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Marjan Orban1, Francois Degorce2 and Jean-Luc
Tardieu2
1BMG LABTECH, Germany; 2Cisbio international, France
Application Note 137
Rev. 12/2005
Introduction
HTRF (homogeneous time-resolved fluorescence) technology, developed
by Cisbio international, is used in assay development and drug screening.
HTRF is based on FRET between a Eu3+ cryptate (donor) and
a second fluorescent label (acceptor). A new acceptor, d2, allows the
introduction of a complete GPCR (G protein-coupled receptor) platform
suitable for drug discovery.
Upon activation, GPCRs carry the information within the cell via two major signalling pathways: the activation of Gαs or Gαi coupled GPCRs results in a variation of the cAMP level, whereas the activation of Gq coupled GPCRs result in a transient increase of intracellular Ca2+ triggered by inositol (1,4,5) tri-phosphate (IP3). Cyclic AMP and IP3 therefore represent two essential secondary messengers for monitoring the activity of most GPCRs.
Concerning the Gq pathway, the precursor molecule for the signaling cascade, IP3, is an extremely instable product (turnover only a few tens of seconds) and its degradation is irreversible. This of course has tended to make its detection highly inaccurate. IP3 induces a transient calcium release in the cell. Calcium sensing, through a very remote indicator, can allow the high throughput investigation of GPCR activity. To date, there has been no widespread use of IP assays in HTS, given its extremely challenging implementation. For lack of better options, the reference method consists of an assay which detects the accumulation of the cascades different inositol phosphates (IP3, IP4, IP2 and IP1) after the radioactive precursors uptake and a separation by affinity.
Recently Cisbio launched a major new kit, HTRF IP-One, for Gq pathway investigation under HTS conditions. The IP-One kit allows the quantification of the cellular accumulation of inositol 1 phosphate (IP1).
This application note focuses on the IP1 assay which has been validated on a broad and representative selection of Gq coupled receptors using the dedicated HTRF plate reader RUBYstar and the multimode plate reader PHERAstar from BMG LABTECH. These HTRF certified compatible readers are capable to simultaneously detect the fluorescence at two wavelengths (620 nm and 665 nm) and further signal ratioing also enables the technology to overcome interference from the medium or from compounds. Such technological efficiency, combined with the use of a high-affinity monoclonal antibody, give the assay all the qualities necessary for an HTS tool, particularly in terms of sensitivity, robustness and reliability.
Assay Principle
As previously mentioned, IP3, though, being the top choice as a secondary
messenger for the Gq signaling pathway, nevertheless does not possess
the necessary profile for an HTS tool because of its overlybrief physiological
existence. Moreover, there is no inhibitor available for the phosphatases
which convert IP3 into IP2 and then into IP1. On the other hand, the final
stage of the cascade when IP1 is transformed into myo-inositol can be
blocked with the use of LiCl, which enables one of the Gq pathways essential
sub-products to be stabilized (figure 1), just as IBMX action prevents
cAMP degradation. In its final configuration, it brings into play a highly
specific MAb coupled to europium cryptate and the IP1 conjugated to the
new HTRF acceptor, d2. This tiny proprietary molecule, with photophysical
characteristics similar to those of XL665 (the reference HTRF acceptor),
improves the technologys performances markedly, particularly in terms
of IC50 stability and of measurement dynamics.
Materials and Methods
Cisbios homogeneous IP-One assay can be carried out in a single microplate,
into which the cells have been dispensed the day before the actual test
is run (figure 2). The cell stimulation conditions meet the particular
characteristics of the cell line used - generally 30 minutes at 37C.
Quantification of the accumulated IP1 is obtained after dispensing the
two diluted conjugates into the lysis solution. Measurements can then
start to be taken after just one hour of incubation, and be repeated as
many times as necessary without impacting final data (e.g. IC50).
The standard curve for the kit was run according to the package insert
protocol in white 384-well plates (COSTAR cat# 3711384) with 20 L
total assay volume. The HTRF signal was read on the PHERAstar and the
RUBYstar.
