The N-terminal of Icatibant and bradykinin interact with the same Asp residues in the human B 2 receptor

Abstract

The pharmacology of peptide and non-peptide bradykinin B 2 receptor ligands was evaluated in the inositol phosphate (IP) production assay in CHO cells expressing the human bradykinin B 2 receptor. The effect of single and double alanine mutation of D266 and D284 residues at the human bradykinin B 2 receptor was evaluated on the agonist profile of bradykinin (H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH) and the synthetic agonist FR190997 (8-[2,6-dichloro-3-[ N-methylcarbamoyl)cinnamidoacetyl]- N-methylamino]benzyloxy]-2-methyl-4-(2-pyridylmethoxy)quinoline). Bradykinin potency (EC 50 0.5 nM at the wild-type receptor) was reduced by 16-fold at D266A and D284A mutants and by 2300-fold at the D266A/D284A double mutant. None of the mutants affected the potency or the efficacy of FR190997. Peptide antagonists, Icatibant (H-DArg-Arg-Pro-Hyp-Gly-Thi-Ser-Dtic-Oic-Arg-OH) and MEN11270 (H-DArg-Arg-Pro-Hyp-Gly-Thi-c(Dab-DTic-Oic-Arg)c(7γ-10α)) (100 nM) similarly antagonized the concentration–response curve to bradykinin or FR190997 (pA 2 values 8.5 and 8.4 versus bradykinin and 8.2 and 8.4 versus FR190997) at the wild-type receptor. Non-peptide antagonists FR173657 ((E)-3-(6-acetamido-3-pyridyl)- N-[ N-[2,4-dichloro-3-[(2-methyl-8-quinolinyl) oxymethyl]phenyl]- N-methylaminocarbonyl methyl]acrylamide) and LF16-0687 (1-[[2,4-dichloro-3-[(2,4-dimethylquinolin-8-yl)oxy] methyl]-phenyl]sulfonyl]- N-[3-[[4-(aminoiminomethyl)-phenyl]carbonylamino]propyl]-(S)-pyrrolidine carboxamide) (100 nM) showed an equivalent potency values in blocking the IP production induced by bradykinin or FR190997 (pA 2 values 8.7 and 8.8 versus bradykinin and 8.8 and 8.6 versus FR190997). Whilst the antagonist potency of FR173657 and LF16-0687 was not affected by D266A/D284A double mutation (IP production induced by the synthetic agonist), that of Icatibant and MEN11270 was reduced by 50- and 200-fold. The antagonist potency of [Ala 1]-Icatibant and [Ala 2]-Icatibant (pA 2 values at wild-type 7.7 and 6.4) was significantly less reduced (20-fold and 13-fold, respectively) by the D266A/D284A double mutation. Our results highlight a crucial role for two aspartic residues, D266 and D284, located at the top of transmembrane segments 6 and 7, in the high-affinity interaction of peptide antagonists with the human bradykinin B 2 receptor. An interaction of these receptor residues with the N-terminal basic residues of Icatibant is hypothesized.