Elucidating the specificity of non-heparin-based conformational activators of antithrombin for factor Xa inhibition

Abstract

Introduction: Antithrombin, the principal inhibitor of coagulation proteases, requires allosteric activation by its physiological cofactor, heparin or heparin sulfate to achieve physiologically permissible rates. This forms the basis of heparin's use as a clinical anticoagulant. However, heparin therapy is beset with severe complications, giving rise to the need to search new non-heparin activators of antithrombin, devoid of these complications and with favorable safety profiles.

Materials and methods: We chose some representative organic compounds that have been shown to be involved in coagulation modulation by affecting antithrombin and applied a blind docking protocol to find the binding energy and interactions of the modified (sulfated) versus unmodified organic scaffolds.

Results and conclusion: Increased sulfation plays a key role in shifting the specificity of organic compounds like quercetin, diosmin, rutin, mangiferin, isomangostin, Trapezifolixanthone and benzofuran towards the heparin binding site (HBS). However, in hesperetin and tetrahydroisoquinoline, sulfation shifts the specificity away from HBS. We have further tried to elucidate changes in the binding affinity of quercetin on account of gradual increase in the number of hydroxyl groups being substituted by sulfate groups. The results show gradual increase in binding energy with increase in sulfation. A theoretical screening approach is an ideal mechanism to predict lead molecules as activators of antithrombin and in determining the specificity for antithrombin.

Keywords: Antithrombin; PyMOL; autodock; flavonoids; heparin.

Figure 1

The structure of an antithrombin–thrombin–heparin ternary complex taken from PDB 1TB6 (a) and antithrombin factor Xa–pentasaccharide complex taken from PDB 2GD4 (b) Shows the crystal structures of the Michaelis complex between (a) antithr mbin–thrombin–heparin ternary complex taken from PDB 1TB6 and (b) antithrombin factor Xa–pentasaccharide complex taken from PDB 2GD4. Thrombin inhibition involves non-allosteric activation, figure shows that thrombin inhibition occurs due to the interaction of thrombin and AT with full-length heparin through a bridging mechanism of activation. Some negative charges available at the full-length heparin chain binds non-specifically to the exosite (positively charged region) of thrombin. Inhibition of factor Xa involves allosteric activation by a heparin pentasaccharide. Circulating ATIII interacts with the high affinity pentasaccharide sequence in full-length heparin via the heparin-binding site forming a complex with endothelial heparin, this leads to the exposure of the RCL, which recognizes factor Xa and is known to provide a conformational activtion mechanism. Molecular graphic images were produced using the UCSF Chimera package from the Resource for iocomputing, visualization and informatics at the University of California, San Francisco

Figure 2

Conformational changes in cofactor (heparin) bound antithrombin and residues involved in cofactor interaction. Structures of native and pentasaccharide bound forms of antithrombin. Antithrombin is depicted in cartoon diagram in its (a) native (1E05) (b) and pentasaccharide bound activated (1E03). The native (1E05) circulating antithrombin in blood shows several regions that are important in controlling and modulating conformational changes. The reactive center loop (RCL) is involved in protease recognition and conformational transformation as strand 4A after inhibition. (a, b) Shows the key structural differences between native and pentasaccharide bound states. It illustrates the heparin-dependent conformational changes in antithrombin, like extension of helix D by forming a 2-turn helix (P helix) at the N-terminal end, a 1.5-turn extension of helix D toward the C-terminal end. Movement of strand 3A and strand 5A and expulsion of RCL. The P1-P1’ (Arg-Ser) residues and the heparin pentasaccharide are shown as balls and sticks, respectively. (b, c) Shows the basic residues in the heparin-binding site (HBS) that interacts with the pentasaccharide (the HBS is indicated by a box in (b)): Lys-11 and Arg-13 in the N-terminal end; Arg-46 and Arg-47 in the helix A; and Lys-114, Phe-121, Phe-122, Lys-125 and Arg-129 in the region of the helix D. The figures were produced using Chimera

Figure 3

Molecular structures of some native and sulfated ligands. The structures were drawn in ChemDraw Ultra 8.0, all the hydroxyl (– OH) groups in the parent compounds were substituted by sulfate (–OSO₃⁻) groups to generate the corresponding polysulfated molecules

Figure 4

Binding affinity and polar contacts of native and sulfated quercetin and diosmin with antithrombin. Binding affinity and polar contacts of native and sulfated quercetin (a, b) and diosmin (c, d) with antithrombin. Images of ligand and antithrombin (1E05 I chain) bound complexes were prepared in PyMOL program and polar contacts between them were noted down. The structures were drawn in ChemDraw Ultra8.0. All the hydroxyl groups in each parent are substituted by a sulfate group (OSO3⁻) to generate the Corresponding polysulfated ligands