Time-Resolved Förster Resonance Energy Transfer-Based Technologies to Investigate G Protein-Coupled Receptor Machinery: High-Throughput Screening Assays and Future Development - Progress in Molecular Biology and Translational Science - page 292

they have well-separated emission wavelengths, thus allowing the detection
of two metabolites from two distinct pathways, at the same time, in the same
well, and upon the same agonist stimulation. 37
Generic screening methods are invaluable strategies for HTS of large
libraries of compounds and can be applied to several GPCR targets to
determine the specificity and the signaling repertoire of each compound.
However, it is not always possible to measure all the downstream events with
HTS-compatible assays, and some hits could be missed. Therefore, comple-
mentary methods are needed. These methods are specific for the chosen target
and range from binding assays to internalization assays and also to the complex
analysis of the compound effects on dimeric and oligomeric GPCR entities. 38
Some target-specific TR-FRET-based assays are under development, and
some are under validation for future application in HTS campaigns. Together
with the generic methods, the target-specific methods contribute to the strat-
egy to identify putative therapeutic molecules with reduced side effects.
4. SCREENING WITH TARGET-SPECIFIC METHODS
Two different target-specific TR-FRET methods to study GPCRs have
been reported so far. The first one, a ligand-binding assay, has been validated for
its use in HTS campaigns. The second one is based on the capability of GPCRs
to form oligomers and the capability of the molecule to act on these complexes.
This second method is still under development but would be very useful in the
field of drug discovery because oligomers possess unique properties.
4.1. Drugs targeting a protein: Ligand-binding assay
As previously mentioned, screening for an active molecule for a given
GPCR using functional assays may be rather difficult. Analyzing a single sig-
naling pathway may lead to false negatives and, thus far, some signaling path-
ways remain difficult to screen in HTS format. Therefore, the identification
of molecules that bind to a specific target, in a binding-like format assay, re-
mains relevant, as they could potentially modulate the receptor signaling.
There exist nowadays nonradioactive methods based on TR-FRET to per-
form ligand binding.
4.1.1 TR-FRET-compatible ligands for binding assay
Like in classical radioactive binding assay, these are based on the displace-
ment of a tracer compound that binds specifically and with a high affinity
to the target. The tracer is often obtained by the derivatization of a known