cAMP DETECTION METHODS
IN HTS: SELECTING THE BEST
FROM THE REST
The number of technologies that enable high-throughput functional screening of G-protein-
coupled receptors has expanded markedly over the past 5 years. Consequently, choosing the
most appropriate technology can be a daunting task, particularly for Gi- or Gs-coupled receptors.
The most common systems for cyclic AMP detection are reviewed, highlighting the practical and
theoretical aspects that are important in their application to high-throughput screening. Current
technologies can do the job, but it is likely that the future may require development of technologies
that provide even greater biological information.
Since the pioneering work of Langley1 and Ehrlich2 at cascade have been used by academia and industry alike
A compound that acts on a the beginning of the twentieth century, receptors — (FIG. 1). However, as manufacturers and suppliers have
modulatory binding site on and especially G-protein-coupled receptors (GPCRs) responded to the pharmaceutical industry’s efforts in
a receptor that is topo- — have grown to be one of the most important areas of these areas, recent years have seen a rapid expansion
graphically distinct from
the agonist binding site.
research in the pharmaceutical industry. Analysis has in the technologies that facilitate the high-throughput
indicated that ~45% of currently marketed medicines quantification of these functional responses. As a
are targeted to receptors and that they represent one of thorough assessment of all of these technologies is
the largest druggable gene families3,4. High-throughput beyond the scope of this review, the focus will be on those
screening (HTS) campaigns that seek to identify novel technologies that are presently available for the detection
receptor modulators have been key to the drug discovery of the intracellular signalling molecule 3′,5′-cyclic adeno-
industry in this respect. However, continued success sine monophosphate (cAMP). Particular consideration
in this highly competitive area has seen an evolution in will be given to the practical and scientific implications of
assay technologies and methodologies 5,6 and a re- the methodologies, with the aim of enabling the reader
evaluation of discovery strategies, with emphasis to make an informed choice about their strategy for
moving towards the quality and relevance of the infor- identifying modulators of Gi or Gs GPCRs.
mation generated3,4,7. Consequently, the HTS com-
munity has seen a recent bias towards the use of assays Regulation of intracellular cAMP levels
that measure the downstream effects of receptor activa- As with all signalling molecules, the levels of intracellular
tion8–10. The key advantage of these functional assays is cAMP are tightly regulated. Production of cAMP is
that they facilitate early and direct pharmacological char- controlled through the adenylyl cyclase family of
Hit Discovery Group (HDG), acterization of compounds. More specifically, they could enzymes, which convert adenosine triphosphate (ATP)
Pfizer Global Research and expedite the separation of antagonist and agonist to cAMP and inorganic pyrophosphate. These enzymes
Development, Ramsgate compounds and enable the identification of novel com- are activated or inhibited via direct interaction with
Road, Sandwich, pounds such as ALLOSTERIC MODULATORS or weak-affinity, G-protein α-subunits and, for some isoforms, with Ca2+
Kent CT13 9NJ, UK.
high-potency agonists11,12. For many years, in vitro and calmodulin. Following Gs-coupled GPCR activa-
sandwich.pfizer.com screening methodologies that quantify functional tion, active Gαs molecules exert a positive effect on
doi:10.1038/nrd1306 effects at a number of points along the GPCR signalling adenylyl cyclase catalysis. cAMP is produced and is able
NATURE REVIEWS | DRUG DISCOVERY VOLUME 3 | FEBRUARY 2004 | 1 2 5
α GDP β α GTP + β
PIP2 IP3 b
Adenylyl cyclase DAG
i[cAMP] d i[cAMP] c i[Ca2+]
CREB CREB NFAT
CRE f CRE e NFAT-RE
Figure 1 | Sites on the GPCR signalling cascade commonly employed in in vitro assays of receptor function. The initial
stage of GPCR activation occurs via agonist-induced conformational changes in the receptor to form an active agonist–receptor
complex that is able to interact with heterotrimeric G-proteins and facilitate its activation via exchange of GDP for GTP at the
α-subunit. This G-protein activation can be detected via the use of non-hydrolysable GTP analogues61,62 (a). Following G-protein
activation, the dissociated α-subunit and βγ dimers modulate activity of effector proteins that control the level of intracellular
signalling molecules. Levels of phosphatidylinositol biphosphate (PIP2) and inositol triphosphate (IP3 ) can also be monitored via
the use of radiolabelled precursors63 (b). Changes in the levels of the intracellular signalling molecules Ca2+ and cAMP can be
measured directly via fluorescent calcium-chelating agents64,65 (c) or through labelled cAMP molecules competing for the binding
site of cAMP sequestering antibodies (d). Finally, changes in the levels of these signalling molecules produce alterations in gene
transcription or protein activity and result in the observed functional response of the cell; these events can be measured via
transcription factors such as NFAT (nuclear factor activated in T-cells) or CREB (cAMP response element binding protein) and
reporter genes under the control of appropriate upstream elements28,29 (e and f). CRE, cAMP regulatory element; DAG, diacylglycerol;
NFAT-RE, NFAT (nuclear factor activated in T-cells)-regulatory element.
