title: Design and characterization of hirulogs: a novel class of bivalent peptide inhibitors of thrombin.
abstract: A novel class of synthetic peptides has been designed that inhibit the thrombin catalytic site and exhibit specificity for the anion-binding exosite (ABE) of alpha-thrombin. These peptides, called "hirulogs", consist of (i) an active-site specificity sequence with a restricted Arg-Pro scissile bond, (ii) a polymeric linker of glycyl residues from 6 to 18 A in length, and (iii) an ABE recognition sequence such as that in the hirudin C-terminus. Hirulog-1 ([D-Phe)-Pro-Arg-Pro-(Gly)4-Asn-Gly-Asp-Phe-Glu-Glu-Ile- Pro-Glu-Tyr-Leu] inhibits the thrombin-catalyzed hydrolysis of a tripeptide p-nitroanilide substrate with Ki = 2.3 nM. In contrast, the synthetic C-terminal hirudin peptide S-Hir53-64, which binds to the thrombin ABE, blocked the fibrinogen clotting activity of the enzyme with Ki = 144 nM but failed to inhibit the hydrolysis of p-nitroanilide substrates at concentrations as high as 1 mM. In addition, the pentapeptide (D-Phe)-Pro-Arg-Pro-Gly, which comprises the catalytic-site inhibitor moiety of hirulog-1, was determined to have a Ki for thrombin inhibition greater than 2 microM. Hirulog-1, but not S-Hir53-64, was found to inhibit the incorporation of [14C]diisopropyl fluorophosphate in thrombin. Hirulog-1 appears specific for thrombin as it lacks inhibitory activities toward human factor Xa, human plasmin, and bovine trypsin at inhibitor:enzyme concentrations 3 orders of magnitude higher than those required to inhibit thrombin. The optimal inhibitory activity of hirulog-1 depends upon all three components of its structure.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Biogen Inc., Cambridge, Massachusetts 02142.
authors.forename: J M
authors.initials: JM
authors.lastname: Maraganore
authors.forename: P
authors.initials: P
authors.lastname: Bourdon
authors.forename: J
authors.initials: J
authors.lastname: Jablonski
authors.forename: K L
authors.initials: KL
authors.lastname: Ramachandran
authors.forename: J W
authors.initials: JW
authors.lastname: Fenton
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000925:Anticoagulants; D001665:Binding Sites; D002417:Cattle; D015195:Drug Design; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000494:pharmacology; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000037:antagonists & inhibitors*
chemical_list: D000925:Anticoagulants; D010446:Peptide Fragments; D010455:Peptides; D013917:Thrombin