BACKGROUNDWe have previously reported the design, pharmacological properties and imaging application of bradykinin (BK) B₂ receptor (B₂R) 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.RESULTSThe antagonist B-9430 (D-Arg-[Hyp³,Igl⁵,D-Igl⁷,Oic⁸]-BK) and the agonist B-9972 (D-Arg-[Hyp³,Igl⁵,Oic⁷,Igl⁸]-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 B₂R vs. the parent peptide, whereas the antagonist B-10665 better retained its potency vs. B-9430 (competition of [³H]BK binding to human B₂R, 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 B₂R-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 B₂R-GFP in live cells, but not the antagonist version at 10-25 nM. The Cy7-labeled peptides did not label cells expressing the β₂-adrenoceptor-GFP construction. B-10665 at low nanomolar concentrations was an effective probe for the recombinant B₂Rs in cytofluorometry and macroscopic imaging of cell wells (IVIS imaging system operated for infrared fluorescence detection).CONCLUSIONSDespite a propensity for non-specific binding when used at high concentrations and limited sensitivity, Cy7-conjugated peptidase-resistant B₂R ligands support original imaging and cytofluorometric applications.

01 Mar 2010·Journal of Pharmaceutical and Biomedical AnalysisQ3 · MEDICINE

LC/MS characterization of impurities and degradation products of a potent antitumor peptidic dimer, CU201

Q3 · MEDICINE

Article

Author: Jennie Wang ; Euphemia Wang ; Mingtao Liu ; Michael Gardner ; Phyllis Elkins ; John Hines ; Chun Yang ; Diego Baptista ; Paul Liu ; Vidhya Krishnamoorthi ; Xiaogang Wu

Compound CU201 [SUIM-(d-Arg-Arg-Pro-Hyp-Gly-Igl-Ser-d-Igl-Oic-Arg)(2), where SUIM=suberimidyl; Hyp=trans-4-hydroxyproline; Igl=alpha-(2-indanyl)-glycine; Oic=octahydroindole-2-carboxylic acid], is a dimeric analog of the potent bradykinin antagonist peptide B9430. It blocks the G(alphaq,11) signal of the heterotrimeric G proteins, stimulates c-Jun kinases, and induces apoptosis in lung cancer cells with neuroendocrine features. CU201 shows potent inhibition for small-cell lung cancer cells in vitro (ED(50)=0.15microM), as well as for small-cell lung cancer SHP-77 tumor growth in vivo. An HPLC method was developed, as part of a study supported by the National Cancer Institute's (NCI's) Rapid Access to Interventional Development (RAID) program, to assess the purity and stability of CU201. Impurities and degradation products were characterized by LC/MS. The identity of a major impurity, with 1 mass unit different from CU201, was confirmed by high resolution LC/MS and the investigation of model compounds. Susceptible linkages in the peptide chains were revealed by the degradation study.

01 Sep 2008·PeptidesQ3 · MEDICINE

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

Q3 · MEDICINE

Article

Author: Adam, Albert ; Bawolak, Marie-Therese ; Marceau, Francois ; Bouthillier, Johanne ; Gera, Lajos ; Stewart, John M.

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.

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