to bind to protein kinases within the cell, initiating activation of the cAMP PDEs is phosphorylation by
phosphorylation events that regulate target enzymes cAMP-dependent protein kinases. Given the diversity
and transcription factors. Following Gi-coupled receptor of isoforms and tissue distribution in this enzyme
activation, Gαi molecules are activated and a negative class, the biology is complex (for a review, see
effect is exerted on enzyme catalysis. Further negative Thompson15). However, it is clear that the cAMP PDE
regulation of the enzyme is achieved by the sequestra- enzymes act as an important negative-feedback sys-
tion of active Gαs molecules by the βγi dimers (for more tem on the receptor-mediated signalling cascade, reg-
detail on the regulation and study of adenylyl cyclase ulating the extent of changes in intracellular cAMP
enzymes, see reviews by Hanone13 and Patel14). concentrations.
Degradation of cAMP is controlled by the cAMP
phosphodiesterase (PDE) enzymes, which catalyse the Accumulation assays for cAMP detection
hydrolysis of the 3′ ester bond of cAMP to form 5′ HTS-compatible accumulation assays for cAMP mea-
adenosine monophosphate (AMP). There are various surement follow a general principle, with changes in
PDE isoforms, which are involved in the breakdown intracellular cAMP being detected by the competition
of either cAMP or cGMP. These isoforms can be between cellular cAMP and a labelled form of cAMP
located in different subcellular compartments and dif- for binding to an anti-cAMP antibody. Protocols for
ferent tissue types and are activated by a number of these assays differ markedly, with a whole plethora of
mechanisms. The most widespread mechanism for technologies, ranging from radiometric to enzymatic,
126 | FEBRUARY 2004 | VOLUME 3 www.nature.com/reviews/drugdisc
Absence of cellular cAMP Presence of cellular cAMP
cAMP + cAMP –[125I]
b Slow rotation
Produces more polarized light following excitation Produces less polarized light following excitation
c Long lifetime Long lifetime
at 665 nm at 620 nm
cAMP cAMP + cAMP
at 620 nm
Figure 2 | Schematic representation of common technologies available for the detection of cAMP accumulation.
a | Radiometric proximity methods, such as the Flashplate technology, use microtitre plates coated with scintillant that enable the
detection of specific binding of radiolabelled molecules without the need for separation or a wash step. These plates are coated with
an anti-cAMP antibody and the assay uses [125I]-labelled cAMP as a tracer. In the absence of cellular cAMP, the antibody sequesters
the [125I]-labelled cAMP, bringing it in close enough proximity to the scintillant on the plate, such that light is produced. In the presence
of cellular cAMP, the unlabelled cAMP competes off the [125I]-labelled cAMP and thereby reduces the signal. b | Fluorescence
polarization cAMP assays (available in kit form from companies such as Perkin Elmer and Amersham Biosciences) monitor the light
emitted from a fluorescently tagged cAMP molecule following excitation with a polarized light source. When excited with polarized
light, the emission from labelled cAMP will be depolarized compared to the exciting light owing to the rotation of the molecule during
the time between excitation and emission. When the labelled cAMP is bound to antibody, the rotation during the time between
excitation and emission is reduced. Therefore, a lower polarization value is observed in the presence of cellular cAMP, when the
labelled cAMP is free in solution, compared to the absence of cellular cAMP. c | The HTRF (homogeneous time-resolved
fluorescence) technology uses anti-cAMP antibodies labelled with europium cryptate and cAMP that is labelled with a modified
allophyocyanin. In the absence of cellular cAMP, these two fluorescent molecules are in close proximity, FRET occurs and long
lifetime fluorescence is emitted at two different wavelengths. When the two molecules are separated by competition with cellular
cAMP, no FRET occurs and only emission from the europium is detected.
taking advantage of this simple approach. Several of the minimize compound artefacts — these radiometric
most common or innovative approaches are discussed technologies are being superseded to some extent by
below (summarized in FIGS 2 and 3). safer non-radiometric read-outs, which might be
Homogeneous radiometric assays, such as scintilla- cheaper and more readily miniaturized.
