Antibody Dependent cAMP Detection Assays

These assays are based on the competition between the cellular cAMP and the labeled cAMP with the anti-cAMP antibody. To achieve the best results possible from cAMP assays, it is essential to determine the optimal number of cells per well. The optimal cell concentration should produce a relatively low basal level of cAMP, which will yield a good response to a relatively low concentration of stimulator. When evaluating the inhibitory compounds, the level of stimulator will be as low as possible to maximize the sensitivity of the assay. Homogeneous formats are preferred in HTS due to higher throughput and ease of automation. These kits can determine cAMP levels in cell supernatants in microtiter plate formats using adherent or suspension cells; some of them even work in cell membranes. The results for suspension cells are generally more reproducible because each well contains approximately the same number of cells. All the kits include cAMP standards. A concentration response curve for the cAMP standard is performed to calculate the amounts of cAMP in the unknown samples. Plotting the bound fraction (B/B0) as a function of log cAMP concentration generates a standard curve. There is no linear relationship between the raw data and the amount of cAMP produced. Therefore, one could anticipate a shift in the affinities of compounds based on the analysis criteria (whether the researcher is analyzing data based on raw signal or converted cAMP levels). cAMP assays are typically performed in the presence of a phosphodiesterase inhibitor, IBMX, which prevents the breakdown of cAMP. It also prevents the identification of PDE inhibitors as false positives in a screen for a Gs-coupled receptor agonist.

9.5.2.2.1 Homogeneous Radiometric Assays

Radioactive assays such as PerkinElmer FlashPlates4041 and Biotrack cAMP kits from Amersham4243 are widely used HTS formats (see Section 9.4.1). The FlashPlate technology utilizes a FlashPlate coated with anti-cAMP antibodies that are blocked to avoid nonspecific binding. Cells from tissue culture flasks are directly placed in 96- or 384-well plates and treated with compound or buffer for 30 min. The cells are lysed with lysis buffer containing [125I] cAMP tracer and incubated for 3 h. cAMP bound to the antibody can be detected in close proximity to the scintillation surface on the flash plate. The Biotrack cAMP kit is a scintillation proximity assay that involves the addition of SPA beads, anti-cAMP antibody, and [125I] cAMP tracer to lysed cell samples. The sensitivity of the assay could be improved by acetylation of standards and unknowns prior to assay. Although the Biotrack is an easy method, it can generate false positives caused by color quenching.

9.5.2.2.2 Fluorescent Polarization Assays

Fluorescence polarization (FP) cAMP assays (PerkinElmer and Amersham Biosciences) rely on measurements of the parallel and perpendicular components of fluorescence emission from a fluorescently tagged cAMP molecule such as fluorescein to the plane of a polarized excitation source.44-46 The rotation speed of the labeled cAMP is reduced and therefore possesses a higher polarization value when it is bound to antibody. In the presence of cellular cAMP, labeled cAMP is free in solution and a lower polarization value is observed. An advantage of the FP format is that it enables the utilization of membranes instead of whole cells for cAMP assay. One major disadvantage of the format is the potential interference from compounds as discussed earlier. Alternate probes such as BODIPY TMR, Alexa 647 (PerkinElmer), MR 121, Evoblue, Cy3, and Cy5 could eliminate these artifacts. For example, the Biotrak cAMP FP immunoassay system utilizes Cya3B, a bright version of the standard Cy3 fluorescent dye conjugated to cAMP, to generate a signal window twice that achievable with conventional dyes such as fluorescein.46

9.5.2.2.3 Time-Resolved Fluorescence Assays

The HTRF® cAMP assay (Cis-Bio International, France) is based on the interaction of europium cryptate anti-cAMP antibody and a modified allophyocyanin-labeled cAMP (XL665).4748 In the absence of cellular cAMP, these two fluorescent molecules are in close proximity to allow fluorescence resonance energy transfer (FRET) and the XL665 emits a long-lived fluorescence at 665 nm. In the presence of cellular cAMP, XL665 will be displaced and will not be in close proximity to the europiumlabeled antibody to allow FRET; only emission from the europium will be detected. Because the signal is measured as a ratio of europium emission at 620 nm and XL665 emission at 665 nm (620/665), the assay is unaffected by colored compounds.

9.5.2.2.4 AlphaScreen" (Amplified Luminescent Proximity Assay)

The PerkinElmer AlphaScreen is an assay based on the competition between cellular cAMP and biotinylated cAMP.49 Anti-cAMP antibody-coated acceptor beads capture biotinylated cAMP to form a sandwich with streptavidin-coated donor beads that bring donor and acceptor beads into close proximity. Upon laser excitation, a photo-sensitizer in the streptavidin-coated donor bead converts oxygen to a more excited singlet state. The singlet state oxygen molecules react with thioxene derivative in the anti-cAMP-conjugated acceptor beads, generating a highly amplified signal in the 520- to 620-nm range. The beads are sensitive to temperature and light fluctuations and the experiments should be performed with caution.

