Bawolak Marie-Thérèse, Gera Lajos, Bouthillier Johanne, Stewart John M, Adam Albert, Marceau François
Centre de recherche en rhumatologie et immunologie, Centre Hospitalier Universitaire de Québec, Université Laval, Québec, Canada G1V 4G2.
Peptides. 2008 Sep;29(9):1626-30. doi: 10.1016/j.peptides.2008.05.007. Epub 2008 May 17.
B-9430 (d-Arg-[Hyp3, Igl5, D-Igl7, Oic8]-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 B2 receptor antagonist with effects extended to the B1 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 B2 receptors of the human isolated umbilical vein, pA2 6.83; an insurmountable antagonist at the B2 receptors in the rabbit jugular vein; a weak competitive antagonist of the B1 receptors in the rabbit aorta, pA2 5.95). B-10330 and B-10380 displaced the binding of [3H]bradykinin from rabbit B2 receptors with a potency slightly inferior to that of B-9430 (larger gap at the rat B2 receptor). Treatment with B-10330 and fluorescent streptavidin did not support imaging of recombinant B2 receptors. However, the plasma membrane of HEK 293a cells that transiently expressed recombinant rabbit B2 receptors, but not B1 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 B2 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.
B - 9430（d - Arg - [Hyp3, Igl5, D - Igl7, Oic8] - 缓激肽），其中Hyp为反式 - 4 - 羟基脯氨酸，Igl为α - (2 - 茚满基)甘氨酸，Oic为(3as, 7as) - 八氢吲哚 - 2 - 基 - 羰基，是一种高亲和力的缓激肽B2受体拮抗剂，在高浓度时对B1受体也有作用。B - 9430的N端已用d - 生物素（B - 10330）或5(6) - 羧基荧光素 - ε - 氨基己酰基（B - 10380）进行延伸，以获得荧光受体探针。B - 10380的药理学特性与B - 9430相似，但效力略有下降（在人离体脐静脉的B2受体上是缓激肽的竞争性拮抗剂，pA2为6.83；在兔颈静脉的B2受体上是不可逾越的拮抗剂；在兔主动脉的B1受体上是弱竞争性拮抗剂，pA2为5.95）。B - 10330和B - 10380从兔B2受体上取代[3H]缓激肽结合的效力略低于B - 9430（在大鼠B2受体上差距更大）。用B - 10330和荧光链霉亲和素处理不支持重组B2受体的成像。然而，瞬时表达重组兔B2受体而非B1受体的HEK 293a细胞的质膜用5 - 50 nM B - 10380进行了标记（落射荧光显微镜观察）。在未转染的细胞中未观察到B - 10380染色，并且在用非肽拮抗剂共同处理表达受体的细胞后染色被消除。用荧光团对缓激肽B2受体的强效肽拮抗剂进行N端延伸产生了一种适用于活细胞成像和其他应用的荧光探针，但亲和力略有损失。
