Binding characteristics of [³H]‐JSM10292: a new cell membrane‐permeant non‐peptide bradykinin B₂ receptor antagonist

Abstract

Equilibrium binding, dissociation and competition studies with various B2 receptor ligands and [ 3 H]-JSM10292 were performed at 4°C and 37°C. The experiments were carried out using HEK293 cells stably (over)expressing wild-type and mutant B2 receptors of human and animal origin. H]-BK showed a different affinity profile for the wild-type B2 receptor in different species (man, cynomolgus, rabbit, mouse, rat, dog, pig, guinea pig). Characterization of B2 receptor mutants and species orthologues combined with homology modelling, using the CXCR4 as template, suggests that the binding site of JSM10292 is different from that of BK but overlaps with that of MEN16132, another small non-peptide B2 receptor ligand. KEY RESULTS CONCLUSIONS AND IMPLICATIONS[ 3 H]-JSM10292 is a novel, cell membrane-permeant, high-affinity B2 receptor antagonist that allows direct in detail studies of active, surface and intracellularly located wild-type and mutant B2 receptors. AbbreviationsAA, acetic acid; B2 receptor, bradykinin B2

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

“…Although the molecule shares a common substructure with those molecules used for its design, JSM10292 binds in different way. Analysis of different poses obtained during the docking process and analyzed according with the mutagenesis results available [42], it was selected as putative bound conformation the one shown in The bound conformation of JSM10292 found in the present study differs slightly of the one described in reference 40. The two models actually differ in the conformation of the ligands.…”

“…As result of the different conformation, the trifluromoiety interacts in the present model with Asn 107 whereas in the other model gets close to Ser 111 . In fact, there are not mutagenesis results of the mutation of serine to alanine; however the replacement to lysine provokes a great loss of affinity [42].…”

Interruption of the Ionic Lock in the Bradykinin B2 Receptor Results in Constitutive Internalization and Turns Several Antagonists into Strong Agonists

“…Similarly, E6.30 mutations in the thromboxane prostanoid receptor resulted in more efficient agonist-induced signaling without any increase in basal activity (Ambrosio et al, 2010). Moreover, for the B 2 R, even the small molecule compound JSM10292, which thus far had displayed no partial agonistic activity (Faussner et al, 2012), becomes a full agonist in mutant E6.30R. This might be explained by assuming a semi-active conformation due to repulsion of the two positively charged arginines (Fig.…”

“…[ (Faussner et al, 2012). Nonspecific binding was determined either with a 1000-fold excess of unlabeled JSM10292 (calculated specific binding comprises intracellular and surface receptors) or BK (calculated specific binding covers only surface receptors).…”

“…We previously characterized a novel cell membrane-permeant small molecule, JSM10292, which, 3 Hlabeled, allows the differentiation between surface and intracellularly located wild-type and mutant B 2 Rs, as long as they are binding competent (Faussner et al, 2012). Unlike the B 2 Rwt, which, unstimulated, is located mostly at the cell surface (∼80%), .50% of the single mutant receptor constructs R3.50A and E6.30A were found to be located intracellularly (Table 2).…”