therapies for COVID-19","volume":"44","author":"Blaskovich","year":"2023","journal-title":"Semin. Respir. Crit. Care Med."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"387","DOI":"10.1038\/s41392-022-01249-8","article-title":"Small molecules in the treatment of COVID-19","volume":"7","author":"Lei","year":"2022","journal-title":"Signal Transduct. Target. Ther."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Shoaib, S., Ansari, M.A., Kandasamy, G., Vasudevan, R., Hani, U., Chauhan, W., Alhumaidi, M.S., Altammar, K.A., Azmi, S., and Ahmad, W. (2023). An Attention towards the Prophylactic and Therapeutic Options of Phytochemicals for SARS-CoV-2: A Molecular Insight. Molecules, 28.","DOI":"10.3390\/molecules28020795"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Brogna, C., Costanzo, V., Brogna, B., Bisaccia, D.R., Brogna, G., Giuliano, M., Montano, L., Viduto, V., Cristoni, S., and Fabrowski, M. (2023). Analysis of Bacteriophage Behavior of a Human RNA Virus, SARS-CoV-2, through the Integrated Approach of Immunofluorescence Microscopy, Proteomics and D-Amino Acid Quantification. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms24043929"},{"key":"ref_8","unstructured":"Food and Drug Administration (2023, February 01). Fact Sheet for Healthcare Providers: Emergency Use Authorization for Molnupiravir, Available online: https:\/\/www.fda.gov\/news-events\/press-announcements\/coronavirus-covid-19-update-fda-authorizes-additional-oral-antiviral-treatment-covid-19-certain."},{"key":"ref_9","unstructured":"Food and Drug Administration (2023, February 01). Fact Sheet for Healthcare Providers: Emergency Use Authorization for Remdesivir, Available online: https:\/\/www.fda.gov\/news-events\/press-announcements\/fda-approves-first-treatment-covid-19."},{"key":"ref_10","unstructured":"Food and Drug Administration (2023, February 01). Fact Sheet for Healthcare Providers: Emergency Use Authorization for Nirmatrelvir, Available online: https:\/\/www.fda.gov\/news-events\/press-announcements\/coronavirus-covid-19-update-fda-authorizes-first-oral-antiviral-treatment-covid-19."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Waidha, K., Zurgil, U., Ben-Zeev, E., Gopas, J., Rajendran, S., and Golan-Goldhirsh, A. (2021). Inhibition of Cysteine Proteases by 6,6\u2032-Dihydroxythiobinupharidine (DTBN) from Nuphar lutea. Molecules, 26.","DOI":"10.3390\/molecules26164743"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"e06657","DOI":"10.1016\/j.heliyon.2021.e06657","article-title":"Design and identification of novel annomontine analogues against SARS-CoV-2: An in-silico approach","volume":"7","author":"Waidha","year":"2021","journal-title":"Heliyon"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"319","DOI":"10.1038\/s41594-021-00570-0","article-title":"Structural basis for inhibition of the SARS-CoV-2 RNA polymerase by suramin","volume":"28","author":"Yin","year":"2021","journal-title":"Nat. Struct. Mol. Biol."},{"key":"ref_14","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_15","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_16","doi-asserted-by":"crossref","first-page":"297","DOI":"10.1179\/136485909X440827","article-title":"Antileishmanial activity in Israeli plants","volume":"103","author":"Ozer","year":"2009","journal-title":"Ann. Trop. Med. Parasitol."},{"key":"ref_17","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-\u03baB","volume":"126","author":"Ozer","year":"2010","journal-title":"Exp. Parasitol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"62","DOI":"10.1111\/jcpe.13036","article-title":"Nupharidine enhances Aggregatibacter actinomycetemcomitans clearance by priming neutrophils and augmenting their effector functions","volume":"46","author":"Levy","year":"2019","journal-title":"J. Clin. Periodontol."},{"key":"ref_19","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_20","doi-asserted-by":"crossref","unstructured":"Muduli, S., Golan-Goldhirsh, A., Gopas, J., and Danilenko, M. (2022). Cytotoxicity of Thioalkaloid-Enriched Nuphar lutea Extract and Purified 6,6\u2032-Dihydroxythiobinupharidine in Acute Myeloid Leukemia Cells: The Role of Oxidative Stress and Intracellular Calcium. Pharmaceuticals, 15.","DOI":"10.3390\/ph15040410"},{"key":"ref_21","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_22","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_23","doi-asserted-by":"crossref","first-page":"112420","DOI":"10.1016\/j.biopha.2021.112420","article-title":"Molecular pathways involved in COVID-19 and potential pathway-based therapeutic targets","volume":"145","author":"Farahani","year":"2022","journal-title":"Biomed. Pharmacother."