Energy transfer assays example

Compounds that interfere with the detection mechanism of the HTS assay wiU, in many cases, be detected as highly potent actives [31]. One example would be compounds that intrinsically emit or absorb light at the wavelengths used in a fluorescence-based assay such as fluorescence resonance energy transfer (FRET). In an HTS screen using a fluorescence-based assay at Wyeth, 1.2% of the samples tested showed not just high fluorescence but the maximum possible initial (time 0) reading on the fluorimeter. 

Another example of improved sensitivity due to modulation of lanthanide photophysics by ancillary ligands can be found in the europium and terbiiun chelates used in time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassays (100,101). Due to their line-type emissions and long decay times, the lanthanide chelate is used as a donor, with some visible-absorbing dye such as Alexa 647 or a rhodamine derivative as the acceptor. Without the helper ligand, the lanthanides would be unprotected from solvent and have much shorter decay times, making them unsuitable for such an assay. 

There are other examples of HIV protease substrates that utilize the fluorescence energy transfer technique. Perhaps the strongest characteristic of these assays is that they provide a continuous readout of enzyme activity. Another advantage is their sensitivity (they use small concentrations of enzyme). The disadvantage of these assays is that they are susceptible to interference by some compounds (inhibition artifacts) because of inner and outer filter effects (see Notes 8-10). Other assays, for example, those based on radioactivity or high-performance liquid chromatography (HPLC) analysis of products are tedious to run, but are less susceptible to interference or inhibition artifacts. 

An example of this technology is the HIV protease assay shown in Fig. 9, which was published by Wang and co-workers [67]. The peptide substrate is labeled at the amino terminus with EDANS (5-((2 -aminoethyl)amino)naphthalene-l-sulfonic acid) as a donor fluorophore and at the carboxyl terminus with DABCYL (4-((4 -(di-methylamino)phenyl)azo)benzoic acid) as the acceptor chromophore. In the intact peptide, fluorescence resonance energy transfer (FRET) from EDANS to DABCYL results in quenching of the EDANS fluorescence. On cleavage of the peptide by HIV protease, the fluorescence of EDANS is restored. 

Fig. 3 Examples of solid phase ECL assay formats (a) and (b) dependent on the amount of ruthenium complex integrated into the DNA system, (c) and (d) dependent on the distance of labeled ECL probe and electrode induced by charger transfer, (e) and (f) based on the energy transfer. Reprinted with permission from Ref. 51. Copyright (2003) American Chemical Society.

The most common assays for ion channel HTS are fluorescent dye-based, measuring influx of, for example, calcium into the cell. Fluorescent resonance energy transfer (FRET) may also be used. However, these methods lack control of voltage over the membrane. To investigate state-dependent voltage-gated ion channek methods with much lower throughput need to be applied [136]. 

We then introduce a few examples for the use of SPFS, first in surface hybridization studies and then for antigen-antibody interaction assays. We will give, in particular, examples for different versions of fluorescence spectroscopy making use of, e.g., donor-acceptor energy transfer phenomena between correspondingly labeled binding partners. 

As an example of a similar approach using in vivo SNAAR, Turcatti et aL" " incorporated a fluorescent amino acid, 3-N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl)-2,3-diaminopropionic acid (NBD-Dpr), at specific sites in the tachykinin neurokinin-2 receptor, a member of the large family of seven a-helix G-protein coupled receptors (GPCRs). The expression system, Xenopus oocytes, allowed for functional assays of the receptor. Structural information was obtained by measuring the intermolecular distance between the fluorescent amino acids placed at different sites and a fluorescently labeled heptapeptide antagonist using fluorescence resonance energy transfer (FRET). 

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