Beruflich Dokumente
Kultur Dokumente
Rev. 09/2006
Introduction
G-protein coupled receptors (GPCRs) are cell surface receptors, which
represent the most predominant drug targets. Following stimulation
of these receptors, intracellular signalling pathways are activated and
this leads to a decrease (coupling to a Gi protein) or increase (via Gs or
Gq proteins) in the production of intracellular second messengers. The
common way of determining the activity of compounds is by measuring
the cellular formation of second messengers such as cAMP and calcium.
DiscoveRx assays offer a non-radioactive alternative for the detection
of a decrease or increase in second messenger production in cells.
They can be used with both cell-lines that express native receptors
and cells transfected with a GPCR of interest, and can be employed in
conjunction with high-throughput screening (HTS).
The HitHunter cAMP High Sensitivity (HS) assay is able to measure
low cAMP levels and is, therefore, particularly suitable for cell-lines
that endogenously express receptors at a level much lower than in
transfected cells overexpressing a cloned GPCR.
HitHunter cAMP assays are in vitro-based competitive
immunoassays that rely on enzyme fragment complementation
technology (EFC, Fig. 1).
Signal
cAMP
Antibody
Labeled ED
cAMP
Inactive EFC
Enzyme
[Competing cAMP]
+
EA
Competing
cAMP
Substrate
EA
Active EFC
Enzyme
ED
Hydrolyzed
Substrate
Signal
Free cAMP molecules from cell lysates compete for antibody binding
with a labelled enzyme donor (ED)-cAMP conjugate, which contains
a small peptide fragment of -galactosidase. In the absence of free
cAMP, the ED-cAMP conjugates are captured by the cAMP-specific
antibody and are unavailable for complementation with the enzyme
acceptor (EA), resulting in a low signal. In the presence of free cAMP,
antibody sites are occupied, allowing the ED-cAMP conjugate to
complement with EA, forming an active -galactosidase enzyme;
substrate hydrolysis by this enzyme produces a chemiluminescent
signal. The signal generated is in direct proportion to the amount of
free cAMP bound by the antibody (Eglen, 2002). Any luminescence
reader used with the HitHunter cAMP HS assay has to be sensitive
enough to detect small changes in cAMP levels. The BMG LABTECH
FLUOstar OPTIMA microplate reader has this required sensitivity.
CGS21680 alone
Plate
0.2 sec
1
1
lens
3000
1 sec
RLU
2000
1000
500
OGS21680
+10-7M ZM241385
basal -10
-9
-8
-7
-6
-5
-4
log [CGS21680,M]
Conclusion
Fig. 2: Screenshot of the settings window from the FLUOstar OPTIMA multimode reader for the HitHunter cAMP HS assay
Both agonist and antagonist data can be generated, using the HitHunter cAMP HS assay, in conjunction with the FLUOstar OPTIMA
microplate reader.
The FLUOstar OPTIMA, which can also be used for absorbance and
fluorescence detection, was used here in luminescence mode. It offers user-friendly software for both protocol set up and data analysis.
It is very amenable for use in an academic environment (e.g. 96-well
format) but can also be employed in all formats up to 1536-well
plates for HTS of library compounds.
Fig. 3: cAMP standard curve for the HitHunter cAMP HS assay (standards
3000
pEC50 = 8(9.7nM)
S/B = 11
References
- Eglen, R. M. (2002). Enzyme fragment complementation: a flexible
high throughput screening assay technology. ASSAY and Drug Development Technologies 1, 97-104.
- Florio, C., Frausin, F., Vertua, R., Gaion, R. M. (1999). Amplification
of cyclic AMP response to forskolin in pheochromocytoma PC12
cells through adenosine A2A purinoceptors. Journal of Pharmacology and Experimental Therapeutics 290, 817-824.
RLU
2000
The views expressed herein are those of the authors and do not necessarily represent those of Nottingham Trent University.
1000
-10
-9
-8
log [cAMP, M]
-7
-6
-5
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