Cells were kindly provided by the Institut de Genomique Fonctionnelle
(IGF), Montpellier, France and Euroscreen, Gosselie, Belgium. IP-One
kit was supplied from Cisbio international (France). The dedicated
plate reader RUBYstar and the multimode plate reader PHERAstar
were purchased from BMG LABTECH (Germany).
Cell lines (see table 1) expressing the GPCR target of interest were used for measuring IP1 production by stimulation of the ligand. In a 384-well format, the cell suspension was dispensed at 15,000 cells/ 20 L/well. After incubation at 37C, the culture supernatants were completely discarded. Then immediately after, 10 L of stimulation buffer containing various concentrations of ligand were added. After incubation at 37C for 1hr, 5 L IP1-d2 conjugate followed by 5 L of Eu-cryptate labeled anti-IP1 antibody were added. Time-resolved fluorescence at 620 nm and 665 nm were measured with PHERAstar and RUBYstar after incubation at 4C overnight, and the ratios of the signals and Delta F were calculated.
Delta F % = [(Standard or sample Ratio Rationeg ) / Rationeg ] x 100
Results and Discussion
As shown in table 1, IP-One has already been validated on different models
and targets - even if the cell lines involved were not specifically optimized
for IP-One assay.
The validations also conclusively demonstrate the assays performances
in the presence of cell lines using Gα16 or Gqi9 type chimeric constructions.
The IP-One assay showed equally good performance on different cellular
backgrounds, stable or transient transfected cells, and chimeric constructs.
There is a strong correlation with reference methods and no cross reactivity
with 50 M of the following (phospho) inositides phosphates could
be observed: Myo-inositol, PIP2, PIP3, IP2, IP3 and IP4.
In addition, for a direct performance comparison of BMG LABTECHs PHERAstar with the RUBYstar the IP-One assay was applied upon the 1321N1-CCK1 cell line together with the agonist CCK8-sulfated. The experiment was performed in 96-well plates resulting in very close EC50 values and a very good correlation regarding the %inhibition / basal curve (figure 3). Z' calculations at an agonist concentration equal to EC80 resulted in Z' > 0.78 for both plate readers.
But reaching beyond these expected results, the IP-One assay has already
proved its worth in the field of screening for inverse agonist compounds.
Looked at closely, and given its transitory characteristics, calcium measurement
is often not sensitive enough to specifically detect the inhibition of
the GPCR constitutive activities, whereas a modulation in the concentration
of IP1 is perfectly able to show this. Using the IP-One assay therefore
represents a tempting new alternative when investigating Gq-coupled receptors
via the quantification of a new secondary messenger for this pathway,
IP1. The messengers stability opens the possibility of the IP-One assays
application to cases where other HTS technologies fail to provide a satisfactory
solution such as in the detection of inverse agonist activities. The
assay also profits from the special qualities of d2, one of the latest
improvements implemented in HTRF technology. Given its performance, d2
is now included in all HTRF cAMP assays. The new HTRF platform for GPCR
screening will thus incorporate a series of HTS assays, all using similar
protocols and a standard robotics and detectors. Such a platform obviously
holds all that is needed to provide test solutions which are particularly
well-adapted to meeting the challenges of the different stages of drug
screening.
Conclusion
Cisbios new IP-One assay, which is based on its proprietary HTRF technology,
is the first high throughput system that can easily detect inositol(1)phosphate
(IP1), one of the major products of the phosphatidyl inositol cascade,
which tightly correlates with Gq-coupled activity. In association with
Cisbios cAMP assay, it allows the establishment of a complete HTRF based
GPCRs platform fully covering the different signalling pathways triggerred
by GPCRs. All HTRF IP-One assay results were produced on BMG LABTECHs
dedicated HTRF plate reader RUBYstar and the multimode plate reader PHERAstar.
The readers showed in a comparison strongly correlating results in terms
of EC50 and Z' values.
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