Thrombin is a key enzyme targeted by the majority of current anticoagulants that are direct inhibitors. Allosteric inhibition of thrombin may offer a major advantage of finely tuned regulation. We present here sulfated benzofurans as the first examples of potent, small allosteric inhibitors of thrombin. A sulfated benzofuran library of 15 sulfated monomers and 13 sulfated dimers with different charged, polar and hydrophobic substituents was studied in this work. Synthesis of the sulfated benzofurans was achieved through a multiple step, highly branched strategy, which culminated with microwave-assisted chemical sulfation. Of the 28 potential inhibitors, eleven exhibited reasonable inhibition of human α-thrombin at pH 7.4. Structure activity relationship analysis indicated that sulfation at the 5-position of the benzofuran scaffold was essential for targeting thrombin. A t-butyl 5-sulfated benzofuran derivative was found to be the most potent thrombin inhibitor with an IC50 of 7.3 μM under physiologically relevant conditions. Michaelis-Menten studies showed an allosteric inhibition phenomenon. Plasma clotting assays indicate that the sulfated benzofurans prolong both the activated partial thromboplastin time and prothrombin time. Overall, this work puts forward sulfated benzofurans as the first small, synthetic molecules as powerful lead compounds for the design of a new class of allosteric inhibitors of thrombin.

Antithrombin is a key regulator of coagulation and prime target of heparins, clinically used anticoagulants. Heparins induce a two-step conformational activation of antithrombin, a process that has remained challenging to target with molecules devoid of the antithrombin-binding pentasaccharide DEFGH. Computational screening of a focused library led to the design of two tetra-sulfated N-arylacyl tetrahydroisoquinoline variants as potential non-saccharide activators of antithrombin. A high yielding synthetic scheme based on Horner-Wadsworth-Emmons or Pictet-Spengler reactions was developed to facilitate the functionalization of the tetrahydoisoquinoline ring, which upon further amidation, deprotection, and sulfation gave the targeted non-saccharide activators. Spectrofluorometric measurement of affinity displayed antithrombin binding affinities in the low to high micromolar range at pH 6.0, I 0.05, 25 ºC. Measurement of second-order rate constants of antithrombin inhibition of factor Xa in the presence and absence of the designed activators showed antithrombin activation in the range of 8 – 80-fold in the pH 6.0 buffer. This work puts forward 20c, a novel tetra-sulfated N-arylacyl tetrahydroisoquinoline-based molecule, that activates AT only 3.8-fold less than that achieved with DEFGH suggesting a strong possibility of rationally designing sulfated organic molecules as clinically relevant AT activators.

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.

Antithrombin, a plasma glycoprotein serpin, requires conformational activation by heparin to induce an anticoagulant effect, which is mediated through accelerated factor Xa inhibition. Heparin, a highly charged polymer and an allosteric activator of the serpin, is associated with major adverse effects. To design better, but radically different activators of antithrombin from heparin, we utilized a pharmacophore-based approach. A tetrahydroisoquinoline-based scaffold was designed to mimic four critical anionic groups of the key trisaccharide DEF constituting the sequence-specific pentasaccharide DEFGH in heparin. Activator IAS5 containing 5,6-disulfated tetrahydroisoquinoline and 3,4,5-trisulfated phenyl rings was found to bind antithrombin at pH 7.4 with an affinity comparable to the reference trisaccharide DEF. IAS5 activated the inhibitor nearly 30-fold, nearly 2 to 3-fold higher than our first generation flavan-based designs. This work advances the concept of antithrombin activation through non-saccharide, organic molecules and pinpoints a direction for the design of more potent molecules.

Designing non-saccharide functional mimics of heparin is a major challenge. In this work, a library of small, aromatic molecules based on the sulfated DHP scaffold was synthesized and screened against thrombin and factor Xa. The results reveal that i) selected monomeric benzofuran derivatives inhibit the two enzymes, albeit weakly; ii) the two enzymes recognize different structural features in the benzofurans studied suggesting significant selectivity of recognition; and iii) the mechanism of inhibition is allosteric. The molecules represent the first allosteric small molecule inhibitors of the two enzymes.