A fluorescent version of the bradykinin B2 receptor antagonist B-9430:: Pharmacological characterization and use in live cell imaging

B-9430 (D-Arg-[Hyp(3), Igl(5), D-Igl(7), Oic(8)]-bradykinin), where Hyp is trans-4-hydroxyproline, Igl is alpha-(2-indanyl)glycine and Oic is (3as, 7as)-octahydroindol-2-yl-carbonyl is a high affinity bradykinin B-2 receptor antagonist with effects extended to the B-1 receptors at high concentrations. The N-terminus of B-9430 has been extended with D-biotinyl (B-10330) or 5(6)-carboxyfluorescein-epsilon-aminocaproyl (B-10380) to derive fluorescent receptor probes. The pharmacological profile of B-10380 was similar to that of B-9430 with a minor loss of potency (a competitive antagonist of bradykinin at the B-2 receptors of the human isolated umbilical vein, pA(2) 6.83; an insurmountable antagonist at the B-2 receptors in the rabbit jugular vein; a weak competitive antagonist of the B-1 receptors in the rabbit aorta, pA(2) 5.95). B-10330 and B-10380 displaced the binding of [H-3]bradykinin from rabbit B-2 receptors with a potency slightly inferior to that of B-9430 (larger gap at the rat B-2 receptor). Treatment with B-10330 and fluorescent streptavidin did not support imaging of recombinant B-2 receptors. However, the plasma membrane of HEK 293a cells that transiently expressed recombinant rabbit B-2 receptors, but not B, receptors, was labeled with 5-50 nM B-10380 (epifluorescence microscopy). B-10380 staining was not observed in nontransfected cells and was abolished by co-treating receptor-expressing cells with a nonpeptide antagonist. The N-terminal extension of a potent peptide antagonist of the bradykinin B-2 receptor with a fluorophore produced a fluorescent probe suitable for live cell imaging and other applications at the expense of a minor loss of affinity. (C) 2008 Elsevier Inc. All rights reserved.