tion proximity assays (SPA, Amersham Biosciences) and The application of fluorescence polarization (FP)
Flashplate technology (NEN/Perkin Elmer) enable the technology to cAMP assays is based on a decrease in the
direct detection of [125I]-labelled cAMP once it is in extent of molecular rotation of a fluorescently labelled
close proximity to a solid scintillant surface16,17. The cAMP that occurs following binding to the larger
Flashplate technology (represented in FIG. 2a) has been anti-cAMP antibody (FIG. 2b). When excited with polar-
successfully used in HTS18 and offers distinct advantages ized light, the emission from labelled cAMP will be
RED-SHIFTED PLATES over more traditional methods in terms of convenience depolarized compared to the exciting light owing to the
Shifting the assay to the red (stimulation and detection can be carried out in the rotation of the molecule in the time between excitation
range ensures the most sensitive
imaging cameras can be used
same well), time and reproducibility. However, although and emission. However, when labelled cAMP is bound
and reduces interference from advances are still being made in this area — for example, to an antibody, the rotation during excitation and
yellow/brown compounds. with the development of RED-SHIFTED PLATES that aim to emission is reduced, leading to a reduction in the
NATURE REVIEWS | DRUG DISCOVERY VOLUME 3 | FEBRUARY 2004 | 1 2 7
Absence of cellular cAMP Presence of cellular cAMP
a Oxygen radical Luminescence
Acceptor No signal
Biotin Acceptor Donor
Donor cAMP bead Streptavidin
Streptavidin Biotin bead
Chemical No light
Figure 3 | Schematic representation of common technologies available for the detection of cAMP accumulation.
a | The amplified luminescence assay, ALPHAScreen, uses a donor and acceptor bead. When the donor bead is excited with light
at 680 nm, a photosensitizer in the donor bead coverts O2 to its free-radical form. When the two bead types are sufficiently close
together, this free-radical oxygen is able to produce a chemiluminescent signal within the acceptor bead, which in turn activates
fluorophores that are also contained within the acceptor bead, thereby amplifying the signal. In cAMP assays, the two beads can
be held in close proximity by biotinylated cAMP molecules as the donor bead is coated with streptavidin and the acceptor bead is
coated with an anti-cAMP antibody. Therefore, in the absence of cellular cAMP, signal amplification is observed, but in the presence
of cellular cAMP the two beads are not held in close proximity and no signal is generated. b | The enzyme complementation
technology available from DiscoveRx uses inactive enzyme donor and acceptor components, which, when combined, form an
active β-galactosidase tetramer. cAMP is labelled with the enzyme donor in such a way that it can still be recognized by an anti-
cAMP antibody. In the absence of cellular cAMP, the conjugated cAMP is sequestered by the antibody and no active enzyme can
be formed. In the presence of cellular cAMP, the enzyme donor conjugates are able to recombine with the enzyme acceptor moieties.
Enzymatic activity of the β-galactosidase formed can then be detected using substrates that are converted to either fluorescent or
luminescent products. c | The electrochemiluminescence technology available from Meso Scale Discovery uses cAMP labelled
with a ruthenium derivative. In the absence of cellular cAMP, anti-cAMP antibodies bound to the proprietary carbon electrode plates
sequester the labelled cAMP. Following addition of substrate and an electrical charge, electrochemical reactions are initiated, which
result in the production of light. However, in the presence of cellular cAMP, the labelled cAMP is no longer in close proximity to the
electrode and no light can be produced.
observed depolarization. Therefore, a higher polariza- potential artefacts owing to a statistically significant inten-
tion (P) value is observed for the labelled cAMP sity difference between the free and bound fluorescein-
bound to antibody than the free labelled cAMP. This labelled cAMP entities19. However, these artefacts could
technique has been successfully applied to high- be eliminated by further investigation of compounds
throughput screening with whole cells in miniaturized displaying percentage activities outside the expected
formats, which is consistent with other homogeneous range20. Alternatively, dyes such as Bodipy-TMR,
approaches in which the cytoplasmic cAMP content is MR121, Alexa, Cy3 and Cy5 could have been used, as a
exposed by cell lysis19. It has also been successfully significant reduction in compound interference has
applied to a less frequently used methodology using cell been observed in FP binding assays with these more
membranes20. However, one disadvantage of this tech- red-shifted dyes21.
nology is the potential for compound interference. Data Time-resolved fluorescence resonance energy transfer
indicate that some artefacts, at least up to a final assay (TR-FRET) has also been applied to the measurement
concentration of 10 µM, were observed using the fluo- of cAMP, via the HTRF (homogeneous time-resolved
rescein-labelled cAMP19,20. Analysis of parallel intensity fluorescence) technology (CIS Bio, France) (FIG. 2c). This
readings were not used as an immediate flag of these technique enables time-resolved, ratiometric data analysis
128 | FEBRUARY 2004 | VOLUME 3 www.nature.com/reviews/drugdisc
and facilitates discrimination of the fluorescent signal interference, although a potential disadvantage of the
from interference due to compounds or the influence of Meso Scale technology is that the plates cannot be re-read
reagent properties such as ionic strength and pH (see once electrically stimulated. However, like the HTRF
the CIS Bio International HTRF web site in online links technology, data from all three of these technologies
box). In this technology, the anti-cAMP antibodies indicate that they provide an additional order of magni-
are labelled with europium cryptate and the cAMP is tude of cAMP detection. Levels of ≤ 10 fmol cAMP per
labelled with a modified allophyocyanin. When these well have been quoted for these novel technologies,
two fluorescent molecules are in close proximity, FRET compared to other homogeneous techniques such as the
occurs and long lifetime fluorescence is emitted at two FlashPlate and FP technologies, which have limits in
different wavelengths. When the two molecules are sep- the order of 50–100 fmol cAMP per well20,22. Although
arated by competition with cellular cAMP, no FRET this level of sensitivity can be achieved using other
occurs and only emission from the europium is luminescent methods26, these novel technologies do
detected. Like the FP technology, this method is readily not require any wash steps and are compatible with
amenable to miniaturization. Owing to the time- miniaturization, which provides significant advantages.
