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HTRF: A technology tailored for drug discovery - a review of theoretical aspects and recent applications.

Degorce F, Card A, Soh S, Trinquet E, Knapik GP, Xie B - Curr Chem Genomics (2009)

Bottom Line: Buffer and media interference is dramatically reduced by dual-wavelength detection, and the final signal is proportional to the extent of product formation.Terbium cryptate possesses different photophysical properties compared to Europium, including increased quantum yield and a higher molar extinction coefficient.This review addresses the general principles of HTRF and its current applications in drug discovery.

View Article: PubMed Central - PubMed

Affiliation: Cisbio Bioassays, 30204 Bagnols-Sur-Cèze, France. fdegorce@cisbio.com

ABSTRACT
HTRF (Homogeneous Time Resolved Fluorescence) is the most frequently used generic assay technology to measure analytes in a homogenous format, which is the ideal platform used for drug target studies in high-throughput screening (HTS). This technology combines fluorescence resonance energy transfer technology (FRET) with time-resolved measurement (TR). In TR-FRET assays, a signal is generated through fluorescent resonance energy transfer between a donor and an acceptor molecule when in close proximity to each other. Buffer and media interference is dramatically reduced by dual-wavelength detection, and the final signal is proportional to the extent of product formation. The HTRF assay is usually sensitive and robust that can be miniaturized into the 384 and 1536-well plate formats. This assay technology has been applied to many antibody-based assays including GPCR signaling (cAMP and IP-One), kinases, cytokines and biomarkers, bioprocess (antibody and protein production), as well as the assays for protein-protein, proteinpeptide, and protein-DNA/RNA interactions.Since its introduction to the drug-screening world over ten years ago, researchers have used HTRF to expedite the study of GPCRs, kinases, new biomarkers, protein-protein interactions, and other targets of interest. HTRF has also been utilized as an alternative method for bioprocess monitoring. The first-generation HTRF technology, which uses Europium cryptate as a fluorescence donor to monitor reactions between biomolecules, was extended in 2008 through the introduction of a second-generation donor, Terbium cryptate (Tb), enhancing screening performance. Terbium cryptate possesses different photophysical properties compared to Europium, including increased quantum yield and a higher molar extinction coefficient. In addition to being compatible with the same acceptor fluorophors used with Europium, it can serve as a donor fluorophore to green-emitting fluors because it has multiple emission peaks including one at 490 nm. Moreover, all Terbium HTRF assays can be read on the same HTRF-compatible instruments as Europium HTRF assays.Overall, HTRF is a highly sensitive, robust technology for the detection of molecular interactions in vitro and is widely used for primary and secondary screening phases of drug development. This review addresses the general principles of HTRF and its current applications in drug discovery.

No MeSH data available.


Related in: MedlinePlus

Features of donor cryptate. a. Structure of cryptate trisbipyridine (TBP). Lambda max absorption: 305nm, molar extinction coefficient at 305nm: 30000 M-1 cm-1, molar extinction coefficient 337nm: 4500 M-1 cm-1. b. Detection wavelengths for Europium and Terbium cryptate. Both Europium and Terbium have emission spectra at 620 nm so that these signals can be used as reference for data analysis.
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Figure 3: Features of donor cryptate. a. Structure of cryptate trisbipyridine (TBP). Lambda max absorption: 305nm, molar extinction coefficient at 305nm: 30000 M-1 cm-1, molar extinction coefficient 337nm: 4500 M-1 cm-1. b. Detection wavelengths for Europium and Terbium cryptate. Both Europium and Terbium have emission spectra at 620 nm so that these signals can be used as reference for data analysis.

Mentions: Four specific fluorophores are used in HTRF forming different TR-FRET systems. The central element, the energy donor, is either Europium cryptate (Eu3+ cryptate) (Fig. 3a) [5] or Lumi4-Tb (Tb2+ cryptate) (unpublished structure) (Fig. 3b). These rare earth complexes, based on the work from Nobel Prize laureate, Professor Jean-Marie Lehn, and from Professor Raymond respectively, have a macrocycle within which a Eu3+ ion or Tb2+ is tightly embedded. These ions are not fluorescent on their own; they need a light-collection device (i.e. the cage) to be excited. Similar to functions of the chelate in other luminescent lanthanide technologies, this cage acts as an antenna and allows both energy collection and transfer to the ions, which ultimately releases this energy with a specific fluorescent pattern. In particular, these cryptates are not subject to the photo-bleaching that affects a number of more conventional fluorophores, and the ions are almost inseparable from their macrocycles [5]. However, due to the unique caged structure, the kinetics stability of the cryptate is extremely higher than the one of those lanthanide chelates [3]. Rare earth chelates would be dissociated and unstable in acidic media or in the presence of divalent ions like Mn2+, while rare earth cryptates are extraordinarily stable under wide range of chemical conditions, like reverse phase chromatography in the presence of trifluoroacetic acid, and are not affected by the presence of divalent ions in the media [4]. It therefore allows for enhanced assay performance in terms of sensitivity, assay window, and robustness, without compromising the features and benefits that HTRF brings to assays: a ‘mix and measure’ non-radioactive format, miniaturizable to uHTS formats, showing excellent robustness and a low compound interference rate (Fig. 3). Lumi4-Tb’s structure, a lanthanide tightly embedded in a surrounding macrocycle, remains very much in line with that of previous HTRF cryptates and shows superior stability compared to other Terbium complexes. Terbium is exceptionally bright – 10 to 20 times brighter than Europium that significantly increases the detection sensitivity in assays such as the exploration of cell surface receptors.


