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antibodies labeled with HTRF
Ò
-compatible fluorophores to enter the
cells, the authors could detect
b
-arrestin and G protein recruitment to
PAR1 upon activation. If this format of assay is not compatible with
HTS, it indicates nevertheless that HTRF
Ò
is perfectly compatible with
the detection of intracellular events in living cells.
3.4. The future of HTS generic assays
In the past two decades, the complexity of GPCR pharmacology has dramat-
ically increased with new concepts challenging the classic scheme of one re-
ceptor activating one specific G protein. Receptor homo- and/or
heterodimerization, allosterism, biased signaling, agonist-dependent traffick-
ing, and G protein-independent signaling are all relatively new concepts in
the GPCR field, which have to be taken into consideration when looking
for new ligands.
7,8
The issue would now be more a question of which
pathway is the therapeutically pertinent one to look at when talking about
a specific disease and, thus, which assay reflects the best what occurs in this
pathway under physiological conditions. Biased signaling, for example, is a
phenomenon in which certain agonists display better efficacies in activating
one pathway over others. Thus, another important aspect to take into
account is whether we can limit adverse effects by designing drugs
activating only one specific pathway and not all the pathways the GPCR
can couple to. For example, Lefkowitz and colleagues have demonstrated
that the angiotensin II type 1 receptor (AT
1
), the target of angiotensin-
receptor blockers for the treatment of hypertension, can activate both
Gq/11-type protein pathway and
b
-arrestin-induced pathway upon
activation by the endogenous ligand angiotensin II.
34
On the contrary,
biased ligands such as Sar1, Ile4, and Ile8-AngII (SII) stimulate
b
-arrestin
signals, such as ERK1/2 activation, in the absence of detectable G protein
agonism.
35
In cardiomyocytes, for example, in the absence of G protein
activation, SII could stimulate proliferation, but not hypertrophy observed
when the Gq/11 protein pathway is also activated.
36
Thus, if a single functional assay capturing only one signaling pathway is
selected for screening compound libraries, potentially valuable compounds
could be missed if the compound does display biased activity. Therefore,
multiplexing assays capable of detecting several signaling pathways may be
the future methods to use in HTS. In TR-FRET-based assays, such a mul-
tiplexing may be possible (at least to detect simultaneously two secondary
messengers). Indeed, terbium used as a donor can be associated with differ-
ent acceptors, a fluorescein and a d2 near-infrared dye, for example, because
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