resolved fluorescence, read-out might not be compatible There are various technologies for the detection of
with all reader types; however, as it might have reduced cAMP accumulation that are amenable to miniaturized,
compound interference and lower cAMP detection homogeneous HTS. Methods that use cell membranes,
limits, it could provide significant advantages over the such as the FP technology, offer advantages over those
FP technology. using whole cells because they minimize the impact of
Recently, three innovative technologies have emerged identifying compounds that act on the downstream sig-
that also aim to provide non-radiometric high-sensitivity nalling pathways and they facilitate use of cell lines that
assays. The first of these — ALPHAScreen (amplified are not readily amenable to large-scale continuous
luminescent proximity homogeneous assay; Packard culture (for example, those containing endogenous
Bioscience/Perkin Elmer) — is a homogeneous assay receptors such as SKNMC cells and human airway
format that uses proximity interactions between beads. smooth-muscle cells)26,27. However, as indicated above,
Following excitation of a ‘donor’ bead, oxygen is con- selection of a given technology might also require evalu-
verted to the more reactive radical state, which reacts ation of other parameters, such as detection limits, cost
with thioxene derivatives in an ‘acceptor’ bead. This reac- and the number of addition steps (summarized in TABLE 1).
tion produces a chemiluminescent signal that initiates an On the basis of this information, the HTRF and Dis-
amplification cascade by activating fluors that are also coveRx technologies might prove advantageous owing to
contained within the ‘acceptor’ bead22,23 (FIG. 3a). their sensitivity, compatibility with multiple readers and
The second system — an enzyme complementation the minimal impact of compound interference.
technology from DiscoveRx (Fremont, California) — uses
a cAMP molecule tagged with an inactive β-galactosidase Reporter-gene assays for cAMP detection
component. When mixed with its counterpart enzyme Reporter-gene assays follow a general principle, whereby
components, this cAMP-tagged enzyme fragment receptor-mediated changes in intracellular cAMP con-
recombines to form an active multimeric enzyme centrations are detected via changes in the expression
complex, the activity of which can then be detected via level of a particular gene (the reporter), the transcrip-
the generation of either a fluorescent or luminescent tion of which is regulated by the transcription factor
product24 (FIG. 3b). cAMP response-element binding protein (CREB) bind-
The third system uses electrochemiluminescence ing to upstream cAMP response elements (CREs).
detection and is a technology available from Meso Scale Specific protocols for these assays differ in the number
Discovery (Gaithersburg, Maryland). In this case, the of CREs used, the choice of reporter gene and the par-
cAMP, which is tagged with a ruthenium derivative, is ticular method for measuring activity of that reporter
captured onto proprietary multi-array plates via anti- gene. Although all reporter genes share common prop-
cAMP antibodies. Following addition of a chemical erties in that they are normally absent from the cell type
substrate and electrical stimulation, an electrochemical considered and a relatively small number of active
reaction cascade is initiated, which results in the pro- molecules are required for detection, they represent the
duction of light from the labelled cAMP (see FIG. 3c and most significant factor when considering which assay
Meso Scale Discovery in online links box). system to choose.
The ALPHAScreen and Meso Scale technologies are Various reporter genes have been used by academics
simple and relatively cheap, but both currently require a and industry for use in in vitro and in vivo studies,
specific reader. By contrast, the DiscoveRx technology is including β-galactosidase, green fluorescent protein
compatible with a variety of readers, and although early (GFP), luciferase and β-lactamase28–31. The key advan-
versions of the kit were cumbersome, later releases have tage for β-galactosidase systems are that there are a wide
been simplified, making the assay readily compatible variety of detection methods that enable the develop-
with automated HTS. The ALPHAScreen technology is ment of simple, cheap and robust assays with colori-
sensitive to COLOUR QUENCH from green and blue com- metric, fluorescent or luminescent read-outs. GFP and
An inappropriate decrease in an
pounds25 and can be sensitive to light and temperature its derivatives have also been extensively used, a key
assay signal due to the presence fluctuations. Conversely, the DiscoveRx and Meso Scale advantage being that these proteins require only oxygen
of a coloured entity. technologies might be less sensitive to compound and no co-factors for fluorescence, allowing detection
NATURE REVIEWS | DRUG DISCOVERY VOLUME 3 | FEBRUARY 2004 | 1 2 9
Table 1 | Summary of important parameters to consider in selection of an accumulation assay technology
Parameter Radiometric Fluorescence Time-resolved Amplified Enzyme Electro-
proximity assay polarization FRET luminescence complementation chemiluminescence
Robustness (Z′) 0.5–0.6 0.5–0.7 0.7–0.8 0.5–0.7 0.7–0.8 ~0.7
Number of additions 3 4 4 4 4 5
1,536 compatibility Not for Flashplate Yes Yes Yes Yes Yes
Detection limit ~100 50–100 <10 ~10 ~10 ~10
Instrument requirement Generic Generic Restricted Specific Generic Specific
Price (pence/384 well)* 99–136 40–80 18–30 7–15 7–40 10–14
Data are obtained from manufacturer web sites, product brochures and the references cited in the text. *Price is dependent on quantities ordered and further discounts might
be available following discussion with manufacturers. FRET, fluorescence resonance energy transfer.
