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Eventually, a further development would be to link a specific conformational
change to a specific property of the molecules (activator, inhibitor, modulator,
etc.). Such a perspective could require the need to implement more local label-
ing and thus smaller tags, and a promising solution could be found in the non-
natural amino acids.
79,80
So far, only nonpermeant TR-FRET substrates have
been used for GPCR labeling, which did not permit the direct kinetic detection
ofalltheintracellulareventssuchasGproteinand
b
-arrestin recruitment on
intact cells as opposed to what is done with classic FRET or BRET assays.
As already mentioned, a proof-of-concept study using antibodies reported
intracellulareventsinrealtimeby TR-FRET but hardly compatible with
HTS.
33
However, cell-permeant substrates for SNAP and CLIP are now
available (yet not derivatized with TR-FRET donors) and would allow the
labeling of intracellular GPCR partners and even of the intracellular part of
the receptor itself, which would permit
the conception of new HTS-
compatible TR-FRET-based assays.
5.2. Detecting GPCR internalization
A second axis for further development of tests would be to implement an
internalization assay. Indeed, after activation, most GPCRs are internalized,
and that property has already been used to screen for active compounds for a
given receptor. At present, some solutions based on internalization are com-
mercially available, but they rely on imaging and semiautomated analysis of
the images and therefore not suitable for HTS campaigns (Innoprot). A re-
cent article reports the use of SNAP and CLIP-tagged receptors to monitor
GPCR internalization using time-resolved fluorescence measurement.
81
Cells expressing the receptor of interest are submitted to drug treatment
at various concentrations and followed by labeling of the SNAP or CLIP
with terbium. After a washing step, the amount of SNAP labeling is quan-
tified relative to untreated cells. Once again, the format of this assay makes it
rather ineligible for HTS. However, the development of a TR-FRET in-
ternalization assay that allows a direct measurement of the kinetics would
surely complete the offer available so far for GPCR analysis.
5.3. Multiplexing: Simultaneous analysis of a compound effect
in several signaling pathways
As mentioned previously, GPCR signaling is a complex phenomenon.
Depending on the receptor, the ligand, the localization, and the oligomer-
ization, the repertoire of activated signaling cascades differs. To better
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