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

Abstract

BACKGROUND AND PURPOSEIcatibant 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. EXPERIMENTAL APPROACHRate 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. KEY RESULTSRecovery of BK-induced responses was slower in cells pretreated with MEN16132 than in those treated with icatibant. The affinity of icatibant (for the [ 3 H]-BK or the B2 receptor antagonist [ 3 H]-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 AND IMPLICATIONSMEN16132 dissociated from the B2 receptor compartment more slowly than icatibant and interacted at a deeper level in transmembrane regions of the receptor.

New insights into the stereochemical requirements of the bradykinin B2 receptor antagonists binding

“…Moreover, this idea also explains the observed effect of the mutation Asn 297 Ala in close contact with Trp 256 [39]. Unfortunately there are no results available on the mutation of Ile 110 or Phe 259 on the affinity of the ligand, but according to the present modeling study it is expected one order of magnitude decrease as shown in fasitibant [31]. On the other side of the molecule anatibant and fasitibant exhibit differential stereochemical features that force them to bind in a differential way.…”

“…The final complex considered for refinement was selected based on the degree of fulfilment of diverse site-directed mutagenesis studies. Special attention was given to residues Trp 86 , Ile 110 , Trp 256 , Asp 266 and Tyr 295 [31].…”

“…Thus, for example the affinity of FR173657 is not affected by the mutation of Asp 266 and Asp 284 [36] or Ser 111 and Trp 256 [37], suggesting that these residues do not act as anchoring points in the bound conformation of the compound. In contrast, Trp 86 and Tyr 295 must be actively involved since their mutation to alanine decreases the affinity of the compound about 500 times [31]. Bearing these results in mind, several docking attempts were carried out, obtaining diverse alternative poses.…”

New insights into the stereochemical requirements of the bradykinin B1 receptor antagonists binding

“…Fasitibant (MEN16132) (1, in Figure 1) is a potent antagonist of the BK B2 receptor with a Ki of 0.09 nM [31]. The structure of fasitibant bound to the model receptor from the present docking study is shown in Figure 5.…”

“…The choice of this ligand was due to the abundant information available from site directed mutagenesis experiments [31]. Finally, the ligand-receptor complex was embedded in a lipid bilayer and refined using molecular dynamics.…”