without cell/tissue destruction. However, although they substrate, CCF2/AM36 (FIG. 4b). First, because the sub-
have been successfully used to monitor receptor- strate is cell-permeable and non-toxic, no cell disruption
mediated cAMP changes32,33, both of these systems have is required for quantitation, enabling rapid development
several caveats. First, GFP fluorescence is non-amplifiable of recombinant cell reagents and development of simpler,
and dependent on intracellular pH, which could result automation-compatible assay protocols. Second, as the
in poor sensitivity or misinterpretation of the data substrate contains a FRET pair cleaved on activation,
obtained. Second, both β-galactosidase and GFP have a ratiometric data analysis is possible, thereby minimizing
relatively long half-life, which means that they might be the potential impact of fluorescent artefacts and well-to-
less sensitive owing to higher background ‘noise’. well variations, without the need for two separate
Luciferase enzymes have shorter half-lives than reporter constructs37. In addition, recent data also indicate
β-galactosidase and GFP, providing some advantage and that this system might provide improved sensitivity over
making them a popular choice in screening campaigns10. other reporter-gene systems38.
The most commonly used luciferase enzyme is that The reporter-gene assays described above are all
from the firefly (Photinus pyralis), which catalyses the homogeneous and readily amenable to detection using
oxidation of luciferin to produce oxyluciferin and standard readers. The assays are simple, compatible
light (FIG. 4a). The advantage of using this particular with miniaturization to 1,536 wells (or further) and
enzyme is that there are various reagents available. reagents are extremely cost effective, particularly when
Some of these reagents can prolong the lifetime of the compared with many of the accumulation technolo-
light emitted, making it detectable for several hours gies. As generation of a recombinant cell line is required
and improving automation compatibility, although it for reporter-gene screens, the use of the β-lactamase
is prudent to note that luciferin analogues that are technology provides significant advantage. This
present in the compound collection will interfere with reporter-gene method is compatible with screening for
the assay regardless of reagent choice. The sea pansy activity in live cells, enabling transfected cell popula-
(Renilla reniformis) luciferase enzyme, which catalyses tions to be enriched by FACS (fluorescence-activated
the oxidation of coelenterazine to coelenteramide and cell sorting). This can significantly improve the time
light (480 nm), can also be used as a reporter and has taken to generate the stable cell line, reducing reagent
been used in conjunction with the firefly enzyme as a generation times from months to weeks36,38,39. How-
means of controlling well-to-well variations or simul- ever, large-scale provision of these cell reagents is a
taneous screening of two different targets34. Recently, consideration with whole-cell functional assays, partic-
the family of luciferase enzymes has been reviewed, ularly for HTS. Although the improved sensitivity
highlighting key residues that are involved in both afforded by a reporter-gene assay can result in lower
substrate binding and bioluminescence colour35. This cell requirements than whole-cell accumulation
could provide valuable information for the develop- assays40, it is commonly recognized that significant tissue
ment of improved luciferase reagents that produce culture resources can be required. Recent studies have
stable signals at alternative wavelengths, maximizing highlighted that some cell types can be used from sus-
HTS compatibility and minimizing the potential for pension cultures, making ‘manual’ large-scale supply
compound interference. less labour intensive as well as potentially reducing
In recent years, reporter-gene systems using β-lacta- assay variability24,41. However, a major advance, partic-
mase enzymes have become very popular. β-Lactamases ularly for adherent cell assays, has been the introduc-
are a family of bacterial enzymes that catalyse hydro- tion of novel robotic systems that enable automated
lysis of β-lactam substrates in antibiotics such as peni- routine cell culture maintenance and plate preparation
cillin and cephalosporin. Like the luciferases, these (for example, SELECT Technology; The Automation
enzymes have a relatively short half-life; however, they Partnership). As the cell lines commonly used in
have several advantages due to the development of a reporter-gene assays — for example, Chinese hamster
non-toxic, cell-permeant FRET-paired fluorescent ovary cells (CHO) and human embryonic kidney cells
130 | FEBRUARY 2004 | VOLUME 3 www.nature.com/reviews/drugdisc
a The luciferase reporter-gene system
Lyse cells and
Incubate with agonist incubate with substrate Detect luminescence
Luciferin Oxyluciferin + Light
b The β-lactamase reporter-gene system Read fluorescence at
447 nm and 520 nm to detect
green and blue cells
Incubate with agonist Incubate with CCF2/AM
FRET = green
Live cells Live cells No FRET = blue
Figure 4 | Schematic representation of the luciferase and β-lactamase reporter-gene technologies. a | Cells containing
the luc gene under the control of cAMP regulatory elements (CREs) are incubated with agonist. Following this receptor activation,
increases in cAMP activate the cAMP-dependent protein kinase (PKA), which then phosphorylates the transcription factor
CRE-binding protein (CREB). Phosphorylated CREB binds to the CREs, mediates transcription and functional luciferase is created.
