Infrared-emitting, peptidase-resistant fluorescent ligands of the bradykinin B(2) receptor: application to cytofluorometry and imaging

BACKGROUND: We have previously reported the design, pharmacological properties and imaging application of bradykinin (BK) B(2) receptor (B(2)R) ligands conjugated with fluorophores such as fluorescein derivatives at their N-terminus. To take advantage of the high penetration of infrared light into living tissues and their low autofluorescence in this region of the spectrum, additional probes conjugated with cyanine dye 7 (Cy7) were synthesized and characterized. RESULTS: The antagonist B-9430 (D-Arg-[Hyp(3),Igl(5),D-Igl(7),Oic(8)]-BK) and the agonist B-9972 (D-Arg-[Hyp(3),Igl(5),Oic(7),Igl(8)]-BK) were N-terminally extended with the infrared fluorophore Cy7, producing the peptides B-10665 and B-10666, respectively. Pharmacological studies indicated that the agonist B-10666 lost much affinity for the B(2)R vs. the parent peptide, whereas the antagonist B-10665 better retained its potency vs. B-9430 (competition of [(3)H]BK binding to human B(2)R, contractility of the human isolated umbilical vein for which potency losses were more important in each case). Both probes stained HEK 293 cells that expressed the B(2)R-green fluorescent protein (GFP) construction in a specific manner (confocal microscopy) and with very extensive co-localization of the green and infrared fluorescence in either case. The agonist B-10666 at 100 nM promoted the endocytosis of B(2)R-GFP in live cells, but not the antagonist version at 10–25 nM. The Cy7-labeled peptides did not label cells expressing the β(2)-adrenoceptor-GFP construction. B-10665 at low nanomolar concentrations was an effective probe for the recombinant B(2)Rs in cytofluorometry and macroscopic imaging of cell wells (IVIS imaging system operated for infrared fluorescence detection). CONCLUSIONS: Despite a propensity for non-specific binding when used at high concentrations and limited sensitivity, Cy7-conjugated peptidase-resistant B(2)R ligands support original imaging and cytofluorometric applications. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13104-016-2258-1) contains supplementary material, which is available to authorized users.