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

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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 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 Ki, 0.9–2.2 μM; kinin B1 receptor binding Ki, 0.5–1.5 nM), and this may be an advantage for specific therapeutic applications (e.g., inhibition of angiogenesis).