Identification of the critical residues of bradykinin receptor B1 for interaction with the kinins guided by site-directed mutagenesis and molecular modeling.

We report the critical residues for the interaction of the kinins with human bradykinin receptor 1 (B1) using site-directed mutagenesis in conjunction with molecular modeling of the binding modes of the kinins in the homology model of the B1 receptor. Mutation of Lys118 in transmembrane (TM) helix 3, Ala270 in TM6, and Leu294 in TM7 causes a significant decrease in the affinity for the peptide agonists des-Arg10kallidin (KD) and des-Arg9BK but not the peptide antagonist des-Arg10Leu9KD. In contrast, mutations in TM2, TM3, TM6, and TM7 cause a significant decrease in the affinity for both the peptide agonists and the antagonist. These data indicate that the B1 bradykinin binding pocket for agonists and antagonists is similar, but the manners in which they interact with the receptor do not completely overlap. Therefore, there is a potential to influence the receptor's ligand selectivity.

Binding modes of dihydroquinoxalinones in a homology model of bradykinin receptor 1.

We report the first homology model of human bradykinin receptor B1 generated from the crystal structure of bovine rhodopsin as a template. Using an automated docking procedure, two B1 receptor antagonists of the dihydroquinoxalinone structural class were docked into the receptor model. Site-directed mutagenesis data of the amino acid residues in TM1, TM3, TM6, and TM7 were incorporated to place the compounds in the binding site of the homology model of the human B1 bradykinin receptor. The best pose in agreement with the mutation data was selected for detailed study of the receptor-antagonist interaction. To test the model, the calculated antagonist-receptor binding energy was correlated with the experimentally measured binding affinity (K(i)) for nine dihydroquinoxalinone analogs. The model was used to gain insight into the molecular mechanism for receptor function and to optimize the dihydroquinoxalinone analogs.

Pharmacological characterization and radioligand binding properties of a high-affinity, nonpeptide, bradykinin B1 receptor antagonist.

Compound A (N-[2-[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]ethyl]-2-[(2R)-1-(2-napthylsulfonyl)-3-oxo-1,2,3,4-tetrahydroquinoxalin-2-yl]acetamide) is a member of a new class of aryl sulfonamide dihydroquinoxalinone bradykinin B1 receptor antagonists that should be useful pharmacological tools. Here we report on some of the pharmacological properties of compound A as well as the characterization of [35S]compound A as the first nonpeptide bradykinin B1 receptor radioligand. Compound A inhibited tritiated peptide ligand binding to the cloned human, rabbit, dog, and rat bradykinin B1 receptors expressed in CHO cells with Ki values of 0.016, 0.050, 0.56, and 29 nM, respectively. It was inactive at 10 microM in binding assays with the cloned human bradykinin B2 receptor. In functional antagonist assays with the cloned bradykinin B1 receptors, compound A inhibited agonist-induced signaling with activities consistent with the competition binding results, but had no antagonist activity at the bradykinin B2 receptor. Compound A was also found to be a potent antagonist in a rabbit aorta tissue bath preparation and to effectively block des-Arg9 bradykinin depressor responses in lipopolysaccharide-treated rabbit following intravenous administration. The binding of [35S]compound A was evaluated with the cloned bradykinin B1 receptors. In assays with human, rabbit, and dog receptors, [35S]compound A labeled a single site with Kd values of 0.012, 0.064, and 0.37 nM, respectively, and with binding site densities equivalent to those obtained using the conventional tritiated peptide ligands. Binding assays with the cloned rat bradykinin B1 receptor were not successful, presumably due to the low affinity of the ligand for this species receptor. There was no specific binding of the ligand detected in CHO cells expressing the human bradykinin B2 receptor. In assays with the cloned human bradykinin B1 receptor, the pharmacologies of the binding of [35S]compound A and [3H][Leu9]des-Arg10-kallidin were the same. The high signal-to-noise ratio obtained with [35S]compound A will allow this ligand to be a very useful tool for future investigations of the bradykinin B1 receptor.

Generation and characterization of a human bradykinin receptor B1 transgenic rat as a pharmacodynamic model.

Antagonists of the B1 bradykinin receptor (B1R) offer the promise of novel therapeutic agents for the treatment of inflammatory and neuropathic pain. However, the in vivo characterization of the pharmacodynamics of B1R antagonists is hindered by the low level of B1R expression in healthy tissue and the profound species selectivity exhibited by many compounds for the human B1R. To circumvent these issues, we generated a transgenic rat expressing the human B1R under the control of the neuron-specific enolase promoter. Membranes prepared from whole brain homogenates of heterozygous transgenic rats indicate a B1R expression level of 30 to 40 fmol/mg; there is no detectable B1R expression in control nontransgenic rats. The pharmacological profile of the B1R expressed in the transgenic rat matches that expected of the human, but not the rat receptor. The mapping of the transgene insertion site to rat chromosome 1 permitted the development of a reliable assay for the identification of homozygous transgenic rats. Significantly, homozygous transgenic rats express 2-fold more B1R than heterozygous animals. Autoradiographic analyses of tissue sections from transgenic rats reveal that the B1R is broadly expressed in both the brain and spinal cord. The human B1R expressed in the transgenic rat functions in an in vitro contractile assay and thus has the potential to elicit a functional response in vivo. Using the humanized B1R transgenic rat, an assay was developed that is suitable for the routine evaluation of a test compound's ability to occupy the human B1R in the central nervous system.