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

Department of Medicinal Chemistry and ‡Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University , Richmond, Virginia 23219, United States.
Journal of Medicinal Chemistry (Impact Factor: 5.45). 07/2012; 55(15):6888-97. DOI: 10.1021/jm300670q
Source: PubMed


Earlier, we reported on the design of sulfated benzofuran dimers (SBDs) as allosteric inhibitors of thrombin (Sidhu et al. J. Med. Chem.201154 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 IC(50) 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 IC(50). Docking studies suggested a hydrophobic patch around Arg173 as a plausible site of SBD binding to thrombin. The absence of the Arg173-like residue in factor Xa supported the observed selectivity of inhibition by SBDs. Cellular toxicity studies indicated that SBDs are essentially nontoxic 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.

Download full-text


Available from: Preetpal S. Sidhu, Jan 04, 2015
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The design of sulfated, small, nonsaccharide molecules as modulators of proteins is still in its infancy as standard drug discovery tools such as library of diverse sulfated molecules and in silico docking and scoring protocol have not been firmly established. Databases, such as ZINC, contain too few sulfate-containing nonsaccharide molecules, which severely limits the identification of new hits. Lack of a generally applicable protocol for scaffold hopping limits the development of sulfated small molecules as synthetic mimetics of the highly sulfated glycosaminoglycans. We explored a sequential ligand-based (LBVS) and structure-based virtual screening (SBVS) approach starting from our initial discovery of monosulfated benzofurans to discover alternative scaffolds as allosteric modulators of thrombin, a key coagulation enzyme. Screening the ZINC database containing nearly 1 million nonsulfated small molecules using a pharmacophore developed from the parent sulfated benzofurans followed by a genetic algorithm-based dual-filter docking and scoring screening identified a group of 10 promising hits, of which three top-scoring hits were synthesized. Each was found to selectively inhibit human alpha-thrombin suggesting the possibility of this approach for scaffold hopping. Michaelis-Menten kinetics showed allosteric inhibition mechanism for the best molecule and human plasma studies confirmed good anticoagulation potential as expected. Our simple sequential LBVS and SBVS approach is likely to be useful as a general strategy for identification of sulfated small molecules hits as modulators of glycosaminoglycan-protein interactions.
    Bioorganic & medicinal chemistry letters 10/2012; 23(1). DOI:10.1016/j.bmcl.2012.10.079 · 2.42 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Inhibition of factor XIa (FXIa) is a novel paradigm for developing anticoagulants without major bleeding consequences. We present the discovery of sulfated pentagalloylglucoside (6) as a highly selective inhibitor of human FXIa. Biochemical screening of a focused library led to the identification of 6, a sulfated aromatic mimetic of heparin. Inhibitor 6 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 6 reduced VMAX of FXIa hydrolysis of chromogenic substrate without affecting the KM suggesting an allosteric mechanism. Competitive studies showed that 6 bound in the heparin-binding site of FXIa. No allosteric small molecule has been discovered to date that exhibits equivalent potency against FXIa. Inhibitor 6 is expected to open up a major route to allosteric FXIa anticoagulants with clinical relevance.
    Journal of Medicinal Chemistry 01/2013; DOI:10.1021/jm301338q · 5.45 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: To discover promising sulfated allosteric modulators (SAMs) of glycosaminoglycan-binding proteins (GBPs), such as human factor XIa (FXIa), we screened a library of 26 synthetic, sulfated quinazolin-4(3H)-ones (QAOs) resulting in the identification of six molecules that reduced the VMAX of substrate hydrolysis without influencing the KM. Mutagenesis of residues of the heparin-binding site of FXIa introduced a nearly 5-fold loss in inhibition potency supporting recognition of an allosteric site. Fluorescence studies showed a sigmoidal binding profile indicating highly cooperative binding. Competition with a positively-charged, heparin-binding polymer did not fully nullify inhibition suggesting importance of hydrophobic forces to binding. This discovery suggest the operation of a dual-element recognition process, which relies on an initial Coulombic attraction of anionic SAMs to the cationic HBS of FXIa that forms a locked complex through tight interaction with an adjacent hydrophobic patch. The dual-element strategy may be widely applicable for discovering SAMs of other GBPs.
    Journal of Medicinal Chemistry 03/2013; 56(6). DOI:10.1021/jm301757v · 5.45 Impact Factor
Show more