A dual-step fluorescence resonance energy transfer-based quenching assay for screening of caspase-3 inhibitors

Valanne, Antti; Malmi, Pekka; Appelblom, Heidi; Niemelä, Pauliina; Soukka, Tero

doi:10.1016/j.ab.2007.12.032

Cited by 22 publications

(22 citation statements)

References 47 publications

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“…All the assays gave similar IC 50 values, with an average of 13 nM. This is in good agreement with the previous reports on the Z-DEVD-FMK inhibitor, where Valanne et al (2008) and Gopalakrishnan et al (2002) obtained IC 50 values of 12 nM and 4 nM, respectively. The highest signal-to-background ratios (compared to reactions with no enzyme) were approximately 45, obtained with the standard 96-well plate.…”

Section: Homogeneous Enzyme Activity Assays (Iii Iv)supporting

confidence: 91%

“…The inhibitor dose-response curves are presented in Figure 24. The observed IC 50 values (61.2 nM and 60.7 nM for terbium and energy transfer data, respectively) were somewhat larger compared to the previously reported values for the ZDEVD-FMK inhibitor, where Valanne et al (2008) and Gopalakrishnan et al (2002) obtained IC 50 values of 12 nM and 4 nM, respectively. It has to be noted, however, that the selection of reagents and their concentrations have an impact on the observed values.…”

Section: Homogeneous Enzyme Activity Assays (Iii Iv)contrasting

confidence: 71%

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Luminescent lanthanide reporters: new concepts for use in bioanalytical applications

Vuojola

Soukka

2014

Methods Appl. Fluoresc.

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Lanthanides represent the chemical elements from lanthanum to lutetium. They intrinsically exhibit some very exciting photophysical properties, which can be further enhanced by incorporating the lanthanide ion into organic or inorganic sensitizing structures. A very popular approach is to conjugate the lanthanide ion to an organic chromophore structure forming lanthanide chelates. Another approach, which has quickly gained interest, is to incorporate the lanthanide ions into nanoparticle structures, thus attaining improved specific activity and a large surface area for biomolecule immobilization. Lanthanide-based reporters, when properly shielded from the quenching effects of water, usually express strong luminescence emission, multiple narrow emission lines covering a wide wavelength range, and exceptionally long excited state lifetimes enabling time-gated luminescence detection. Because of these properties, lanthanide-based reporters have found widespread applications in various fields of life. This review focuses on the field of bioanalytical applications. Luminescent lanthanide reporters and assay formats utilizing these reporters pave the way for increasingly sensitive, simple, and easily automated bioanalytical applications.

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Section: Homogeneous Enzyme Activity Assays (Iii Iv)supporting

confidence: 91%

Section: Homogeneous Enzyme Activity Assays (Iii Iv)contrasting

confidence: 71%

Luminescent lanthanide reporters: new concepts for use in bioanalytical applications

Vuojola

Soukka

2014

Methods Appl. Fluoresc.

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“…111 This study utilized lanthanide-containing inorganic nanocrystals, which have a unique ability to emit light in the visible spectrum when excitated by near-infrared wavelengths, thus eliminating autofluorescence of biological samples. The assay technique is similar to the one described below 112 and relies on dual-step energy transfer, however here Black Hole Quencher 3 (BHQ-3) was used as a quencher. Its main advantage (over the BlackBerry Quencher 650) is a good stability in the presence of reducing agents (DTT).…”

Section: Caspase Substratesmentioning

confidence: 99%

Small Molecule Active Site Directed Tools for Studying Human Caspases

et al. 2015

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Caspases are proteases of clan CD and were described for the first time more than two decades ago. They play critical roles in the control of regulated cell death pathways including apoptosis and inflammation. Due to their involvement in the development of various diseases like cancer, neurodegenerative diseases or autoimmune disorders, caspases have been intensively investigated as potential drug targets, both in academic and industrial laboratories. This review presents a thorough, deep, and systematic assessment of all technologies developed over the years for the investigation of caspase activity and specificity using substrates and inhibitors, as well as activity based probes, which in recent years have attracted considerable interest due to their usefulness in the investigation of biological functions of this family of enzymes.

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“…A FRET-based quenching assay for screening of caspase-3 inhibitors has been proposed using Eu(III)-chelate doped NP donors coated with streptavidin in conjunction with a dual-labeled (the N-terminal Alexa Fluor 680 fluorescent acceptor and the C-terminal BlackBerry quencher 650) caspase-3-specific peptide substrate modified with a biotinyl moiety [55]. In inhibitory conditions, the substrate remains intact and the sensitized acceptor emission is attenuated by the quencher dye.…”

Section: Individual Assaysmentioning

confidence: 99%

High Throughput Bioassays Using Nanoparticles

Gómez‐Hens¹,

Fernández-Romero²,

Aguilar-Caballos³

2010

CCHTS

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An overview of the usefulness of different nanoparticles to improve the features of high throughput separation and individual and multiplexed detection bioassays is presented. Although the development of microarray and microfluidic systems has expanded the capabilities of these high throughput assays, the combined use of NPs and these devices has provided them with new applications in drug discovery, proteomic and genomic studies, and clinical diagnosis. This article reviews the wide application field of magnetic, gold, silver, semiconductor and other nanoparticles in high throughput bioassays. Also, the versatility of the detection systems described shows that NPs are useful alternatives to fluorescent dyes, which are often used in these assays.

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A dual-step fluorescence resonance energy transfer-based quenching assay for screening of caspase-3 inhibitors

Cited by 22 publications

References 47 publications

Luminescent lanthanide reporters: new concepts for use in bioanalytical applications

Luminescent lanthanide reporters: new concepts for use in bioanalytical applications

Small Molecule Active Site Directed Tools for Studying Human Caspases

High Throughput Bioassays Using Nanoparticles

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