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These primarily operate through inhibition of the central coagulation protease thrombin by binding to the active site and either exosite I or exosite II. Herein, we describe the rational design of a novel class of trivalent thrombin inhibitors that simultaneously block both exosites as well as the active site. These engineered hybrids were synthesized using tandem diselenide\u2010selenoester ligation (DSL) and native chemical ligation (NCL) reactions in one\u2010pot. The most potent trivalent inhibitors possessed femtomolar inhibition constants against \u03b1\u2010thrombin and were selective over related coagulation proteases. A lead hybrid inhibitor possessed potent anticoagulant activity, blockade of both thrombin generation and platelet aggregation in vitro and efficacy in a murine thrombosis model at 1\u2005mg\u2009kg<jats:sup>\u22121<\/jats:sup>. The rational engineering approach described here lays the foundation for the development of potent and selective inhibitors for a range of other enzymatic targets that possess multiple sites for the disruption of protein\u2013protein interactions, in addition to an active site.<\/jats:p>","DOI":"10.1002\/anie.202015127","type":"journal-article","created":{"date-parts":[[2020,12,21]],"date-time":"2020-12-21T12:28:58Z","timestamp":1608553738000},"page":"5348-5356","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":15,"title":["Potent Trivalent Inhibitors of Thrombin through Hybridization of Salivary Sulfopeptides from Hematophagous Arthropods"],"prefix":"10.1002","volume":"60","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5973-272X","authenticated-orcid":false,"given":"Stijn M.","family":"Agten","sequence":"first","affiliation":[{"name":"School of Chemistry and ARC Centre of Excellence for Innovations in Peptide and Protein Science The University of Sydney  Sydney 2006 NSW Australia"},{"name":"Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM) Maastricht University  Universiteitssingel 50 6229 ER Maastricht The Netherlands"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5946-473X","authenticated-orcid":false,"given":"Emma E.","family":"Watson","sequence":"additional","affiliation":[{"name":"School of Chemistry and ARC Centre of Excellence for Innovations in Peptide and Protein Science The University of Sydney  Sydney 2006 NSW Australia"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7235-317X","authenticated-orcid":false,"given":"Jorge","family":"Ripoll\u2010Rozada","sequence":"additional","affiliation":[{"name":"IBMC\u2014Instituto de Biologia Molecular e Celular and Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade Universidade do Porto  4200-135 Porto Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9728-5166","authenticated-orcid":false,"given":"Luke J.","family":"Dowman","sequence":"additional","affiliation":[{"name":"School of Chemistry and ARC Centre of Excellence for Innovations in Peptide and Protein Science The University of Sydney  Sydney 2006 NSW Australia"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4039-4409","authenticated-orcid":false,"given":"Mike C. 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