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

J Pharmacol Exp Ther. 2006 Apr;317(1):300-8. doi: 10.1124/jpet.105.095661. Epub 2005 Dec 20.

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).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding, Competitive
  • Bradykinin / analogs & derivatives*
  • Bradykinin / pharmacology
  • Bradykinin B1 Receptor Antagonists*
  • CD13 Antigens / antagonists & inhibitors*
  • Cell Membrane / drug effects
  • Cell Membrane / enzymology
  • Cell Membrane / metabolism
  • Cells, Cultured
  • Dose-Response Relationship, Drug
  • Enzyme Inhibitors / pharmacology*
  • Humans
  • Muscle Contraction / drug effects
  • Myocytes, Smooth Muscle / drug effects*
  • Myocytes, Smooth Muscle / enzymology
  • Myocytes, Smooth Muscle / metabolism
  • Rabbits
  • Radioligand Assay
  • Substrate Specificity

Substances

  • B-9958
  • Bradykinin B1 Receptor Antagonists
  • Enzyme Inhibitors
  • CD13 Antigens
  • Bradykinin