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                              cAMP DETECTION METHODS
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                            Agonist




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                                   Absence of cellular cAMP                                                     ...
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                            Absence of cellular cAMP                                                          Pr...
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                                   and facilitates discrimination of the fluorescent signal      interference, a...
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Table 1 | Summary of important parameters to consider in selection of an accumulation assay technology
Parameter...
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                        a The luciferase reporter-gene system


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                               a greater number of amplification steps compared to              membrane-based a...
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                         Table 2 | Advantages and disadvantages of accumulation and reporter-gene systems
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                                            assay and a cAMP reporter-gene assay enabling con-                  ...
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56. Conway, B. R. et al. Quantification of G-protein coupled       62. Milligan, G. Principles: extending the ut...
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C Amp Detection Methods In Hts

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C Amp Detection Methods In Hts

  1. 1. REVIEWS cAMP DETECTION METHODS IN HTS: SELECTING THE BEST FROM THE REST Christine Williams 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. ALLOSTERIC MODULATOR 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. e-mail: chris_williams@ 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
  2. 2. REVIEWS Agonist γ γ α GDP β α GTP + β a 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
  3. 3. REVIEWS Absence of cellular cAMP Presence of cellular cAMP a [125I]– cAMP cAMP + cAMP –[125I] Scintillant coated plate Light b Slow rotation Fast rotation cAMP + cAMP cAMP Produces more polarized light following excitation Produces less polarized light following excitation c Long lifetime Long lifetime fluorescence fluorescence at 665 nm at 620 nm cAMP cAMP + cAMP Long lifetime fluorescence at 620 nm FRET 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
  4. 4. REVIEWS Absence of cellular cAMP Presence of cellular cAMP a Oxygen radical Luminescence Acceptor No signal bead cAMP + Biotin Acceptor Donor Donor cAMP bead Streptavidin Streptavidin Biotin bead bead cAMP b Enzyme donor Fluorescent/ luminescent product cAMP cAMP + + cAMP cAMP Enzyme acceptor Active enzyme c Light Chemical No light cAMP cAMP cAMP Carbon electrode plate Electrical 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
  5. 5. REVIEWS 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 COLOUR QUENCH 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
  6. 6. REVIEWS 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 (including cells) 1,536 compatibility Not for Flashplate Yes Yes Yes Yes Yes Detection limit ~100 50–100 <10 ~10 ~10 ~10 (fmol/384 well) 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
  7. 7. REVIEWS a The luciferase reporter-gene system Lyse cells and Incubate with agonist incubate with substrate Detect luminescence produced from substrate conversion Luciferase Dead cells Live cells 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
  8. 8. REVIEWS 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
  9. 9. REVIEWS Table 2 | Advantages and disadvantages of accumulation and reporter-gene systems Advantages Disadvantages Reporter-gene system 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 Accumulation system 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
