title: Designing allosteric regulators of thrombin. Exosite 2 features multiple subsites that can be targeted by sulfated small molecules for inducing inhibition.
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 homologues of the parent designed dimers were synthesized in seven to eight 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 γ'-fibrinogen peptide (exosite 2 ligands) demonstrated exosite 2 recognition in a manner different from that of 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.
authors.affiliation: Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, Virginia 23219, USA.
authors.forename: Preetpal Singh
authors.initials: PS
authors.lastname: Sidhu
authors.forename: May H
authors.initials: MH
authors.lastname: Abdel Aziz
authors.forename: Aurijit
authors.initials: A
authors.lastname: Sarkar
authors.forename: Akul Y
authors.initials: AY
authors.lastname: Mehta
authors.forename: Qibing
authors.initials: Q
authors.lastname: Zhou
authors.forename: Umesh R
authors.initials: UR
authors.lastname: Desai
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects; D001572:Benzofurans / Q000737:chemistry; D001665:Binding Sites; D001777:Blood Coagulation / Q000187:drug effects; D019281:Dimerization; D015195:Drug Design; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D015842:Serine Proteinase Inhibitors / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D013379:Substrate Specificity; D013431:Sulfates / Q000737:chemistry*; D013917:Thrombin / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001572:Benzofurans; D015842:Serine Proteinase Inhibitors; D013431:Sulfates; D013917:Thrombin; C105430:benzofuran