title: Potent bivalent thrombin inhibitors: replacement of the scissile peptide bond at P(1)-P(1)' with arginyl ketomethylene isosteres.
abstract: We have designed highly potent synthetic bivalent thrombin inhibitors, which consist of an active site blocking segment, a fibrinogen recognition exosite blocking segment, and a linker connecting these segments. The bivalent inhibitors bind to the active site and the fibrinogen recognition exosite simultaneously. As a result, the inhibitors showed much higher affinity for thrombin than the individual blocking segments. Various arginyl ketomethylene isosteres ArgPsi[CO-CH(2)-X]P(1)' were incorporated into the bivalent inhibitors as P(1)-P(1)' segment to eliminate the scissile bond. The P(1)' residue is a natural or unnatural amino acid; specifically, the incorporation of mercaptoacetic acid exhibited superiority in synthesis and affinity for thrombin. Inhibitor 16, (D-cyclohexylalanine)-Pro-ArgPsi[CO-CH(2)-S]Gly-(Gly)(4)-Asp-Tyr-G lu- Pro-Ile-Pro-Glu-Glu-Tyr-cyclohexylalanine-(D-Glu)-OH, showed the lowest K(i) value of 3.5 +/- 0.5 x 10(-13) M, which is comparable to that (K(i) = 2.3 x 10(-13) M) of recombinant hirudin. Consequently we successfully reduced the size of the inhibitor from approximately 7 kDa of recombinant hirudin to approximately 2 kDa without losing the affinity.
authors.affiliation: Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Avenue, Montreal, Quebec, Canada H4P 2R2. H7V 4A7.
authors.forename: T
authors.initials: T
authors.lastname: Steinmetzer
authors.forename: B Y
authors.initials: BY
authors.lastname: Zhu
authors.forename: Y
authors.initials: Y
authors.lastname: Konishi
mesh_terms: D000818:Animals; D002417:Cattle; D005337:Fibrin / Q000737:chemistry; D006629:Hirudins / Q000737:chemistry; D006801:Humans; D010455:Peptides; D011485:Protein Binding; D011506:Proteins / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000138:chemical synthesis* / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013917:Thrombin / Q000037:antagonists & inhibitors* / Q000737:chemistry
chemical_list: D006629:Hirudins; D010455:Peptides; D011506:Proteins; D015842:Serine Proteinase Inhibitors; C120837:cyclohexylalanine-prolyl-arginyl-psi(COCH2S)glycyl-glycyl-glycyl-glycyl-glycyl-aspartyl-tyrosyl-glutamyl-prolyl-isoleucyl-prolyl-glutamyl-glutamyl-tyrosyl-cyclohexylalanine-glutamic acid; D005337:Fibrin; D013917:Thrombin