Aiye Liang

Rational Design of Potent, Small, Synthetic Allosteric Inhibitors of Thrombin

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, 11 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 tert-butyl 5-sulfated benzofuran derivative was found to be the most potent thrombin inhibitor with an IC(50) 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.

First steps in the direction of synthetic, allosteric, direct inhibitors of thrombin and factor Xa

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.

Interaction of Antithrombin with Sulfated, Low Molecular Weight Lignins: OPPORTUNITIES FOR POTENT, SELECTIVE MODULATION OF ANTITHROMBIN FUNCTION*

Antithrombin, a major regulator of coagulation and angiogenesis, is known to interact with several natural sulfated polysaccharides. Previously, we prepared sulfated low molecular weight variants of natural lignins, called sulfated dehydrogenation polymers (DHPs) (Henry, B. L., Monien, B. H., Bock, P. E., and Desai, U. R. (2007) J. Biol. Chem. 282, 31891–31899), which have now been found to exhibit interesting antithrombin binding properties. Sulfated DHPs represent a library of diverse noncarbohydrate aromatic scaffolds that possess structures completely different from heparin and heparan sulfate. Fluorescence binding studies indicate that sulfated DHPs bind to antithrombin with micromolar affinity under physiological conditions. Salt dependence of binding affinity indicates that the antithrombin-sulfated DHP interaction involves a massive 80–87% non-ionic component to the free energy of binding. Competitive binding studies with heparin pentasaccharide, epicatechin sulfate, and full-length heparin indicate that sulfated DHPs bind to both the pentasaccharide-binding site and extended heparin-binding site of antithrombin. Affinity capillary electrophoresis resolves a limited number of peaks of antithrombin co-complexes suggesting preferential binding of selected DHP structures to the serpin. Computational genetic algorithm-based virtual screening study shows that only one sulfated DHP structure, out of the 11 present in a library of plausible sequences, bound in the heparin-binding site with a high calculated score supporting selectivity of recognition. Enzyme inhibition studies indicate that only one of the three sulfated DHPs studied is a potent inhibitor of free factor VIIa in the presence of antithrombin. Overall, the chemo-enzymatic origin and antithrombin binding properties of sulfated DHPs present novel opportunities for potent and selective modulation of the serpin function, especially for inhibiting the initiation phase of hemostasis.

Designing nonsaccharide, allosteric activators of antithrombin for accelerated inhibition of factor Xa.

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 nonsaccharide 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 nonsaccharide activators. Spectrofluorometric measurement of affinity displayed antithrombin binding affinities in the low to high micromolar range at pH 6.0, I 0.05, 25 °C. 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.

On designing non-saccharide, allosteric activators of antithrombin

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 IAS(5) 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. IAS(5) activated the inhibitor nearly 30-fold, nearly 2- to 3-fold higher than our first generation flavanoid-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.

Study of physico-chemical properties of novel highly sulfated, aromatic, mimetics of heparin and heparan sulfate

Heparin (H) and heparan sulfate (HS) play major roles in a number of biological processes. Yet, H/HS-based pharmaceutical agents are also associated with multiple adverse effects. This has led to the concept of designing noncarbohydrate, aromatic mimetics that modulate H/HS function. In this work, we study a library of synthetic, aromatic H/HS mimetics for their capillary electrophoretic profiles, the acid and base stability, and aqueous-organic partitioning property. The nonsugar H/HS mimetics exhibit electrophoretic properties similar to sulfated oligosaccharides suggesting that the mimetics can be rapidly and quantitatively analyzed. Stability studies show that the mimetics are essentially stable under neutral and basic conditions in a manner similar to the heparins, but are considerably unstable under acidic conditions in contrast to heparins. The measurement of partition coefficients show major differences within the sulfated mimetics as well as between the measured and calculated log P values. Understanding these physico-chemical properties is expected to have significant implications in the pharmaceutical development of this growing class of molecules.

Capillary Electrophoretic Study of Small, Highly Sulfated, Non- Sugar Molecules Interacting With Antithrombin

Affinity CE (ACE) was used to study interactions of small, highly sulfated, aromatic molecules with antithrombin (AT). The high charge density of the small molecules induces differential migration of the complex resulting in a versatile method of assessing binding affinities, nature of interactions and site of binding on the inhibitor. Scatchard analysis of the interaction of three tetrahydroisoquinoline‐based polysulfated molecules with AT results in monophasic profiles with affinities in the range of 40–60 μM in 20 mM sodium phosphate buffer, pH 7.4. For a pentasulfated molecule, a biphasic profile with affinities of 4.7 and 30 μM was observed. Measurement of KD as a function of ionic strength of the medium indicated that ionic and non‐ionic forces contribute 2.4 and 1.9 kcal/mol, respectively, at pH 7.4 and 100 mM NaCl. Competitive binding studies showed that the tetrahydroisoquinoline‐based molecules do not compete with a high‐affinity heparin pentasaccharide. In contrast, the affinity of these tetrahydroisoquinoline derivatives decreases dramatically in the presence of an extended heparin‐binding site ligand. Overall, ACE analysis of small, sulfated aromatic molecules interacting with AT is relatively easy and obviates the need for an external signal, e.g. fluorescence, for monitoring the interaction. In addition to affording biochemical knowledge, the small sample requirement and fast analysis time of ACE could be particularly advantageous for high‐throughput screening of potential anticoagulants.

Studying Glycosaminoglycan–Protein Interactions Using Capillary Electrophoresis

Methods for studying interactions between glycosaminoglycans (GAGs) and proteins have assumed considerable significance as their biological importance increases. Capillary electrophoresis (CE) is a powerful method to study these interactions due to its speed, high efficiency, and low sample/reagent consumption. In addition, CE works effectively under a wide range of physiologically relevant conditions. This chapter presents state-of-the-art on CE methods for studying GAG-protein interactions including affinity capillary electrophoresis (ACE), capillary zone electrophoresis (CZE), frontal analysis (FA)/frontal analysis continuous capillary electrophoresis (FACCE), and capillary electrokinetic chromatography (CEC) with detailed experimental protocols for ACE and CZE methods.

Dynamic affinity chromatography in the separation of sulfated lignins binding to thrombin

Sulfated low molecular weight lignins (LMWLs), a mixture of chemo-enzymatically prepared oligomers, have been found to be potent antagonists of coagulation. However, structures that induce anticoagulation remain unidentified. The highly polar sulfate groups on these molecules and the thousands of different structures present in these mixtures make traditional chromatographic resolution of sulfated LMWLs difficult. We performed dynamic thrombin affinity chromatography monitored using chromogenic substrate hydrolysis assay to isolate sulfated LMWL fractions that differed significantly in their biophysical and biochemical properties. Three fractions, I(35), I(55) and Peak II, were isolated from the starting complex mixture. Independent plasma clotting assays suggested that I(35) possessed good anticoagulation potential (APTT=4.2μM; PT=6.8μM), while I(55) and Peak II were approximately 10- and 100-fold less potent. The ESI-MS spectrum of this oligomeric fraction showed multiple peaks at 684.8, 610.6, 557.4, 541.4, 536.5, and 519.4m/z, which most probably arise from variably functionalized β-O4β-β-linked trimers and/or a β-O4β-O4-linked dimers. The first direct observation of these structures in sulfated LMWLs will greatly assist in the discovery of more potent sulfated LMWL-based anticoagulants.