Designing allosteric regulators of thrombin. Monosulfated benzofuran dimers selectively interact with Arg173 of exosite 2 to induce inhibition.
ABSTRACT 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.
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ABSTRACT: 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 homologs of the parent designed dimers were synthesized in 7-8 steps and found to exhibit a wide range of potencies. Among these, trimer 9a 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 '-fibrinogen peptide (exosite 2 ligands), demonstrated exosite 2 recognition in a manner dramatically different from the parent dimers. Alanine scanning mutagenesis of 12 Arg/Lys residues of exosite 2 revealed a defect in 9a 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.Journal of Medicinal Chemistry 05/2013; · 5.61 Impact Factor
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ABSTRACT: In-Silico drug design tools are extensively used to improve the drug discovery program by reducing the cost associated with initial screening, exploring larger chemical space, and eliminating the non drug-like molecules at very initial stages of discovery. 1-3 With advancement in the computational field and development of various computational chemistry software suites, in-silico approach emerges with greater predictive ability in identifying the initial lead candidate. 4-5 Molecular docking and virtual screening are more commonly used these days to identify and design novel inhibitors with a rational design approach. Availability of crystal structures of various proteins via open sources has made structure-based drug design approach increasingly common. In-silico approach has been successfully applied to the discovery of inhibitors for enzymes such as proteases and kinases targeting numerous diseases. Here, we specifically discuss two cases of virtual screening approach to discover direct and allosteric inhibitors of thrombin. 6-9 Thrombin is a key enzyme involved in regulation of the coagulation cascade. Quick search on the NCBI database reveals more than 900 structures of wild or mutant-type thrombin. A thorough understanding of Thrombin structure will facilitate designing an inhibitor targeting the active site and exosites of thrombin. 10-16 Several successful attempts have been made to design such inhibitors using ligand or structure-based drug design approach using various in-silico tools. Not only the active site of thrombin, but also the allosteric sites were potential targets for an efficient inhibitor design. However, rationally designing allosteric inhibitors is still difficult due to the lack of understanding of allosteric connections and shallow nature of its exosites. 8-9 Recent work by Sidhu et al 13 shows that allosteric inhibitors of thrombin were discovered using ligand-based virtual screening approach. In their study, pharmacophore model was generated from a library of allosteric inhibitors of thrombin targeting the exosite II of thrombin. The important features required for mediating allosteric inhibition of thrombin were identified. The generated pharmacophore was used to perform virtual screening on Zinc Database containing a collection of approximately one million compounds. The hit molecules identified from virtual screening were modified to resemble the actual structures. Two-step docking filter was applied using GOLD docking software to select the hits that fit best in binding site. In the first filter, docking studies were performed to identify hit molecules that bind in a defined binding region with high binding score. In the second-step, molecules selected from the first filter were docked in triplicate. The binding modes generated from all three independent runs were compared. The molecules with similar binding pose in three independent runs were selected based on R 2 value. The ten molecules that were chosen from the library showed good binding score and consistent binding poses. Three molecules were purchased and sulfated using microwave-based sulfation protocol. All three molecules showed good thrombin inhibition potency and were found to be allosteric in nature. Also, the plasma clotting studies showed good anti-coagulation activity. The present work is a novel in-silico approach to identify allosteric inhibitors of thrombin.Journal of Postdoctoral Research. 12/2013; 1(12):46.
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ABSTRACT: Growing evidence suggests that plasmin is involved in a number of physiological processes in addition to its key role in fibrin cleavage. Plasmin inhibition is critical in preventing adverse consequences arising from plasmin overactivity, e.g., blood loss that may follow cardiac surgery. Aprotinin was widely used as an antifibrinolytic drug before its discontinuation in 2008. Tranexamic acid and ε-aminocaproic acid, two small molecule plasmin inhibitors, are currently used in the clinic. Several molecules have been designed utilizing covalent, but reversible, chemistry relying on reactive cyclohexanones, nitrile warheads, and reactive aldehyde peptidomimetics. Other major classes of plasmin inhibitors include the cyclic peptidomimetics and polypeptides of the Kunitz and Kazal-type. Allosteric inhibitors of plasmin have also been designed including small molecule lysine analogs that bind to plasmin's kringle domain(s) and sulfated glycosaminoglycan mimetics that bind to plasmin's catalytic domain. Plasmin inhibitors have also been explored for resolving other disease states including cell metastasis, cell proliferation, angiogenesis, and embryo implantation. This review highlights functional and structural aspects of plasmin inhibitors with the goal of advancing their design.Medicinal Research Reviews 03/2014; · 9.58 Impact Factor