  10. 10. REVIEWS 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. 1. Langley, J. N. On nerve endings and on special excitable A good example of a thorough evaluation of a 39. Whitney, M. et al. A genome-wide functional assay of substances in cells. Proc. Roy. Soc. B78, 170–194 (1906). cAMP accumulation assay. Highlights some key signal transduction in living mammalian cells. Nature 2. Ehrlich, P. Chemotherapeutics: scientific principles, methods considerations for accumulation technologies in Biotech. 16, 1329–1333 (1998). and results. Lancet 2, 445–451 (1913). general, in addition to specific aspects of the use 40. George, S. E. Evaluation of a CRE-directed luciferase 3. Bleicher, K. H. et al. Hit and lead generation: beyond of membranes and the fluorescence polarization reporter gene assay as an alternative to measuring cAMP high-throughput screening. Nature Rev. Drug Disc. 2, technology in these assays. accumulation. J. Biomol. Screen. 2, 235–240 (1997). 369–378 (2003). 21. Banks, P. et al. Impact of a red-shifted dye label for high An early but important study highlighting some of 4. Hopkins, A. L. & Groom, C. R. The druggable genome. throughput fluorescence polarisation assays of G-protein the benefits and pitfalls in using reporter-gene Nature Rev. Drug. Disc. 1, 727–730 (2002). coupled receptors. J. Biomol. Screen. 5, 329–334 (2000). assays. Specifically, it directly compares 5. Sittampalam, G. S. et al. High-throughput screening: 22. Packard Bioscience. Whole cell cAMP Functional Assay accumulation and reporter-gene assays for the advances in assay technologies. Curr. Opin. Chem. Biol. 13, (Technical Note, AN002–ASc). (Packard Instrument study of agonism at Gi- and Gs-coupled receptors 384–391 (1997). Company, Meriden, Connecticut, 2000). that are expressed endogenously in CHO cells. 6. Hemmila, I. A. & Hurskainen, P. Novel detection 23. Bouchard, N. et al. cAMP alphascreen assay: a method 41. Goetz, A. S. et al. Development of a facile method for high strategies for drug discovery. Drug Disc. Today 7, for the pharmacological characterisation and screening of throughput screening with reporter gene assays. J. Biomol. S150–S156 (2002). GαI-coupled receptors in whole cells. (Perkin Elmer, Screen. 5, 377–384 (2000). 7. Walters, W. P. & Namchuk, M. Designing screens: how to Montreal, Canada, 2002). 42. Kemp, D. M. et al. The effect of ICER on screening make your hits a hit. Nature Rev. Drug. Disc. 2, 259–266 24. Golla, R. & Seethala, R. A homogeneous enzyme fragment methods involving CRE-mediated reporter gene expression. (2003). complementation cyclic AMP screen for GPCR agonists. J. Biomol. Screen. 7, 141–148 (2002). 8. Croston, G. E. Functional cell-based uHTS in chemical J. Biomol. Screen. 7, 515–525 (2002). 43. Kemp, D. M. et al. Partial agonism at serotonin 5-HT1B and genomic drug discovery. Trends Biotech. 20, 110–115 25. Packard Bioscience. Analysis of potential compound dopamine D2L receptors using a luciferase reporter gene (2002). interference of ALPHAScreen Signal (Application Note, assay. Eur. J. Pharmacol. 373, 215–222 (1999). 9. Hertzberg, R. P. & Pope, A. J. High-throughput screening: ASC–012). (Packard Bioscience Company Inc., Meriden, 44. Terstappen, G. C. et al. Development of a functional new technology for the 21st century. Curr. Opin. Chem. Biol. Connecticut, 2001). reporter gene HTS assay for the identification of mGluR7 4, 445–451 (2000). 26. Chiulli, A. C. et al. A novel high throughput chemilumi- modulators. J. Biomol. Screen. 5, 255–261 (2000). 10. Johnston, P. A. & Johnston, P. A. Cellular platforms for nescent assay for the measurement of cellular cyclic 45. Baker, J. et al. Influence of agonist efficacy and receptor HTS: three case studies. Drug Disc. Today 7, 353–363 adenosine monophosphate levels. J. Biomol. Screen. 5, phosphorylation on antagonist affinity measurements: (2002). 239–247 (2000). differences between second messenger and reporter gene Highlights some of the benefits and challenges of 27. Scott, M. G. H. et al. Effects of a range of β2-adrenoceptor responses. Mol. Pharmacol. 64, 679–688 (2003). using cell-based assays in screening campaigns by agonists on changes in intracellular cyclic AMP and on cyclic A recent paper comparing antagonist data providing data from three functional assays that AMP driven gene expression in cultured human airway generated via accumulation and reporter-gene have been used in HTS. Specifically, it discusses a smooth muscle cells. Br. J. Pharmacol. 128, 721–729 (1999). technologies with a recombinantly expressed luciferase reporter-gene assay and a FLIPR Ca2+ 28. Hill, S. J. et al. Reporter-gene systems for the study of Gs-coupled receptor. It highlights that assay for GPCR targets as well as a radioligand- G-protein-coupled receptors. Curr. Opin. Pharmacol. 1, desensitization might be an important factor uptake assay for a transporter target. 526–532 (2001). influencing the data observed. 11. Christopoulos, A. Allosteric binding sites on cell-surface 29. Wood, K. V. Marker proteins for gene expression. Curr. Opin. 46. Nagmani, R. Evaluation of beta-adrenergic receptor receptors: novel targets for drug discovery. Nature Rev. Drug Biotechnol. 6, 50–58 (1995). subtypes in the human prostate cancer cell line-LNCaP. Disc. 1, 198–210 (2002). 30. Southward, C. M. & Surett, M. G. The dynamic microbe: Biochem. Pharmacol. 65, 1489–1494 (2003). 12. Kenakin, T. 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