Comparison of the molecular interactions of two antagonists, MEN16132 or icatibant, at the human kinin B₂ receptor

Icatibant is a well-known kinin B&sub2; receptor antagonist currently used for angiooedema attacks. MEN16132 is a non-peptide B&sub2; receptor antagonist, more potent and long lasting than icatibant in different models. Here we studied the reasons for these differences between the two antagonists. Experimental approach: Rate of reversibility (over about 3 h) of the functional receptor blockade exerted by the antagonists was compared (inositol phosphates accumulation assay) in CHO cells expressing the human B&sub2; receptor and in human synovial cells. Antagonist pretreated cells were washed with medium and the time taken to restore bradykinin (BK) response measured. Antagonist affinity was measured by radioligand binding to wild type and mutated B&sub2; receptors. Key

Icatibant is a well-known kinin B&sub2; receptor antagonist currently used for angiooedema attacks. MEN16132 is a non-peptide B&sub2; receptor antagonist, more potent and long lasting than icatibant in different models. Here we studied the reasons for these differences between the two antagonists. Experimental approach: Rate of reversibility (over about 3 h) of the functional receptor blockade exerted by the antagonists was compared (inositol phosphates accumulation assay) in CHO cells expressing the human B&sub2; receptor and in human synovial cells. Antagonist pretreated cells were washed with medium and the time taken to restore bradykinin (BK) response measured. Antagonist affinity was measured by radioligand binding to wild type and mutated B&sub2; receptors. Key

Recovery of BK-induced responses was slower in cells pretreated with MEN16132 than in those treated with icatibant. The affinity of icatibant (for the [³H]-BK or the B&sub2; receptor antagonist [³H]-MEN11270 binding site) was compared to that of MEN16132 using a panel of point-mutated receptors with mutations located at the transmembrane regions of the B&sub2; receptor, previously shown to decrease MEN16132 high affinity interaction. No consistent decrease of icatibant affinity was observed. From the different affinity of MEN16132 derivatives at wild type and W86A (transmembrane 2 region) receptors, and by evaluating its antagonist profile at the D266A/D284A double mutant receptor, a model of the MEN16132-B&sub2; receptor complex is proposed. Conclusions and implications: MEN16132 dissociated from the B&sub2; receptor compartment more slowly than icatibant and interacted at a deeper level in transmembrane regions of the receptor.