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Proc Natl Acad Sci U S A. 2019 Jul 9;116(28):13873-13878. doi: 10.1073/pnas.1905177116. Epub 2019 Jun 20.

Rapid assembly and profiling of an anticoagulant sulfoprotein library.

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School of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia.
Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135 Porto, Portugal.
Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal.
Central Clinical School, Heart Research Institute, Newtown, NSW 2042, Australia.
Charles Perkins Centre, The University of Sydney, Sydney, NSW 2006, Australia.
Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, 4200-135, Porto, Portugal.
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037.
School of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia;


Hematophagous organisms produce a suite of salivary proteins which interact with the host's coagulation machinery to facilitate the acquisition and digestion of a bloodmeal. Many of these biomolecules inhibit the central blood-clotting serine proteinase thrombin that is also the target of several clinically approved anticoagulants. Here a bioinformatics approach is used to identify seven tick proteins with putative thrombin inhibitory activity that we predict to be posttranslationally sulfated at two conserved tyrosine residues. To corroborate the biological role of these molecules and investigate the effects of amino acid sequence and sulfation modifications on thrombin inhibition and anticoagulant activity, a library of 34 homogeneously sulfated protein variants were rapidly assembled using one-pot diselenide-selenoester ligation (DSL)-deselenization chemistry. Downstream functional characterization validated the thrombin-directed activity of all target molecules and revealed that posttranslational sulfation of specific tyrosine residues crucially modulates potency. Importantly, access to this homogeneously modified protein library not only enabled the determination of key structure-activity relationships and the identification of potent anticoagulant leads, but also revealed subtleties in the mechanism of thrombin inhibition, between and within the families, that would be impossible to predict from the amino acid sequence alone. The synthetic platform described here therefore serves as a highly valuable tool for the generation and thorough characterization of libraries of related peptide and/or protein molecules (with or without modifications) for the identification of lead candidates for medicinal chemistry programs.


blood clotting; ligation; protein; synthesis; thrombin


Conflict of interest statement

The authors declare no conflict of interest.

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