Designing Allosteric Regulators of Thrombin. Monosulfated Benzofuran Dimers Selectively Interact With Arg173 of Exosite 2 to Induce Inhibition

Abstract

Earlier, we reported on the design of sulfated benzofuran dimers (SBDs) as allosteric inhibitors of thrombin (Sidhu et al. (2011) J Med Chem 54: 5522-5531). To identify the site of binding of SBDs, we studied thrombin inhibition in the presence of exosite 1 and 2 ligands. Whereas hirudin peptide and heparin octasaccharide did not affect the IC50 of thrombin inhibition by a high affinity SBD, the presence of full-length heparin reduced inhibition potency by 4-fold. The presence of γ’ fibrinogen peptide, which recognizes Arg93, Arg97, Arg173, Arg175 and other residues, resulted in a loss of affinity that correlated with the ideal Dixon-Webb competitive profile. Replacement of several arginines and lysines of exosite 2 with alanine did not affect thrombin inhibition potency, except for Arg173, which displayed a 22-fold reduction in IC50. Docking studies suggested a hydrophobic patch around Arg173 as a plausible site of SBD binding to thrombin. Absence of Arg173-like residue in factor Xa supported the observed selectivity of inhibition by SBDs. Cellular toxicity studies indicated that SBDs are essentially non-toxic to cells at concentrations as high as 250 mg/kg. Overall, the work presents the localization of the SBD binding site, which could lead to allosteric modulators of thrombin that are completely different from all clinically used anticoagulants.

On scaffold hopping: Challenges in the discovery of sulfated small molecules as mimetics of glycosaminoglycans

“…In these assays, the concentration of an anticoagulant required to double the clotting time of human plasma is measured using a fibrometer, as described in our earlier work. 12 The best hit, 1 , was selected for these studies. A 2-fold increase in PT and APTT required 606 and 550 μM concentrations of 1 , respectively suggesting useful anticoagulant potency of this initial hit (Fig.. 7).…”

“…Enzyme kinetic studies showed an allosteric inhibition phenomenon, while later studies with site-directed thrombin mutants identified the site of binding to a hydrophobic region close to Arg173, a site encompassed within exosite II. 12 This site of binding is a new discovery and is likely to be a promising avenue for anticoagulant drug discovery.…”

“…17–22 We have previously explored both SBVS and LBVS, individually, to discover sulfated small molecules and understand their interaction with proteins. 11,12,23,24 Whereas SBVS of sulfated small molecules has proved to be challenging because of lack of robust scoring functions used in the docking, LBVS has been found to be beset with inability to consider the shape of the binding site. 25 LBVS is useful when applied to large libraries but such libraries are not available for sulfated small molecules.…”

“…First we performed LBVS on a library of nearly 1 million non -sulfated molecules using a pharmacophore query developed on the basis of our mono sulfated benzofuran results. 11,12 The hits generated from the LBVS study were then screened by docking onto thrombin to identify a smaller group of 10 hits. Of these, three top-scoring molecules were selected for thrombin inhibition studies.…”

Discovery of Allosteric Modulators of Factor XIa by Targeting Hydrophobic Domains Adjacent to Its Heparin-Binding Site

“…27 This gave rise to the possibility of designing molecules that specifically target the hydrophobic (hP) domain surrounding the heparin-binding site (HBS) of proteins. In fact, coagulation enzymes possess hP domains of varying sizes around their strongly electropositive HBSs.…”