Cell-based β2-adrenergic receptor–ligand binding assay using synthesized europium-labeled ligands and time-resolved fluorescence

Martikkala, Eija; Lehmusto, Mirva; Lilja, Minna K.; Rozwandowicz-Jansen, Anita; Lunden, Jenni; Tomohiro, Takenori; Hänninen, Pekka; Petäjä-Repo, Ulla E.; Härmä, Harri

doi:10.1016/j.ab.2009.05.022

Cited by 17 publications

(23 citation statements)

References 22 publications

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“…This η sens parameter is sizeable (30-40%) and can be compared favorably with the value of 43% calculated for the 12-membered polyaza-macrocyclic ligand incorporating a diethylenetriacetic core and an intracyclic pyridine moiety (Eu . PCTA [12]) (51). The main factor that limits the observed luminescence quantum yield is the rather low efficiency of metal-centered luminescence.…”

Section: Luminescence Studiessupporting

confidence: 70%

Bifunctional Gd(III) and Tb(III) chelates based on a pyridine–bis(iminodiacetate) platform, suitable optical probes and contrast agents for magnetic resonance imaging

Laurent

Elst

Galaup

et al. 2014

Contrast Media & Molecular

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To study the physicochemical properties of lanthanide complexes derived from a bifunctional chelating agent based on a PMN-tetraacetic acid moiety {PMN-tetraacetic acid (1): [2,6-pyridinediylbis(methylene nitrilo)-tetraacetic acid]}, 4-carboxylic acid substituted pyridine derivative (2) was synthesized. This ligand forms heptadentate (N3 O4 ) Ln(III) complexes (Ln = Gd, Eu, Tb), with two water molecules completing the inner coordination sphere of the metal. The parameters that govern the relaxivity of the Gd(III) complex and the luminescence of Eu(III) and Tb(III) complexes were obtained by (17) O and (1) H NMR studies and time-resolved fluorescence experiments, respectively. The gadolinium and terbium complexes show interesting properties either for MRI or FOR optical imaging; that is, for the Gd complex, a high proton relaxivity (r1 = 6.4 s(-1) mM(-1) at 20 MHz) with short water residence time (τM = 38.5 ns); for the Tb complex, a luminescence lifetime of 1.22 ms at room temperature and a luminescence quantum yield of 10%. The kinetic stability of these complexes toward blood protein, cation or bioactive oxyanion was also examined. The Gd(2)(H2O)2 complex does not interact with human serum albumin, but undergoes a transmetalation reaction with Zn(II) in a phosphate buffer solution (pH 7.4), rather similar to that of Gd-DTPA-BMA(H2 O). On the other hand, as observed for Eu and Tb complexes, these chelates do not form ternary complexes with bidentate anions such as l-lactate, citrate or carbonate. Finally, a phosphatidylserine-specific hexapeptide (TLVSSL) was grafted on Gd or Tb chelates, and the Gd-peptide conjugate was used in vitro for targeting apoptotic cells.

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Section: Luminescence Studiessupporting

confidence: 70%

Bifunctional Gd(III) and Tb(III) chelates based on a pyridine–bis(iminodiacetate) platform, suitable optical probes and contrast agents for magnetic resonance imaging

Laurent

Elst

Galaup

et al. 2014

Contrast Media & Molecular

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“…(33) In addition, a europium-chelated fluorescent derivative of pindolol for use in time-resolved receptor–ligand binding assays has also been described, but its full structure was not revealed. (34)…”

Section: Introductionmentioning

confidence: 99%

Synthesis and Characterization of High-Affinity 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene-Labeled Fluorescent Ligands for Human β-Adrenoceptors

et al. 2011

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The growing practice of exploiting noninvasive fluorescence-based techniques to study G protein-coupled receptor pharmacology at the single cell and single molecule level demands the availability of high-quality fluorescent ligands. To this end, this study evaluated a new series of red-emitting ligands for the human β-adrenoceptor family. Upon the basis of the orthosteric ligands propranolol, alprenolol, and pindolol, the synthesized linker-modified congeners were coupled to the commercially available fluorophore BODIPY 630/650-X. This yielded high-affinity β-adrenoceptor fluorescent ligands for both the propranolol and alprenolol derivatives; however, the pindolol-based products displayed lower affinity. A fluorescent diethylene glycol linked propranolol derivative (18a) had the highest affinity (log KD of −9.53 and −8.46 as an antagonist of functional β2- and β1-mediated responses, respectively). Imaging studies with this compound further confirmed that it can be employed to selectively label the human β2-adrenoceptor in single living cells, with receptor-associated binding prevented by preincubation with the nonfluorescent β2-selective antagonist 3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol (ICI 118551) (J. Cardiovasc. Pharmacol.1983, 5, 430–437.)

