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Thus, the combination of both strategies should provide a powerful approach to drug discovery. Here, we investigate protein\u2010templated Ugi 4CR quantitatively using a fluorescence\u2010based enzyme assay, HPLC\u2010QTOF mass spectrometry (MS), and native protein MS with SARS\u2010CoV\u20102 main protease as template. Ugi reactions were analyzed in aqueous buffer at varying pH and fragment concentration. Potent inhibitors of the protease were formed in presence of the protein via Ugi 4CR together with Ugi three\u2010component reaction (Ugi 3CR) products. Binding of inhibitors to the protease was confirmed by native MS and resulted in the dimerization of the protein target. Formation of Ugi products was, however, more efficient in the non\u2010templated reaction, apparently due to interactions of the protein with the isocyanide and imine fragments. Consequently, in\u2010situ ligation screening of Ugi 4CR products was identified as a superior approach to the discovery of SARS\u2010CoV\u20102 protease inhibitors.<\/jats:p>","DOI":"10.1002\/chem.202303940","type":"journal-article","created":{"date-parts":[[2024,1,21]],"date-time":"2024-01-21T22:34:28Z","timestamp":1705876468000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Protein\u2010Templated Ugi Reactions versus In\u2010Situ Ligation Screening: Two Roads to the Identification of SARS\u2010CoV\u20102 Main Protease Inhibitors"],"prefix":"10.1002","volume":"30","author":[{"given":"Rebekka","family":"Wamser","sequence":"first","affiliation":[{"name":"Department of Biology Chemistry and Pharmacy Institute of Pharmacy Medicinal Chemistry Freie Universit\u00e4t Berlin  K\u00f6nigin-Luise-Str.2+4 14195 Berlin Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xinting","family":"Zhang","sequence":"additional","affiliation":[{"name":"Department of Biology Chemistry and Pharmacy Institute of Pharmacy Medicinal Chemistry Freie Universit\u00e4t Berlin  K\u00f6nigin-Luise-Str.2+4 14195 Berlin Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Benno","family":"Kuropka","sequence":"additional","affiliation":[{"name":"Department of Biology Chemistry and Pharmacy Institute of Chemistry and Biochemistry Freie Universit\u00e4t Berlin  Thielallee 63 14195 Berlin Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Christoph","family":"Arkona","sequence":"additional","affiliation":[{"name":"Department of Biology Chemistry and Pharmacy Institute of Pharmacy Medicinal Chemistry Freie Universit\u00e4t Berlin  K\u00f6nigin-Luise-Str.2+4 14195 Berlin Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6678-3165","authenticated-orcid":false,"given":"J\u00f6rg","family":"Rademann","sequence":"additional","affiliation":[{"name":"Department of Biology Chemistry and Pharmacy Institute of Pharmacy Medicinal Chemistry Freie Universit\u00e4t Berlin  K\u00f6nigin-Luise-Str.2+4 14195 Berlin Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2024,1,31]]},"reference":[{"key":"e_1_2_9_1_1","unstructured":"\u00a0"},{"key":"e_1_2_9_2_2","doi-asserted-by":"publisher","DOI":"10.1002\/anie.201610372"},{"key":"e_1_2_9_3_2","doi-asserted-by":"publisher","DOI":"10.1002\/1521-3773(20020315)41:6<1053::AID-ANIE1053>3.0.CO;2-4"},{"key":"e_1_2_9_4_1","unstructured":"\u00a0"},{"key":"e_1_2_9_5_2","doi-asserted-by":"publisher","DOI":"10.1002\/anie.200704594"},{"key":"e_1_2_9_6_2","doi-asserted-by":"publisher","DOI":"10.1002\/anie.200901647"},{"key":"e_1_2_9_7_2","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms6170"},{"key":"e_1_2_9_8_2","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms12761"},{"key":"e_1_2_9_9_2","doi-asserted-by":"publisher","DOI":"10.1002\/anie.201611547"},{"key":"e_1_2_9_10_2","doi-asserted-by":"publisher","DOI":"10.1002\/anie.202102074"},{"key":"e_1_2_9_11_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-018-07923-2"},{"key":"e_1_2_9_12_1","unstructured":"\u00a0"},{"key":"e_1_2_9_13_2","first-page":"126","volume":"51","author":"Passerini M.","year":"1921","journal-title":"Gazz. 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