Designing allosteric regulators of thrombin. Monosulfated benzofuran dimers selectively interact with Arg173 of exosite 2 to induce inhibition.
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.
Department of Medicinal Chemistry and ‡Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University , Richmond, Virginia 23219, United States.
May H
MH
Abdel Aziz
Preetpal Singh
PS
Sidhu
Aiye
A
Liang
Ji Yeong
JY
Kim
Philip D
PD
Mosier
Qibing
Q
Zhou
David H
DH
Farrell
Umesh R
UR
Desai
D000494:Allosteric Regulation; D000925:Anticoagulants / Q000138:chemical synthesis* / Q000737:chemistry / Q000633:toxicity; D001120:Arginine / Q000235:genetics*; D001190:Arylsulfonates / Q000138:chemical synthesis* / Q000737:chemistry / Q000633:toxicity; D001572:Benzofurans / Q000138:chemical synthesis* / Q000737:chemistry / Q000633:toxicity; D001665:Binding Sites; D002460:Cell Line; D019281:Dimerization; D065427:Factor Xa Inhibitors; D005340:Fibrinogen / Q000737:chemistry; D006493:Heparin / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics
D000925:Anticoagulants; D001190:Arylsulfonates; D001572:Benzofurans; D065427:Factor Xa Inhibitors; D011994:Recombinant Proteins; D005340:Fibrinogen; D006493:Heparin; D001120:Arginine; D013917:Thrombin