Biology Reference
In-Depth Information
pathways, new partners, etc.) and also on the development of new
technologies.
Because of the increasing number of available compounds to be tested, the
format of HTS assays had to adapt to miniaturized formats (384-, 1536-, and
even 3456-well microplates) and automated platforms. This miniaturization
must come with increased accuracy of the assays to limit false positives. Also,
the will to use environment-friendly tests had required scientists to find alter-
natives to the radioactive tests that generate wastes that have to be handled at
high cost. A good alternative was found in fluorescent probes and fluorescent
assays. The first HTS assays using these probes appeared rather accurate and
almost as good as the previous assays and are now being widely used by phar-
maceutical companies in screening campaigns. However, among the screened
molecules, some are colored and interfere with the detection of the fluores-
cent probe.
1
This can lead to false positives, and these compounds are often
excluded from further fluorescent analysis and thus from further characteriza-
tion. To circumvent this, some solutions are available. Indeed, in time-
resolved F¨rster resonance energy transfer (TR-FRET), which is a specific
type of fluorescent method, the peculiar timeframe of signal recordings per-
mits getting rid of most of fluorescent contamination arising from molecules
and other components of the assay.
2,3
TR-FRET has additional advantages
over the other techniques, such as a higher signal-to-noise ratio, for
example. This technology has been successfully applied to offer kits to
measure the activation of different cellular processes and solutions to
measure the binding of molecules to a specific target. This chapter presents
how TR-FRET and the ratiometric “homogeneous time-resolved
fluorescence” (HTRF
Ò
) technology are an interesting alternative in HTS
assays and provides examples using a model of the targets used in drug
discovery, namely, G protein-coupled receptors (GPCRs).
GPCRs are cell-surface proteins that recognize a large variety of stim-
uli and that are key signaling molecules participating in most cellular pro-
cesses.
4
Today, up to 30% of the therapeutic molecules available on the
market and many drugs of abuse act on GPCRs,
5
and this family of recep-
tors still represents a promising target for the development of new ther-
apeutic solutions for the treatment of several pathologies. The GPCR
family contains more than 850 members in humans classified on the basis
of their sequence homology into several classes, mainly class-A (rhodopsin-
like), class-B (secretin receptor-like), and class-C (metabotropic glutamate re-
ceptor (mGluR)-like) receptors
6
A structural feature common to all GPCRs is
a bundle of seven membrane-spanning
a
-helices with the amino terminus at
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