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"101695","DOI":"10.1016\/j.jbc.2022.101695","article-title":"The spike protein of SARS-CoV-2 induces endothelial inflammation through integrin \u03b15\u03b21 and NF-\u03baB signaling","volume":"298","author":"Robles","year":"2022","journal-title":"J. Biol. Chem."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1038\/s42003-021-02983-5","article-title":"SARS-CoV-2 infection induces a pro-inflammatory cytokine response through cGAS-STING and NF-\u03baB","volume":"5","author":"Neufeldt","year":"2022","journal-title":"Commun. Biol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"L372","DOI":"10.1152\/ajplung.90437.2008","article-title":"SARS-CoV proteins decrease levels and activity of human ENaC via activation of distinct PKC isoforms","volume":"296","author":"Ji","year":"2009","journal-title":"Am. J. Physiol. Lung Cell. Mol. Physiol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"e0105622","DOI":"10.1128\/spectrum.01056-22","article-title":"Protein Kinase C Inhibitors Reduce SARS-CoV-2 Replication in Cultured Cells","volume":"10","author":"Huang","year":"2022","journal-title":"Microbiol. Spectr."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"134","DOI":"10.1038\/s41392-021-00558-8","article-title":"Cathepsin L plays a key role in SARS-CoV-2 infection in humans and humanized mice and is a promising target for new drug development","volume":"6","author":"Zhao","year":"2021","journal-title":"Signal Transduct. Target. Ther."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"589505","DOI":"10.3389\/fcimb.2020.589505","article-title":"Cathepsin L in COVID-19: From Pharmacological Evidences to Genetics","volume":"10","author":"Gomes","year":"2020","journal-title":"Front. Cell. Infect. Microbiol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"3198","DOI":"10.1074\/jbc.M508381200","article-title":"SARS coronavirus, but not human coronavirus NL63, utilizes cathepsin L to infect ACE2-expressing cells","volume":"281","author":"Huang","year":"2006","journal-title":"J. Biol. Chem."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"8887","DOI":"10.1128\/JVI.00415-08","article-title":"Cathepsin L functionally cleaves the severe acute respiratory syndrome coronavirus class I fusion protein upstream of rather than adjacent to the fusion peptide","volume":"82","author":"Bosch","year":"2008","journal-title":"J. Virol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"270","DOI":"10.1038\/s41586-020-2012-7","article-title":"A pneumonia outbreak associated with a new coronavirus of probable bat origin","volume":"579","author":"Zhou","year":"2020","journal-title":"Nature"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1107","DOI":"10.1016\/j.omtn.2021.10.016","article-title":"Dual inhibition of TMPRSS2 and Cathepsin Bprevents SARS-CoV-2 infection in iPS cells","volume":"26","author":"Hashimoto","year":"2021","journal-title":"Mol. Ther. Nucleic Acids"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"2618","DOI":"10.1016\/j.cell.2021.03.051","article-title":"TOP1 inhibition therapy protects against SARS-CoV-2-induced lethal inflammation","volume":"184","author":"Ho","year":"2021","journal-title":"Cell"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"6294","DOI":"10.1038\/s41598-022-10293-x","article-title":"Mitoxantrone modulates a heparan sulfate-spike complex to inhibit SARS-CoV-2 infection","volume":"12","author":"Zhang","year":"2022","journal-title":"Sci. Rep."},{"key":"ref_36","unstructured":"Armed Forces Institute of Pathology (U.S.) (1992). Laboratory Methods in Histotechnology, American Registry of Pathology."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1226","DOI":"10.1038\/s41592-019-0582-9","article-title":"ilastik: Interactive machine learning for (bio)image analysis","volume":"16","author":"Berg","year":"2019","journal-title":"Nat. Methods"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"676","DOI":"10.1038\/nmeth.2019","article-title":"Fiji: An open-source platform for biological-image analysis","volume":"9","author":"Schindelin","year":"2012","journal-title":"Nat. Methods"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/24\/9\/8327\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T19:30:04Z","timestamp":1760124604000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/24\/9\/8327"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,5,5]]},"references-count":38,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2023,5]]}},"alternative-id":["ijms24098327"],"URL":"https:\/\/doi.org\/10.3390\/ijms24098327","relation":{},"ISSN":["1422-0067"],"issn-type":[{"type":"electronic","value":"1422-0067"}],"subject":[],"published":{"date-parts":[[2023,5,5]]}}}