However, the cells need to be lysed to detect the activity of the luciferase enzyme. Following this lysis and addition of substrate,
the enzyme catalyses a mono-oxygenation reaction, which produces oxyluciferin and light (550–570 nm) that can be measured.
The light produced is naturally short-lived; however, reagents are available that prolong this lifetime, making it suitable for automated
high-throughput screening. b | Cells containing the β-lac gene under the control of cAMP regulatory elements (CREs) are
incubated with agonist, to initiate the signalling cascade. The subsequent transcription events produce functional β-lactamase
enzyme that is detected following loading of the cells with the cell-permeant, non-toxic dye CCF2/AM. This dye is formed by
adding 7-hydroxycoumarin and fluorescein fluorophores to the 7 and 3′ positions of cephalosporin. When excited with light at 409
nm, fluorescence resonance energy transfer (FRET) occurs between the two fluorophores. Consequently, the 7-hydroxycoumarin
emission is quenched and fluorescein re-emits the light at 520 nm (green). However, when the dye is cleaved by β-lactamase, the
fluorescein has negligible fluorescence but the 7-hydroxycoumarin-cephalosporin fragment emits light at 447 nm (blue). The
substrate is retained within the cell through enzymatic modification by cytoplasmic esterases and, because it is non-toxic, the cells
remain viable for prolonged periods after dye has been loaded. Furthermore, as fluorescence can be measured at each wavelength,
the ratio of blue to green cells can be calculated, minimizing the impact of well-to-well variations and compound interference.
(HEKs) — are amenable to this automated continuous confirm whether they were competitive or allosteric
culture, whole-cell screening technologies are no longer modulators. In the case of reporter-gene systems, the
so resource-demanding, and cell-based screening can presence of inducible cAMP early repressor (ICER) has
be carried out 5 days per week. also been reported to produce false-positives in some
circumstances. This transcriptional repressor has been
Additional factors in assay choice shown to cause downregulation of cAMP reporter-gene
All functional assays have the potential to identify com- activity in certain cell lines when threshold cAMP levels
pounds that do not act at the level of the receptor. For are exceeded42. However, as ICER activation will be sys-
both accumulation and reporter-gene assays, false- tem dependent and is most likely to impact on systems
positives that act on the downstream signalling cascade that are overexpressed or highly stimulated, the effects
can easily be removed by further evaluation either in an should be overcome by selection of appropriate cell
equivalent assay that uses the same cells expressing lines or optimization of the assay incubation times and
another appropriately coupled receptor (this could be a compound concentrations42.
screening target in its own right), or the host cell line41. The ability to discriminate between different agonist
Those compounds that were found to act at the level of efficacies is also a factor to be considered for all func-
the receptor would also require further downstream tional assays and is dependent on the receptor expression
ligand-binding characterization if it was necessary to level and coupling efficiency12. Reporter-gene assays have
NATURE REVIEWS | DRUG DISCOVERY VOLUME 3 | FEBRUARY 2004 | 1 3 1
a greater number of amplification steps compared to membrane-based accumulation assay can be developed,
accumulation assays, which might improve the apparent it might minimize the tissue culture resource required
coupling efficiency. This increases the potential for and could potentially minimize false-positives that
systems in which the receptor is expressed at higher would otherwise be observed as a result of downstream
levels to identify PARTIAL AGONISTS as full agonists. In accu- activity of compounds.
mulation assays, the ability to distinguish partial and full
agonists can also be limited by the particular assay tech- cAMP assays in screening campaigns
nology and is realized when converting data to absolute Important considerations for any screening campaigns
levels of cAMP. There is a nonlinear relationship between are: the assay tolerances to organic solvents such as
receptor activation and signal detection, which means dimethylsulphoxide (DMSO), the assay robustness and
that relatively large changes in absolute cAMP concen- throughput. Although system-dependent, DMSO toler-
tration can equate to small changes in read-out. This can ances in the order of 1–10% have been reported for the
mask the difference in cAMP modulation between the cAMP assay technologies described here18–20,41,44. In addi-
full and partial agonist and can also significantly affect tion, the assay robustness, as measured via the Z′
the apparent assay performance in terms of robustness20. factor47, indicates that both the accumulation and
Although it is important to highlight these considera- reporter-gene assay technologies that have been
tions, there are examples in which partial agonists can be described are suitable for single-point screening10,18–20,37,41.
