MEN16132, a novel potent and selective nonpeptide antagonist for the human bradykinin B 2 receptor. In vitro pharmacology and molecular characterization

Abstract

The pharmacological characterization of the novel nonpeptide antagonist for the B 2 receptor, namely MEN16132 (4-( S)-Amino-5-(4-{4-[2,4-dichloro-3-(2,4-dimethyl-8-quinolyloxymethyl)phenylsulfonamido]-tetrahydro-2 H-4-pyranylcarbonyl}piperazino)-5-oxopentyl](trimethyl)ammonium chloride hydrochloride) is presented. The affinity of MEN16132 for the bradykinin B 2 receptor has been investigated by means of competition studies at [ 3H]bradykinin binding to membranes prepared from Chinese Hamster Ovary (CHO) cells expressing the human bradykinin B 2 receptor (pK i 10.5), human lung fibroblasts (pK i 10.5), guinea pig airways (pK i 10.0), guinea pig ileum longitudinal smooth muscle (pK i 10.2), or guinea pig cultured colonic myocytes (pK i 10.3). In all assays MEN16132 was as potent as the peptide antagonist Icatibant, and from 3- to 100-fold more potent than the reference nonpeptide antagonists FR173657 or LF16-0687. The selectivity for the bradykinin B 2 receptor was checked at the human bradykinin B 1 receptor (pK i < 5), and at a panel of 26 different receptors and channels. The antagonist potency was measured in functional assays, i.e., in blocking the bradykinin induced inositolphosphates (IP) accumulation at the human (CHO: pK B 10.3) and guinea pig (colonic myocytes: pK B 10.3) B 2 receptor, or in antagonizing the bradykinin induced contractile responses in human (detrusor smooth muscle: pK B 9.9) and guinea pig (ileum longitudinal smooth muscle: pK B 10.1) tissues. In both functional assay types MEN16132 exerted a different antagonist pattern, i.e., surmountable at the human and insurmountable at the guinea pig bradykinin B 2 receptors. Moreover, the receptor determinants important for the high affinity interaction of MEN16132 with the human bradykinin B 2 receptor were investigated by means of radioligand binding studies performed at 24 point-mutated receptors. The results obtained revealed that residues in transmembrane segment 2 (W86A), 3 (I110A), 6 (W256A), and 7 (Y295A, Y295F but not much Y295W), were crucial for the high affinity of MEN16132. In conclusion, MEN16132 is a new, potent, and selective nonpeptide bradykinin B 2 receptor antagonist.