Comparison of the molecular interactions of two antagonists, MEN16132 or icatibant, at the human kinin B₂ receptor.
OBJECTIVE
Icatibant is a well-known kinin B₂ receptor antagonist currently used for angiooedema attacks. MEN16132 is a non-peptide B₂ receptor antagonist, more potent and long lasting than icatibant in different models. Here we studied the reasons for these differences between the two antagonists.


METHODS
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 B₂ 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 B₂ receptors.


RESULTS
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 B₂ 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 B₂ 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-B₂ receptor complex is proposed.


CONCLUSIONS
MEN16132 dissociated from the B₂ receptor compartment more slowly than icatibant and interacted at a deeper level in transmembrane regions of the receptor.
Department of Pharmacology, Menarini Ricerche, Florence, Italy. smeini@menarini-ricerche.it
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D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D001920:Bradykinin / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D065094:Bradykinin B2 Receptor Antagonists; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism / Q000494:pharmacology; D009952:Ornithine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D010456:Peptides, Cyclic / Q000378:metabolism / Q000494:pharmacology; D043782:Receptor, Bradykinin B2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013449:Sulfonamides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*
C506838:(4-amino-5-(4-(4-(2,4-dichloro-3-(2,4-dimethyl-8-quinolyloxymethyl)phenylsulfonamido)tetrahydro-2H-4-pyranoylcarbonyl)piperazino)-5-oxopentyl)(trimethyl)ammonium; D065094:Bradykinin B2 Receptor Antagonists; C119318:MEN 11270; D050505:Mutant Proteins; D009842:Oligopeptides; D010456:Peptides, Cyclic; D043782:Receptor, Bradykinin B2; D011994:Recombinant Proteins; D013449:Sulfonamides; C065679:icatibant; D009952:Ornithine; D001920:Bradykinin