identified in accumulation assays or reporter-gene Furthermore, throughput in the order of 30,000–150,000
systems, indicating that either system can be used under data points per day have been achieved using these
suitable conditions20,23,43,44. Furthermore, given the addi- technologies20,41 — numbers that are compatible with
tional signal amplification afforded by reporter-gene screening large compound files. However, there are
systems and the fact that they generally require longer other factors that might have an influence on the
incubation times to allow for the gene transcription successful identification of hits from screening cam-
event, these systems can provide a significant advantage paigns using these assay technologies that also need to
over accumulation assays for the detection of weak ago- be considered.
nists in screening campaigns, either by generating higher To detect negative regulation of adenylyl cyclase by
potency for full agonists or improving the efficacy of par- Gi-coupled GPCRs, the system must first be active. To
tial agonists28,40. Although such effects might not be achieve this, investigators have used the direct chemical
reproduced in a more physiologically relevant system, modulator of adenylyl cyclase, forskolin. The EC50 value
these compounds might still provide valuable starting for forskolin can vary dependent on the cell type used
points, which would otherwise have been missed, to and might also vary dependent on the type of assay for-
generate more efficacious and potent compounds. mat (that is, accumulation versus reporter gene)41.
Alternatively, identification of partial agonists could Therefore, it is important to determine the potency of
prove beneficial to a project if tolerance is observed with forskolin for the system of choice. In addition, forskolin
chronic dosing of a full agonist. can stimulate cells to produce changes in cAMP that are
Recently, it has also been reported that small but far greater than would be generated via receptor-
significant differences in antagonist affinity were mediated action — therefore, careful evaluation of the
observed when investigating cAMP accumulation and concentrations used is required to ensure that optimum
reporter-gene assays for the β2-adrenoceptor45. These sensitivity is obtained. It is because of this level of com-
observations were linked to the efficacy of the agonist plexity in activating the system that some investigators
used and the levels of receptor desensitization obtained prefer to use chimeric or promiscuous G-proteins to
in the two systems. These changes in affinity data seem study Gi-coupled receptors. These G-proteins enable the
to be supported by other investigators who have con- system to be switched to either the phospholipase
sidered either accumulation or reporter-gene assays, as C/Ca2+ pathway or the stimulatory adenylyl cyclase/
assessed by the affinity of propranolol in the different cAMP pathway48–50. However, such strategies also need
systems20,46. However, as there are many examples of to be used with care because these alternative G-proteins
investigators using reporter-gene assays in which data do not couple effectively to all receptor types, which can
do correlate with other assay formats, it is difficult to lead to either an altered pharmacological profile or to no
assess how generally applicable these findings are. signal being detected50,51.
Given that this is the first report of its kind and this Breakdown of cAMP via PDEs can also affect the
information is contrary to the traditional receptor phar- data that is generated in assays that measure the
macological theories, no doubt other receptor systems adenylyl cyclase pathway. For example, PDE inhibitors
will be under investigation. could appear as false-positives in a screen for agonists
Consideration of the factors discussed in the sections of a Gs-coupled GPCR. To control for such effects, the
above (summarized in TABLE 2), in conjunction with the non-selective PDE inhibitor isobutyl methyl-xanthine
specific requirements of the project, might indicate (IBMX) can be included in the buffer. Dependent on
PARTIAL AGONIST whether an accumulation or reporter-gene technology the system, this might significantly increase basal levels
An agonist that is unable to is more appropriate for a given screening campaign. For of cAMP, but might also improve the observed
induce maximal activation
of a receptor population,
example, if an agonist is required, a reporter-gene system potency of forskolin40; therefore, the impact of the
regardless of the amount of might offer additional sensitivity, thereby maximizing inclusion of IBMX on the assay sensitivity should be
compound applied. the potential of identifying activities. However, if a fully evaluated.
132 | FEBRUARY 2004 | VOLUME 3 www.nature.com/reviews/drugdisc
Table 2 | Advantages and disadvantages of accumulation and reporter-gene systems
Amplification might mean greater sensitivity to weak agonism Amplification means that the compound activity
Assays are generally cheaper than accumulation assays detected might not be physiologically relevant
Assays are compatible with standard plate readers and automation Molecular biology required
Tissue-culture resource might be less than whole-cell Misleading data might arise due to ICER or
accumulation assay receptor desensitization
Some technologies have been successful with membranes Might be less sensitive to weak agonism
that can reduce the impact of false-positives Some technologies might require a specific reader
Molecular biology not required if endogenous receptors are used Accumulation technologies are generally more
Might be more physiologically relevant than reporter systems expensive than reporters
Misleading data might arise due to large changes
in cAMP producing small changes in raw data
ICER, inducible cAMP early repressor.
