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INFLAMMATION AND IMMUNOPHARMACOLOGY

Discovery of a Dual-Function Peptide That Combines Aminopeptidase N Inhibition and Kinin B₁ Receptor Antagonism

Department of Biochemistry, University of Colorado Health Sciences Center, Denver, Colorado (L.G., J.M.S.); Centre de Recherche en Rhumatologie et Immunologie, Centre Hospitalier Universitaire de Québec, Quebec, Canada (J.-P.F., F.M.); and Faculté de Pharmacie, Université de Montréal, Montreal, Quebec, Canada (A.A.)

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 B₁ receptor (agonists or antagonists). In this study, we found that the high-affinity antagonist B-9958 (Lys-Lys-[Hyp³, CpG⁵, D-Tic⁷, CpG⁸]des-Arg⁹-BK; des-Arg⁹-BK, des-arginine⁹-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 pA₂ 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 B₁ receptors (1.2–2.8-fold), as estimated by the displacement of [³H]Lys-des-Arg⁹-BK binding, but were more potent antagonists of des-Arg⁹-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 µM; kinin B₁ receptor binding K_i, 0.5–1.5 nM), and this may be an advantage for specific therapeutic applications (e.g., inhibition of angiogenesis).

Address correspondence to: Dr. François Marceau, Centre de Recherche en Rhumatologie et Immunologie, CHUQ, Pavillon CHUL, T1-49, 2705 Laurier Boulevard, Quebec, Canada G1V 4G2. E-mail: francois.marceau{at}crchul.ulaval.ca

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