Sulfated Pentagalloylglucoside Is a Potent, Allosteric, and Selective Inhibitor of Factor XIa

Inhibition of factor XIa (FXIa) is a novel paradigm for developing anticoagulants without major bleeding consequences. We present the discovery of sulfated pentagalloylglucoside (<b>6</b>) as a highly selective inhibitor of human FXIa. Biochemical screening of a focused library led to the identification of <b>6</b>, a sulfated aromatic mimetic of heparin. Inhibitor <b>6</b> displayed a potency of 551 nM against FXIa, which was at least 200-fold more selective than other relevant enzymes. It also prevented activation of factor IX and prolonged human plasma and whole blood clotting. Inhibitor <b>6</b> reduced <i>V</i>_MAX of FXIa hydrolysis of chromogenic substrate without affecting the <i>K</i>_M, suggesting an allosteric mechanism. Competitive studies showed that <b>6</b> bound in the heparin-binding site of FXIa. No allosteric small molecule has been discovered to date that exhibits equivalent potency against FXIa. Inhibitor <b>6</b> is expected to open up a major route to allosteric FXIa anticoagulants with clinical relevance

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

Targeting the GPIbα Binding Site of Thrombin To Simultaneously Induce Dual Anticoagulant and Antiplatelet Effects

Exosite 2 of human thrombin contributes to two opposing pathways, the anticoagulant pathway and the platelet aggregation pathway. We reasoned that an exosite 2 directed allosteric thrombin inhibitor should simultaneously induce anticoagulant and antiplatelet effects. To assess this, we synthesized SbO4L based on the sulfated tyrosine-containing sequence of GPIbα. SbO4L was synthesized in three simple steps in high yield and found to be a highly selective, direct inhibitor of thrombin. Michelis–Menten kinetic studies indicated a noncompetitive mechanism of inhibition. Competitive inhibition studies suggested ideal competition with heparin and glycoprotein Ibα, as predicted. Studies with site-directed mutants of thrombin indicated that SbO4L binds to Arg233, Lys235, and Lys236 of exosite 2. SbO4L prevented thrombin-mediated platelet activation and aggregation as expected on the basis of competition with GPIbα. SbO4L presents a novel paradigm of simultaneous dual anticoagulant and antiplatelet effects achieved through the GPIbα binding site of thrombin

Chemoenzymatically Prepared Heparan Sulfate Containing Rare 2‑O-Sulfonated Glucuronic Acid Residues

The structural diversity of natural sulfated glycosaminoglycans (GAGs) presents major promise for discovery of chemical biology tools or therapeutic agents. Yet, few GAGs have been identified so far to exhibit this promise. We reasoned that a simple approach to identify such GAGs is to explore sequences containing rare residues, for example, 2-O-sulfonated glucuronic acid (GlcA<i>p</i>2S). Genetic algorithm-based computational docking and filtering suggested that GlcA<i>p</i>2S containing heparan sulfate (HS) may exhibit highly selective recognition of antithrombin, a key plasma clot regulator. HS containing only GlcA<i>p</i>2S and 2-N-sulfonated glucosamine residues, labeled as HS_2S2S, was chemoenzymatically synthesized in just two steps and was found to preferentially bind antithrombin over heparin cofactor II, a closely related serpin. Likewise, HS_2S2S directly inhibited thrombin but not factor Xa, a closely related protease. The results show that a HS containing rare GlcA<i>p</i>2S residues exhibits the unusual property of selective antithrombin activation and direct thrombin inhibition. More importantly, HS_2S2S is also the first molecule to activate antithrombin nearly as well as the heparin pentasaccharide although being completely devoid of the critical 3-<i>O</i>-sulfonate group. Thus, this work shows that novel functions and mechanisms may be uncovered by studying rare GAG residues/sequences