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drugs with improved side-effect profile for treating psychiatric disorders asso-
ciated with dysregulation of dopamine signaling.
4.2.3.2 New strategies to specifically target GPCR heteromers in HTS
TR-FRET assays
To be able to use the TR-FRET assays described above on a specific
heterodimer, it will be first necessary to develop a method to specifically
label the heterodimer. Of course, all the tags described above can be fused
to the receptors to study the complex properties
in vitro
. But even more
interesting, specific antibodies or ligand recognizing specifically the
heterodimer can be developed and later labeled with TR-FRET-
compatible probes. Indeed, Gupta and colleagues generated antibodies that
selectively recognize the MOR-DOR heteromer and blocked the
in vitro
signaling, and could then analyze
in vivo
their increased abundance after
chronic morphine treatment in pain-processing areas.
67
Targeting oligomers represents one of the new challenges in drug discov-
ery. Based on the current knowledge, this strategy could limit the adverse
effects of the molecule when administered to animal models and later to pa-
tients. Indeed, since the oligomers seem to be tissue specific and opposite to
what is observed for a receptor-specific drug, an oligomer-specific drug
would then target the receptor present only in the specific tissue.
5. PERSPECTIVES
The TR-FRET-derived solutions available so far for the analysis of
GPCRmachinery cover the main events linked to their activation: ligand bind-
ing, G protein activation and other signaling cascades, oligomerization, and spe-
cific pharmacology of the oligomers. However, one could imagine extending
further the offer to events such as conformational changes or internalization.
5.1. Detecting GPCR conformational changes upon binding
FRET- or BRET-based assays have been developed to monitor the confor-
mational changes of GPCRs (for review, see Ref.
78
). While these assays are
often developed using microscopy detection, one could imagine developing a
counterpart based on time-resolved FRETbyusingthelanthanideasafluores-
cent donor and a compatible fluorescent acceptor, thus compatible with plate
readers. Such implementation would be useful to detect a compound within a
library that induces conformational changes of a given GPCR. This strategy has
the advantage of detecting all molecules acting on a GPCR without requiring
any knowledge on the signaling pathway activated by the molecules.
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