{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,7,30]],"date-time":"2025-07-30T09:57:15Z","timestamp":1753869435957,"version":"3.41.2"},"reference-count":18,"publisher":"Wiley","issue":"5","license":[{"start":{"date-parts":[[2023,5,23]],"date-time":"2023-05-23T00:00:00Z","timestamp":1684800000000},"content-version":"vor","delay-in-days":22,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Journal of Medical Virology"],"published-print":{"date-parts":[[2023,5]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Host proteases trypsin and trypsin\u2010like proteases have been reported to facilitate the entry of coronavirus SARS\u2010CoV\u20102 in its host cells. These protease enzymes cleave the viral surface glycoprotein, spike, leading to successful cell surface receptor attachment, fusion and entry of the virus in its host cell. The spike protein has protease cleavage sites between the two domains S1 and S2. Since the cleavage site is recognized by the host proteases, it can be a potential antiviral therapeutic target. Trypsin\u2010like proteases play an important role in virus infectivity and the property of spike protein cleavage by trypsin and trypsin\u2010like proteases can be used to design assays for screening of antiviral candidates against spike protein cleavage. Here, we have documented the development of a proof\u2010of\u2010concept assay system for screening drugs against trypsin\/trypsin\u2010like proteases that cleave spike protein between its S1 and S2 domains. The assay system developed uses a fusion substrate protein containing a NanoLuc luciferase reporter protein, the protease cleavage site between S1 and S2 domains of SARS\u2010CoV\u20102 spike protein and a cellulose binding domain. The substrate protein can be immobilized on cellulose via the cellulose binding domain of the substrate. When trypsin and trypsin\u2010like proteases cleave the substrate, the cellulose binding domain remain bound to the cellulose and the reporter protein is dislodged. Reporter assay using the released reporter protein is the\u00a0read out of the protease activity. We have demonstrated the proof\u2010of\u2010concept using multiple proteases like trypsin, TMPRSS2, furin, cathepsin B, human airway trypsin and cathepsin L. A significant increment in fold change was observed with increasing enzyme concentration and incubation time. Introduction of increasing amounts of enzyme inhibitors in the reaction reduced the luminescent signal, thus validating the assay. Furthermore, we used SDS\u2010PAGE and immunoblot analyses to study the cleavage band pattern and re\u2010confirm the cleavage for enzymes tested in the assay. Taken together, we have tested an in\u2010vitro assay system using the proposed substrate for screening drugs against trypsin like protease\u2010based cleavage of SARS\u2010CoV\u20102 spike glycoprotein. The assay system can also be potentially used for antiviral drug screening against any other enzyme that might cleave the used cleavage site.<\/jats:p>","DOI":"10.1002\/jmv.28796","type":"journal-article","created":{"date-parts":[[2023,5,23]],"date-time":"2023-05-23T09:34:31Z","timestamp":1684834471000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A substrate for a cell free in vitro assay system to screen drugs targeting trypsin like protease\u2010based cleavage of SARS\u2010CoV\u20102 spike glycoprotein and viral entry"],"prefix":"10.1002","volume":"95","author":[{"given":"Feroza","family":"Begum","sequence":"first","affiliation":[{"name":"CSIR\u2010Indian Institute of Chemical Biology Kolkata West Bengal India"},{"name":"Academy of Scientific and Innovative Research (AcSIR) Ghaziabad India"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Amit K.","family":"Srivastava","sequence":"additional","affiliation":[{"name":"CSIR\u2010Indian Institute of Chemical Biology Kolkata West Bengal India"},{"name":"Academy of Scientific and Innovative Research (AcSIR) Ghaziabad India"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Prem Prakash","family":"Tripathi","sequence":"additional","affiliation":[{"name":"CSIR\u2010Indian Institute of Chemical Biology Kolkata West Bengal India"},{"name":"Academy of Scientific and Innovative Research (AcSIR) Ghaziabad India"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4783-9295","authenticated-orcid":false,"given":"Upasana","family":"Ray","sequence":"additional","affiliation":[{"name":"CSIR\u2010Indian Institute of Chemical Biology Kolkata West Bengal India"},{"name":"Academy of Scientific and Innovative Research (AcSIR) Ghaziabad India"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2023,5,23]]},"reference":[{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2020.02.052"},{"key":"e_1_2_9_3_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2020.04.022"},{"key":"e_1_2_9_4_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2020.02.058"},{"key":"e_1_2_9_5_1","doi-asserted-by":"publisher","DOI":"10.1021\/acsinfecdis.0c00701"},{"key":"e_1_2_9_6_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-020-15562-9"},{"key":"e_1_2_9_7_1","doi-asserted-by":"publisher","DOI":"10.1111\/tra.12389"},{"key":"e_1_2_9_8_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41392-020-0184-0"},{"key":"e_1_2_9_9_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2002589117"},{"key":"e_1_2_9_10_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00705-021-05343-0"},{"key":"e_1_2_9_11_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41392-021-00558-8"},{"key":"e_1_2_9_12_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.isci.2020.101212"},{"key":"e_1_2_9_13_1","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.05300-11"},{"key":"e_1_2_9_14_1","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.02232-10"},{"key":"e_1_2_9_15_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0076469"},{"key":"e_1_2_9_16_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-018-34859-w"},{"key":"e_1_2_9_17_1","doi-asserted-by":"publisher","DOI":"10.26508\/lsa.202000786"},{"key":"e_1_2_9_18_1","doi-asserted-by":"publisher","DOI":"10.1021\/acschembio.1c00138"},{"key":"e_1_2_9_19_1","doi-asserted-by":"publisher","DOI":"10.1002\/pro.4073"}],"container-title":["Journal of Medical Virology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/jmv.28796","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,1,22]],"date-time":"2024-01-22T11:00:28Z","timestamp":1705921228000},"score":1,"resource":{"primary":{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.28796"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,5]]},"references-count":18,"journal-issue":{"issue":"5","published-print":{"date-parts":[[2023,5]]}},"alternative-id":["10.1002\/jmv.28796"],"URL":"https:\/\/doi.org\/10.1002\/jmv.28796","archive":["Portico"],"relation":{},"ISSN":["0146-6615","1096-9071"],"issn-type":[{"type":"print","value":"0146-6615"},{"type":"electronic","value":"1096-9071"}],"subject":[],"published":{"date-parts":[[2023,5]]},"assertion":[{"value":"2022-12-01","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2023-05-05","order":1,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2023-05-23","order":2,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e28796"}}