Designing Allosteric Regulators of Thrombin. Exosite 2 Features Multiple Subsites That Can Be Targeted by Sulfated Small Molecules for Inducing Inhibition

We recently designed a group of novel exosite-2-directed sulfated, small, allosteric inhibitors of thrombin. To develop more potent inhibitors, monosulfated benzofuran tri- and tetrameric homologues of the parent designed dimers were synthesized in seven to eight steps and found to exhibit a wide range of potencies. Among these, trimer <b>9a</b> was found to be nearly 10-fold more potent than the first generation molecules. Michaelis–Menten studies indicated an allosteric mechanism of inhibition. Competitive studies using a hirudin peptide (exosite 1 ligand) and unfractionated heparin, heparin octasaccharide, and γ′-fibrinogen peptide (exosite 2 ligands) demonstrated exosite 2 recognition in a manner different from that of the parent dimers. Alanine scanning mutagenesis of 12 Arg/Lys residues of exosite 2 revealed a defect in <b>9a</b> potency for Arg233Ala thrombin only confirming the major difference in site of recognition between the two structurally related sulfated benzofurans. The results suggest that multiple avenues are available within exosite 2 for inducing thrombin inhibition