Discovery of a dual-function peptide that combines aminopeptidase N inhibition and kinin B1 receptor antagonism

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-[Hyp3, CpG5, D-Tic7, CpG8] des-Arg9BK; des-Arg9-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 (1.2-2.8fold), as estimated by the displacement of [H-3] 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 mu M; 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).