HTRF: A technology tailored for drug discovery - a review of theoretical aspects and recent applications.

Degorce F, Card A, Soh S, Trinquet E, Knapik GP, Xie B - Curr Chem Genomics (2009)

Features of donor cryptate. a. Structure of cryptate trisbipyridine (TBP). Lambda max absorption: 305nm, molar extinction coefficient at 305nm: 30000 M-1 cm-1, molar extinction coefficient 337nm: 4500 M-1 cm-1. b. Detection wavelengths for Europium and Terbium cryptate. Both Europium and Terbium have emission spectra at 620 nm so that these signals can be used as reference for data analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2802762&req=5

Figure 3: Features of donor cryptate. a. Structure of cryptate trisbipyridine (TBP). Lambda max absorption: 305nm, molar extinction coefficient at 305nm: 30000 M-1 cm-1, molar extinction coefficient 337nm: 4500 M-1 cm-1. b. Detection wavelengths for Europium and Terbium cryptate. Both Europium and Terbium have emission spectra at 620 nm so that these signals can be used as reference for data analysis.
Mentions: Four specific fluorophores are used in HTRF forming different TR-FRET systems. The central element, the energy donor, is either Europium cryptate (Eu3+ cryptate) (Fig. 3a) [5] or Lumi4-Tb (Tb2+ cryptate) (unpublished structure) (Fig. 3b). These rare earth complexes, based on the work from Nobel Prize laureate, Professor Jean-Marie Lehn, and from Professor Raymond respectively, have a macrocycle within which a Eu3+ ion or Tb2+ is tightly embedded. These ions are not fluorescent on their own; they need a light-collection device (i.e. the cage) to be excited. Similar to functions of the chelate in other luminescent lanthanide technologies, this cage acts as an antenna and allows both energy collection and transfer to the ions, which ultimately releases this energy with a specific fluorescent pattern. In particular, these cryptates are not subject to the photo-bleaching that affects a number of more conventional fluorophores, and the ions are almost inseparable from their macrocycles [5]. However, due to the unique caged structure, the kinetics stability of the cryptate is extremely higher than the one of those lanthanide chelates [3]. Rare earth chelates would be dissociated and unstable in acidic media or in the presence of divalent ions like Mn2+, while rare earth cryptates are extraordinarily stable under wide range of chemical conditions, like reverse phase chromatography in the presence of trifluoroacetic acid, and are not affected by the presence of divalent ions in the media [4]. It therefore allows for enhanced assay performance in terms of sensitivity, assay window, and robustness, without compromising the features and benefits that HTRF brings to assays: a ‘mix and measure’ non-radioactive format, miniaturizable to uHTS formats, showing excellent robustness and a low compound interference rate (Fig. 3). Lumi4-Tb’s structure, a lanthanide tightly embedded in a surrounding macrocycle, remains very much in line with that of previous HTRF cryptates and shows superior stability compared to other Terbium complexes. Terbium is exceptionally bright – 10 to 20 times brighter than Europium that significantly increases the detection sensitivity in assays such as the exploration of cell surface receptors.

Bottom Line: Buffer and media interference is dramatically reduced by dual-wavelength detection, and the final signal is proportional to the extent of product formation.Terbium cryptate possesses different photophysical properties compared to Europium, including increased quantum yield and a higher molar extinction coefficient.This review addresses the general principles of HTRF and its current applications in drug discovery.

View Article: PubMed Central - PubMed

Affiliation: Cisbio Bioassays, 30204 Bagnols-Sur-Cèze, France. fdegorce@cisbio.com

ABSTRACT
HTRF (Homogeneous Time Resolved Fluorescence) is the most frequently used generic assay technology to measure analytes in a homogenous format, which is the ideal platform used for drug target studies in high-throughput screening (HTS). This technology combines fluorescence resonance energy transfer technology (FRET) with time-resolved measurement (TR). In TR-FRET assays, a signal is generated through fluorescent resonance energy transfer between a donor and an acceptor molecule when in close proximity to each other. Buffer and media interference is dramatically reduced by dual-wavelength detection, and the final signal is proportional to the extent of product formation. The HTRF assay is usually sensitive and robust that can be miniaturized into the 384 and 1536-well plate formats. This assay technology has been applied to many antibody-based assays including GPCR signaling (cAMP and IP-One), kinases, cytokines and biomarkers, bioprocess (antibody and protein production), as well as the assays for protein-protein, proteinpeptide, and protein-DNA/RNA interactions.Since its introduction to the drug-screening world over ten years ago, researchers have used HTRF to expedite the study of GPCRs, kinases, new biomarkers, protein-protein interactions, and other targets of interest. HTRF has also been utilized as an alternative method for bioprocess monitoring. The first-generation HTRF technology, which uses Europium cryptate as a fluorescence donor to monitor reactions between biomolecules, was extended in 2008 through the introduction of a second-generation donor, Terbium cryptate (Tb), enhancing screening performance. Terbium cryptate possesses different photophysical properties compared to Europium, including increased quantum yield and a higher molar extinction coefficient. In addition to being compatible with the same acceptor fluorophors used with Europium, it can serve as a donor fluorophore to green-emitting fluors because it has multiple emission peaks including one at 490 nm. Moreover, all Terbium HTRF assays can be read on the same HTRF-compatible instruments as Europium HTRF assays.Overall, HTRF is a highly sensitive, robust technology for the detection of molecular interactions in vitro and is widely used for primary and secondary screening phases of drug development. This review addresses the general principles of HTRF and its current applications in drug discovery.

No MeSH data available.


Related in: MedlinePlus