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“…The number of receptors per cell has been previously determined in a saturation ligand-binding assay by washing Eu-C12-pindolol-saturated induced HEK293 cells and subsequently releasing Eu(III) ion to fluorescence enhancement solutions for fluorescence measurement. 17 We found 240,000 receptors per cell and calculated a K d value of 37 nM for the Eu-labeled ligand. A K d value of 1.3 nM was obtained in a radioligand membrane-based saturation assay with [ 3 H]dihydroalprenolol as a radioligand with the same cell line.…”

Section: The Influence Of the Number Of Cells On Eu-c12-pindolol Ligamentioning

confidence: 99%

“…Stably expressed human embryonic kidney 293 cells (HEK293 i ) 17 were grown at an atmosphere of 95% air and 5% CO 2 at 37 °C in growth medium (DMEM high glucose, 10% FBS, 1% penicillin/streptomycin solution, 2 mM L-glutamine, 1 µg ml -1 blasticidin, and 100 µg ml -1 hygromycin). Cells were grown to 80% to 90% confluence in 100-mm culture plates, washed with 1×PBS, and passaged with the use of trypsin/ EDTA solution.…”

Section: Cell Culturementioning

confidence: 99%

A New Simple Cell-Based Homogeneous Time-Resolved Fluorescence QRET Technique for Receptor-Ligand Interaction Screening

Härmä¹,

Rozwandowicz-Jansen²,

Martikkala³

et al. 2009

SLAS Discovery

Self Cite

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In this article, a single-label separation-free fluorescence technique is presented as a potential screening method for cell-based receptor antagonists and agonists.The time-resolved fluorescence technique, quenching resonance energy transfer (QRET), relies on a single-labeled binding partner in combination with a soluble quencher. The quencher efficiently suppresses the luminescence of the unbound labeled ligand, whereas the luminescence of the bound fraction is not affected. This approach allows the development of cell-based screening assays in a simple and cost-effective manner. The authors have applied the technique to the screening of β 2 -adrenoreceptor (β 2 AR) antagonists and agonists in intact human embryonic kidney HEK293 i cells overexpressing human β 2 -adrenergic receptors. Two antagonists (propranolol, alprenolol) and 2 agonists (metaproterenol, terbutaline) for β 2 AR were investigated in a displacement assay using europium(III)-labeled pindolol ligand. The assay Z′ values ranged from 0.68 to 0.78, the coefficient of variation was less than 10%, and the K i values were 19 nM for propranolol and alprenolol and 14 and 5.9 µM for metaproterenol and terbutaline, respectively. The QRET technique with β 2 AR was also applied to LOPAC compound library screening, yielding nearly error-free recognition of known binders. This simple and cost-effective technique can be readily adapted to laboratory and industrial-scale screening. (Journal of Biomolecular Screening 2009:936-943)

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Cell-based β2-adrenergic receptor–ligand binding assay using synthesized europium-labeled ligands and time-resolved fluorescence

Cited by 17 publications

References 22 publications

Bifunctional Gd(III) and Tb(III) chelates based on a pyridine–bis(iminodiacetate) platform, suitable optical probes and contrast agents for magnetic resonance imaging

Bifunctional Gd(III) and Tb(III) chelates based on a pyridine–bis(iminodiacetate) platform, suitable optical probes and contrast agents for magnetic resonance imaging

Synthesis and Characterization of High-Affinity 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene-Labeled Fluorescent Ligands for Human β-Adrenoceptors

A New Simple Cell-Based Homogeneous Time-Resolved Fluorescence QRET Technique for Receptor-Ligand Interaction Screening

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