title: Discovery of a dual-function peptide that combines aminopeptidase N inhibition and kinin B1 receptor antagonism.
abstract: Previous analyses support that aminopeptidase N is a major inactivation pathway for high-affinity peptide ligands of the human and rabbit forms of the kinin B(1) receptor (agonists or antagonists). In this study, we found that the high-affinity antagonist B-9958 (Lys-Lys-[Hyp(3), CpG(5), D-Tic(7), CpG(8)]des-Arg(9)-BK; des-Arg(9)-BK, des-arginine(9)-bradykinin) is an aminopeptidase N substrate based on its capacity to compete for the hydrolysis of the chromogenic substrate L-Ala-p-nitroanilide by membranes isolated from human or rabbit arterial smooth muscle cells, its inactivation in the presence of these membranes (radioreceptor assay) and on its intense potentiation by the aminopeptidase N inhibitor amastatin in the rabbit aorta contractility assay (gain of 0.84 units in the pA(2) scale). Analogs of B-9958 in which the N-terminal Lys residue was substituted by D-Lys or D-Arg (B-10352 and B-10356, respectively) showed reduced affinity at the human or rabbit B(1) receptors (1.2-2.8-fold), as estimated by the displacement of [(3)H]Lys-des-Arg(9)-BK binding, but were more potent antagonists of des-Arg(9)-BK-induced contraction of the rabbit aorta than B-9958 in the absence of amastatin; they were not potentiated by the latter inhibitor. Unexpectedly, B-10356 inhibited L-Ala- p-nitroanilide hydrolysis without being inactivated, suggesting that it is an aminopeptidase N inhibitor. This was verified because B-10356 (but not B-10352) potentiated peptides unrelated to kinins but susceptible to aminopeptidase N inactivation (angiotensin III, thrombin receptor hexapeptide agonist). B-10356 inhibits dual molecular targets (aminopeptidase N enzyme K(i), 0.9-2.2 microM; kinin B(1) receptor binding K(i), 0.5-1.5 nM), and this may be an advantage for specific therapeutic applications (e.g., inhibition of angiogenesis).
authors.affiliation: Department of Biochemistry, University of Colorado Health Sciences Center, Denver, USA.
authors.forename: Lajos
authors.initials: L
authors.lastname: Gera
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Fortin
authors.forename: Albert
authors.initials: A
authors.lastname: Adam
authors.forename: John M
authors.initials: JM
authors.lastname: Stewart
authors.forename: François
authors.initials: F
authors.lastname: Marceau
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D001920:Bradykinin / Q000031:analogs & derivatives* / Q000494:pharmacology; D065169:Bradykinin B1 Receptor Antagonists; D018826:CD13 Antigens / Q000037:antagonists & inhibitors*; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D009119:Muscle Contraction / Q000187:drug effects; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D011817:Rabbits; D011869:Radioligand Assay; D013379:Substrate Specificity
chemical_list: C510436:B-9958; D065169:Bradykinin B1 Receptor Antagonists; D004791:Enzyme Inhibitors; D018826:CD13 Antigens; D001920:Bradykinin