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ligand with a fluorescent moiety. Depending on the receptor, this ligand can
be a small molecule, a peptide, a small protein, or an antibody. The smaller
the ligand, the more difficult its derivatization. Indeed, a major challenge is
to keep high affinity after the fluorescent labeling of the ligand. Though
small proteins and antibodies offer several possibilities to attach the fluo-
rophore, as only part of the molecule interacts directly with the receptor,
the same is not true for small ligands and peptides for which most of the mol-
ecule interacts directly with the receptor upon binding. In the latter case, a
good knowledge of the binding pocket and of the pharmacophore is often
necessary to synthesize high-affinity fluorescent ligands. This helps in the
identification of the part of the molecule to which a linker can be added
and further derivatized with a fluorophore. 39,40 As an example, Baker and
colleagues have performed a systematic analysis of the derivatization of
two adenosine A1 receptor ligands (one agonist and one antagonist) by
different fluorophores and various linkers, and both appeared to affect the
pharmacological and fluorescent properties. 41 Some of these ligands
appeared to be powerful tools to monitor binding events in living cells
using fluorescent correlation spectroscopy. 42 Over the past years, efforts
have been made by several companies and academic laboratories to
develop such ligands. By the end of 2011, fluorescent ligands targeting
more than 70 GPCRs were commercially available from Cisbio
Bioassays, Cellaura, Perkin Elmer, and Abcam.
While these fluorescent ligands can be used directly to perform binding
assays (e.g., see Refs. 42,43 or DELFIA Ò technology by Perkin Elmer), these
still require washing steps to separate the bound from the unbound fractions
of the ligand. In the DELFIA Ò assays, for example, after incubating the cells
with a europium-labeled ligand and the compounds to be screened until
equilibrium is reached, the unbound fraction is washed away and the
leftover fluorescence is read in a time-resolved manner after addition of
an enhancing solution. These washing steps, which often prevent the
miniaturization and automation of an assay, are not compatible with
HTS, and solutions had to be found to circumvent this. HTRF
represents a good alternative to the classic binding-assay format. Indeed,
as already mentioned, the specific properties of the HTRF-compatible
fluorophores allow highly specific signal detection in a homogeneous
solution and prevent the detection of most of the background fluorescence,
especially that coming from unspecific binding. Indeed, the HTRF binding
assays are based on the measurement of the FRET signal between a
fluorophore linked to the receptor and the fluorescent ligand ( Fig. 7.3 ).
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