Bradykinin antagonists in human systems: correlation between receptor binding, calcium signalling in isolated cells, and functional activity in isolated ileum.

The determination of the relationship between ligand affinity and bioactivity is important for the understanding of receptor function in biological systems and for drug development. Several physiological and pathophysiological functions of bradykinin (BK) are mediated via the B2 receptor. In this study, we have examined the relationship between B2 receptor (soluble and membrane-bound) binding of BK peptidic antagonists, inhibition of calcium signalling at a cellular level, and in vitro inhibition of ileum contraction. Only human systems were employed in the experiments. Good correlations between the studied activities of BK antagonists were observed for a variety of different peptidic structures. The correlation coefficients (r) were in the range of 0.905 to 0.955. In addition, we analyzed the effect of the C-terminal Arg9 removal from BK and its analogs on B2 receptor binding. The ratios of binding constants (Ki(+Arg)/Ki(-Arg)) for the Arg9 containing compounds and the corresponding des-Arg9 analogs varied from about 10 to 250,000. These ratios strongly depend on the chemical structures of the compounds. The highest ratios were observed for two natural agonist pairs, BK/des-Arg9-BK and Lys0-BK/des-Arg9-Lys0-BK.

Bradykinin receptor antagonists containing N-substituted amino acids: in vitro and in vivo B(2) and B(1) receptor antagonist activity.

We report a systematic probing of the structural requirements of the bradykinin (BK) type 2 (B(2)) receptor for antagonist activity by incorporating N-alkyl-amino acid residues at positions 7 and 8 of a potent antagonist sequence. Compound 1 (D-Arg(0)-Arg(1)-Pro(2)-Hyp(3)-Gly(4)-Thi(5)-Ser(6)-D-Tic(7)-N-Chg (8)-Arg(9), CP-0597)(1,2) is a potent (pA(2) = 9.3, rat uterus; pK(i) = 9.62, binding, human receptor clone) B(2) receptor antagonist devoid of in vitro B(1) antagonist activity (rabbit aorta). Compound 1 exhibits high potency (ED(50) = 29.2 pmol/kg/min, iv, rabbit) and duration of action when tested in models for in vivo B(2) antagonist activity. Although devoid of activity in a classic B(1) isolated tissue assay, B(1) antagonist activity for 1 was demonstrated in vivo, in a LPS-treated, inducible BK(1) receptor rabbit blood pressure model (ED(50) = 1.7 nmol/kg/min). D-Arg(0) of 1 can be formally replaced by an achiral arginine surrogate, without significant loss in antagonist potency on rat uterus (compound 11, B(2) pA(2) = 9.1). Antagonist 13 (Hyp(2), Nchg(8)), pK(i) = 10.2, and agonist 4 (N-methylcyclohexyl-Gly(8)), pK(i) = 10.1, also exhibited substantial binding to guinea pig ileum membrane receptors as well as a human B(2) receptor clone. Very minor structural changes in the N-alkyl amino acid residues in positions 7 and 8 can modify the activity of this class of compounds from being extremely potent antagonists to tight binding partial or full agonists. These studies have resulted in a series of compounds containing inexpensive amino acid residues but which produce broad spectrum BK receptor blocking potency and exceptional in vivo duration of action.