title: Design and evaluation of novel bivalent thrombin inhibitors based on amidinophenylalanines.
abstract: Two bivalent thrombin inhibitors were synthesized, which consist of a benzamidine-based active-site-blocking segment, a fibrinogen recognition exosite inhibitor and a peptidic linker connecting these fragments. BZA-1 hirulog contains an Nalpha-(2-naphthylsulfonyl)-S-3-amidinophenylalanyl-is onipecotic acid residue connected via the carboxyl group to the linker segment. The active-site-directed moiety of BZA-2 hirulog [Nalpha-(2-naphthylsulfonyl-glutamyl)-R-4-amidinophenylal anyl-piperid ide] was coupled to the linker via the side chain of the glutamic acid. Both BZA-hirulogs contain almost identical linker-exo site inhibitor parts, except for the substitution of a glycine as the first linker residue in BZA-1 hirulog by a gamma-amino butyric acid in BZA-2 hirulog, thus increasing flexibility and linker length by two additional atoms. BZA-1 hirulog showed moderate potency (Ki = 0. 50 +/- 0.14 nM), while BZA-2 hirulog was characterized as a slow, tight binding inhibitor of thrombin (Ki = 0.29 +/- 0.08 pM). The stability in human plasma of both analogs was strongly improved compared with hirulog-1. For BZA-2 hirulog a significantly reduced plasma clearance was observed after intravenous injection in rats compared with BZA-1 hirulog and hirulog-1. The X-ray structure of the BZA-2 hirulog in complex with human alpha-thrombin was solved and confirmed the expected bivalent binding mode.
authors.affiliation: Friedrich-Schiller-Universität, Institut für Biochemie & Biophysik, Jena, Germany. steinmetzer@merlin.biologie.uni-jena.de
authors.forename: T
authors.initials: T
authors.lastname: Steinmetzer
authors.forename: M
authors.initials: M
authors.lastname: Renatus
authors.forename: S
authors.initials: S
authors.lastname: Künzel
authors.forename: A
authors.initials: A
authors.lastname: Eichinger
authors.forename: W
authors.initials: W
authors.lastname: Bode
authors.forename: P
authors.initials: P
authors.lastname: Wikström
authors.forename: J
authors.initials: J
authors.lastname: Hauptmann
authors.forename: J
authors.initials: J
authors.lastname: Stürzebecher
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000991:Antithrombins / Q000737:chemistry*; D001550:Benzamidines / Q000737:chemistry*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D006146:Guanidines / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010455:Peptides / Q000138:chemical synthesis; D010649:Phenylalanine / Q000031:analogs & derivatives*; D051381:Rats
chemical_list: D000991:Antithrombins; D001550:Benzamidines; D004791:Enzyme Inhibitors; D006146:Guanidines; D010455:Peptides; D010649:Phenylalanine