Screening Scheme Based on Measurement of Fluorescence Lifetime in the Nanosecond Domain

Hoefelschweiger, Bianca K.; Wolfbeis, Otto S.

doi:10.1177/1087057105277493

Cited by 14 publications

(18 citation statements)

References 33 publications

(35 reference statements)

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“…The fluorescence lifetime of the fluorescent labels L-1, Py-1, Py-4, and Py-6 was used as a parameter to measure binding between biotin and streptavidin and biotinylated bovine serum albumin and streptavidin [93]. The assay was performed in a microplate using diode laser excitation.…”

Section: Rb-646mentioning

confidence: 99%

Long-wavelength fluorescence lifetime labels

et al. 2011

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Section: Rb-646mentioning

confidence: 99%

Long-wavelength fluorescence lifetime labels

et al. 2011

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“…In addition, others have previously shown that modification of this nitrogen results in a compound that maintains its ability to bind to a range of kinases. 12,13 Because we 18 and others [19][20][21] have previously shown that fluorophores conjugated to small-molecule ligands for protein targets can undergo substantial changes in fluorescence lifetime between the free and bound state, we reasoned that by coupling a fluorescent molecule to staurosporine at this position, we could prepare a fluorescence lifetime-based probe that could be used to identify and characterize ATP-competitive inhibitors for a wide range of kinases.…”

Section: Introductionmentioning

confidence: 99%

A Fluorescence Lifetime–Based Binding Assay to Characterize Kinase Inhibitors

Lebakken¹,

Kang²,

Vogel³

2007

SLAS Discovery

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The authors present a fluorescence lifetime-based kinase binding assay that identifies and characterizes compounds that bind to the adenosine triphosphate (ATP)-binding pocket of a range of tyrosine and serine/threonine kinases. The assay is based on displacement of an Alexa Fluor 647 conjugate of staurosporine from the ATP-binding site of a kinase, which is detected by a change in the fluorescence lifetime of the probe between the free (displaced) and kinase-bound states. The authors screened 257 kinases for specific binding and displacement of the Alexa Fluor 647-staurosporine probe and found that approximately half of the kinases tested could potentially be assayed with this method. They present inhibitor binding data against 4 selected serine/threonine kinases and 4 selected tyrosine kinases, using 6 commonly used kinase inhibitors. Two of these kinases were chosen for further studies, in which inhibitor binding data were compared to inhibition of kinase activity using 2 separate activity assay formats. Rank-order potencies of compounds were similar, but not identical, between the binding and activity assays. It was postulated that these differences could be caused by the fact that the assays are measuring distinct phenomena, namely, activity versus binding, and in a purified recombinant kinase preparation, there can exist a mixture of active and nonactivated kinases. To explore this possibility, the authors compared binding affinity for the probe using 2 kinases in their respective nonactivated and activated (phosphorylated) forms and found a kinase-dependent difference between the 2 forms. This assay format therefore represents a simple method for the identification and characterization of small-molecule kinase inhibitors that may be useful in screening a wide range of kinases and may be useful in identifying small molecules that bind to kinases in their active or nonactivated states.

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“…9 Prompted by the development of relatively inexpensive diodebased pulsed lasers, fluorescence lifetime measurement has been added to the portfolio of assay formats available in HTS, with the anticipation that many of the compound interference problems could be solved. 10,11 In this article, we aim to establish the most appropriate conditions for using lifetime measurements in the context of reducing the effects of compound interference.…”

Section: He Shift From Radiochemical-to Fluorescence-basedmentioning

confidence: 99%

“…10,11 In this article, we aim to establish the most appropriate conditions for using lifetime measurements in the context of reducing the effects of compound interference. Mixtures of fluorophores will be used to evaluate how the choice of reporter fluorophore and data acquisition conditions influences the ability to resolve lifetimes of multiple fluorescing components.…”

mentioning

confidence: 99%

The Application of Fluorescence Lifetime Readouts in High-Throughput Screening

Moger¹,

Gribbon²,

Sewing³

et al. 2006

SLAS Discovery

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Measurement of fluorescence lifetime is a well-established technique, which has recently been introduced into the portfolio of assay formats used in high-throughput screening (HTS). This investigation establishes appropriate conditions for using lifetime measurements to reduce the impact of compound interference effects during large-scale HTS of corporate screening files. Experimental data on mixtures of standard fluorophores and interfering compounds (from 5 HTS campaigns) have been combined with a theoretical model to identify the minimum data quality required, defined by the photon count in the peak channel, for discrimination of biological activity. Single-component fluorophore lifetimes can be recovered with an error of 1%, with a peak photon count of 10 2 , but the same accuracy with a 2-component decay requires a peak photon count of 10 3 . When a 3rd component is introduced, the minimum peak count increases to 10 4 . The influence of scattered light on lifetime determination was investigated using an emulsion (diameters 25-675 nm). The measured decays of interfering compounds, identified as autofluorescent, show that the vast majority have a very short lifetime that can readily be resolved from the reporter fluorophore, using appropriate data-fitting methods. ( analytical methods has given benefits of increased sensitivity and speed along with reduced reaction volume and environmental impact, especially in the application of highthroughput screening (HTS) of large corporate compound collections.1 However, compared to radiometric assay formats, fluorescence methods introduce increased opportunities for compound interference effects.2 The fraction of compounds in an HTS campaign that interfere with bioassays in a deleterious manner may be significant compared to those having genuine biological activity leading to a greater burden on compound logistics in hit follow-up.3 More important, however, a significant proportion of reproducible false positives can cause difficulties in the analysis of the screen output, with the worst-case scenario being when a compound with genuine biological activity is overlooked in favor of interfering compounds. 4 Several modes of action have been described for interfering compounds, which are a consequence of their underlying physicochemical properties. Weakly or insoluble compounds can interfere with optical detection by generating light-scattering aggregates. More subtly, aggregates themselves can act as adsorption centers and sequestrate biomolecules in a manner that mimics a true inhibition-type response. 5,6 Other optical effects include light absorption due to colored compounds, quenching by a compound of the fluorescent emission of a reporter fluorophore, and compounds having intrinsic fluorescence over the spectral range of the reporter fluorophore.2,7 To attempt to address these deficiencies, the HTS community has advanced assay and detection technology, with approaches such as time-resolved fluorescence resonant energy transfer, now available.8 Although some success ...

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Screening Scheme Based on Measurement of Fluorescence Lifetime in the Nanosecond Domain

Cited by 14 publications

References 33 publications

Long-wavelength fluorescence lifetime labels

Long-wavelength fluorescence lifetime labels

A Fluorescence Lifetime–Based Binding Assay to Characterize Kinase Inhibitors

The Application of Fluorescence Lifetime Readouts in High-Throughput Screening

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