The configuration of a sensitive cAMP assay is also The accumulation and reporter-gene systems high-
dependent on whether the screen aims to identify lighted here have been applied to the investigation of
agonists or antagonists of the receptor-mediated Gs- and Gi-coupled GPCRs, can be used with recom-
response. Whether the target receptor is coupled to Gi binant or non-recombinant receptors in a variety of
or Gs G-proteins, it is advisable to use agonist concen- cell types and are compatible with robust (Z′) and
trations that are submaximal (EC50–EC80) to obtain miniaturized screens in either 384 or 1,536 wells, high-
the most sensitive antagonist screens. Although at lighting that there should be an assay that is compati-
maximal and supramaximal concentrations of agonist ble with any given project requirements18,19,37,40,41,43,44.
the Z′ for the assay will probably be improved, the Compound interference is a consideration with many of
observed IC50 values of antagonists will begin to the fluorescent technologies and there are many
diverge from their affinity constants (Ki). Although advances in the methodologies described that are
this divergence can be corrected via derivations of the aimed at minimizing this (for example, red-shifted
Cheng–Prussoff equation52,53 for full dose–response flashplates and red-shifted dye labels). Although cost
evaluations of competitive antagonists, if single-point and simplicity will inevitably be drivers for many
testing is being carried out, this decrease in detected investigators to seek new methods of doing things,
activity could have a detrimental effect on the number particularly with many companies generating ever
of hits observed. Although Gs-coupled receptors rep- larger compound files, there seem to be few issues that
resent an apparently simpler system, as they do not remain to be resolved.
require pre-activation of adenylyl cyclase, data from
agonist screens for either Gs- or Gi-coupled GPCRs Outlook
needs to be interpreted with caution. For example, a Although there might be limited benefit to the investi-
test compound producing a 50% response relative to gator in any endeavours to develop cAMP detection
controls (usually a maximal concentration of stan- technologies similar to those described, real benefit
dard agonist), could represent the maximal effect of could come from those technologies that provide even
a potent partial agonist or a sub-maximal effect of a greater information about the target or the com-
less-potent full agonist. Therefore, it is essential to gener- pound’s action at that target. For example, the industry
ate dose–response curves to differentiate compounds has also seen the development of many technologies
of further interest. that enable high-content screening. Investigators can
now gain information about the size, shape and
Conclusion translocation events of their given cell line in response
There is a place for cell-based assays in HTS, with to test compounds, which provides more detailed eval-
competitive advantages being provided by the fact uation of drug–drug interactions54, effects on receptor
that they have the ability to identify more/novel com- dimerization55 and internalization/desensitization6,56,57.
pounds and to obtain additional information about Although some of these technologies are becoming
the action of a compound. However, to be successful, the truly amenable to high-throughput screening, addi-
cell-based assay technologies used need to be homo- tional information can still be gained from further
geneous, miniaturizable, readily compatible with development of these and similar technologies.
automation and must be sufficiently sensitive to For example, the complexity of cellular signalling can
detect the desired activity. The assay technologies that result in agonist-specific trafficking in which the ability
have been described here are compatible with HTS to discriminate the actions of different compounds on
campaigns, being miniaturizable to 384-well formats these pathways could be important in a given disease58.
and fully automation compatible. Most are also non- Detection of multiple pathways can be achieved in HTS,
radiometric, thereby providing additional safety benefit. with the temporal difference in a calcium mobilization
NATURE REVIEWS | DRUG DISCOVERY VOLUME 3 | FEBRUARY 2004 | 1 3 3
assay and a cAMP reporter-gene assay enabling con- nucleotide gated ion channel (CNG), resulting in a sys-
current analysis of a phospholipase-C- and adenylyl- tem that is capable of real-time kinetic measurement of
cyclase-mediated signal using FLIPR and TopCount cAMP in live cells. High-content screening systems
instruments, respectively 59. However, this is not simulta- similar to this that could be combined to achieve high-
neous detection of two pathways, and the concurrent throughput simultaneous detection and separation of
processing that is required affects the throughput that multiple signalling pathways, preferably without the
can be achieved. However, a variety of fluorescent tools demand for extensive molecular biology or use of large
or biosensors are available for the study of signalling tags, could provide the additional benefit that investiga-
networks60. Of particular interest in this respect is a tors require. They would not only provide benefit for
novel cAMP biosensor system that has been developed known receptor screening programmes, but might
by Atto Bioscience (see online links box). This technology also prove more appropriate for the investigation of
combines the use of a cAMP sensor, similar to those used orphan receptors compared with current techniques
for Ca2+ assays, and the expression of a modified cyclic that generally use promiscuous G-proteins.
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