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

Harri Härmä, Anita Rozwandowicz-Jansen, Eija Martikkala, Heini Frang, Ilkka Hemmilä, Niko Sahlberg, Vidal Fey, Merja Perälä, Pekka Hänninen

Research output: Contribution to journalArticleScientificpeer-review

30 Citations (Scopus)

Abstract

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 β2-adrenoreceptor (β2AR) antagonists and agonists in intact human embryonic kidney HEK293i cells overexpressing human β2-adrenergic receptors. Two antagonists (propranolol, alprenolol) and 2 agonists (metaproterenol, terbutaline) for β2AR 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 Ki values were 19 nM for propranolol and alprenolol and 14 and 5.9 µM for metaproterenol and terbutaline, respectively. The QRET technique with β2AR 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.
Original languageEnglish
Pages (from-to)936-943
JournalJournal of Biomolecular Screening
Volume14
Issue number8
DOIs
Publication statusPublished - 2009
MoE publication typeA1 Journal article-refereed

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Fluorescence Resonance Energy Transfer
Alprenolol
Metaproterenol
Energy transfer
Terbutaline
Quenching
Screening
Fluorescence
Energy Transfer
Ligands
Luminescence
Propranolol
Assays
Europium
Pindolol
Costs and Cost Analysis
Adrenergic Receptors
Kidney
Binders
Labels

Cite this

Härmä, H., Rozwandowicz-Jansen, A., Martikkala, E., Frang, H., Hemmilä, I., Sahlberg, N., ... Hänninen, P. (2009). A New Simple Cell-Based Homogeneous Time-Resolved Fluorescence QRET Technique for Receptor-Ligand Interaction Screening. Journal of Biomolecular Screening, 14(8), 936-943. https://doi.org/10.1177/1087057109341657
Härmä, Harri ; Rozwandowicz-Jansen, Anita ; Martikkala, Eija ; Frang, Heini ; Hemmilä, Ilkka ; Sahlberg, Niko ; Fey, Vidal ; Perälä, Merja ; Hänninen, Pekka. / A New Simple Cell-Based Homogeneous Time-Resolved Fluorescence QRET Technique for Receptor-Ligand Interaction Screening. In: Journal of Biomolecular Screening. 2009 ; Vol. 14, No. 8. pp. 936-943.
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abstract = "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 β2-adrenoreceptor (β2AR) antagonists and agonists in intact human embryonic kidney HEK293i cells overexpressing human β2-adrenergic receptors. Two antagonists (propranolol, alprenolol) and 2 agonists (metaproterenol, terbutaline) for β2AR 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 Ki values were 19 nM for propranolol and alprenolol and 14 and 5.9 µM for metaproterenol and terbutaline, respectively. The QRET technique with β2AR 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.",
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Härmä, H, Rozwandowicz-Jansen, A, Martikkala, E, Frang, H, Hemmilä, I, Sahlberg, N, Fey, V, Perälä, M & Hänninen, P 2009, 'A New Simple Cell-Based Homogeneous Time-Resolved Fluorescence QRET Technique for Receptor-Ligand Interaction Screening', Journal of Biomolecular Screening, vol. 14, no. 8, pp. 936-943. https://doi.org/10.1177/1087057109341657

A New Simple Cell-Based Homogeneous Time-Resolved Fluorescence QRET Technique for Receptor-Ligand Interaction Screening. / Härmä, Harri; Rozwandowicz-Jansen, Anita; Martikkala, Eija; Frang, Heini; Hemmilä, Ilkka; Sahlberg, Niko; Fey, Vidal; Perälä, Merja; Hänninen, Pekka.

In: Journal of Biomolecular Screening, Vol. 14, No. 8, 2009, p. 936-943.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - A New Simple Cell-Based Homogeneous Time-Resolved Fluorescence QRET Technique for Receptor-Ligand Interaction Screening

AU - Härmä, Harri

AU - Rozwandowicz-Jansen, Anita

AU - Martikkala, Eija

AU - Frang, Heini

AU - Hemmilä, Ilkka

AU - Sahlberg, Niko

AU - Fey, Vidal

AU - Perälä, Merja

AU - Hänninen, Pekka

PY - 2009

Y1 - 2009

N2 - 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 β2-adrenoreceptor (β2AR) antagonists and agonists in intact human embryonic kidney HEK293i cells overexpressing human β2-adrenergic receptors. Two antagonists (propranolol, alprenolol) and 2 agonists (metaproterenol, terbutaline) for β2AR 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 Ki values were 19 nM for propranolol and alprenolol and 14 and 5.9 µM for metaproterenol and terbutaline, respectively. The QRET technique with β2AR 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.

AB - 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 β2-adrenoreceptor (β2AR) antagonists and agonists in intact human embryonic kidney HEK293i cells overexpressing human β2-adrenergic receptors. Two antagonists (propranolol, alprenolol) and 2 agonists (metaproterenol, terbutaline) for β2AR 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 Ki values were 19 nM for propranolol and alprenolol and 14 and 5.9 µM for metaproterenol and terbutaline, respectively. The QRET technique with β2AR 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.

U2 - 10.1177/1087057109341657

DO - 10.1177/1087057109341657

M3 - Article

VL - 14

SP - 936

EP - 943

JO - Journal of Biomolecular Screening

JF - Journal of Biomolecular Screening

SN - 1087-0571

IS - 8

ER -