Exploring Structure−Activity Relationships by Engineering Modified Thrombin Inhibitors from the Flea Xenopsylla cheopis

Engineering of post-translationally sulfated polypeptides from hematophagous organisms has shown promise for the development of novel anticoagulants with improved therapeutic potential. Here, we describe the development of a high-throughput peptide engineering workflow to facilitate rapid assembly of libraries of modified peptide analogues. Library members were designed from the privileged chimeric anticoagulant sulfo-XC43-Hiru, comprised of a peptide fragment of XC43 (from the rat flea Xenopsylla cheopis) and a sulfopeptide fragment of hirudin (from the medicinal leech, Hirudo medicinalis). Focus was placed on exploring the influence of non-native structural motifs through the incorporation of non-canonical amino acids at a number of strategic sites. The thrombin inhibitory activity and cleavage resistance of each analogue is profiled and provides valuable insight into the relationship between the sequence and biological activity of these molecules.