9.5.2.2.5 Enzyme Complementation Technology

The HitHunter® assay from DiscoveRx (Sunnyvale, California) utilizes p-galactosi-dase fragment complementation. Inactive fragments, enzyme acceptor (EA), and enzyme donor (ED) complement to form an active enzyme. In the EFC-cAMP (enzyme fragment complementation) assay, the p-galactosidase donor fragmentcAMP (ED-cAMP) conjugate binds with the EA fragment to form an active p-galactosidase enzyme. Binding of ED-cAMP conjugate to the anti-cAMP antibody prevents its complementation with the EA fragment to form the active enzyme. The amount of p-galactosidase formed is thus proportional to the cAMP concentration in the cell lysate. Enzyme activity is subsequently detected using a chemiluminescent or fluorescent substrate.50,51 This technology is less prone to compound artifacts and easily amenable to miniaturization and automation.

9.5.2.2.6 Electrochemiluminescence Technology

Electrochemiluminescence technology available from MesoScale Discovery (Gaith-ersburg, Maryland) uses cAMP labeled with a ruthenium derivative. In the absence of cellular cAMP, labeled cAMP is bound by an anti-cAMP antibody on the surface of a disposable carbon electrode. When a potential is applied to the electrode, labeled cAMP in close proximity to the electrode emits light. One disadvantage of the MesoScale technology is that the plate cannot be read more than once due to significant reduction in signal. Also, the limited availability of specifically designed instruments to read this technology has hindered its acceptance in the HTS field.

All the homogeneous methods offer the advantages of easy automation and feasibility for a robust HTS screen. However, the sensitivity of the assay to detect cAMP can vary from one technology to another and a researcher must select a format based on the level of sensitivity needed and the automation and miniaturization capabilities available in his or her laboratory. The nonhomogeneous formats such as the DELFIA® assay of PerkinElmer, the chemiluminescence assay of PE Biosystem, and the Catchpoint cAMP kit from Molecular Devices (Sunnyvale, California) are also in limited use, primarily because of the washing steps that cannot be easily automated. Compounds that interact with the cAMP antibody can appear as false positives in all the cAMP detection assays. However, performing the assay in the absence of cells can easily identify these false positives.

9.5.2.3 Antibody-Free Methods for Measuring cAMP

9.5.2.3.1 Reporter Gene Assays

The most common reporter genes utilized in assays for cAMP are based on the expression of p-lactamase or luciferase. These assays are based on a reporter construct consisting of promoter and reporter genes that are stably incorporated into the genome of the cell (or transiently transfected). The activity of the reporter gene is controlled by the transcriptional regulation of the promoter element, which is activated directly by the receptor present on the cell membrane. For example, receptors coupling to Gs activation have usually been monitored with reporter constructs containing cAMP-responsive elements (CREs) Upon stimulation of the Gs-coupled receptors, the elevated levels of cAMP lead to activation of protein kinase A (PKA).

The catalytic subunit of PKA translocates to the nucleus where it phosphorylates and activates the cAMP response element binding protein (CREB) bound to an upstream CRE.52,53 Reporter gene assays are well suited for high-throughput screening approaches in various assay formats investigating full or partial agonists, antagonists, or inverse agonists.

Other reporter systems available include p-galactosidase, ^-glucuronidase (GUS), secreted alkaline phosphatase (SEAP), human growth hormone (hGH), green fluorescent protein (GFP), and chloramphenicol acetyl transferase (CAT). All these reporter gene assays have inherent advantages and disadvantages.54

Overall, reporter gene assays are comparable to second messenger assays in sensitivity but are typically much more amenable for HTS. In order to analyze reporter gene activity at a single cell level, p-galactosidase and p-lactamase have been used.55 The p-lactamase assay is simple and rapid and does not pose any background problems due to the lack of endogenous p-lactamase activity in mammalian cells. p-Lactamase assays based on the calcium-sensitive transcription factor, nuclear factor of activated T-cell (NFAT), are used to study Gaq, Gaq chimeric proteins, and promiscuous Ga15/16 G-proteins. p-Lactamase assays utilize the fluorescent probe CCF2/4, which is composed of two fluorescent dyes, 7-hydroxycou-marin-3-carboxamide and fluorescein. Once CCF2/4-AM enters the cell, endogenous esterases cleave the ester to CCF2/4, which produces a green fluorescence due to the FRET at an emission wavelength of fluorescein at 530 nm. This provides the additional benefit of detecting cytotoxic compounds that inhibit esterase activity in cells. A lower fluorescence will be observed because the ester groups quench the fluorescence of CCF2/4.

Cleavage of CCF2/4 by p-lactamase causes the loss of FRET, resulting in blue fluorescence (l = 460 nm). The signal is detected by using the ratio of two fluorescence wavelengths, rather than absolute changes in the fluorescence signal. This significantly reduces assay variability. Other substrates for p-lactamase are being generated to provide additional flexibility.56

GPCRs transduce signals from cell surfaces to intracellular effectors through various G proteins. As with other G proteins, a reporter gene assay could be used to characterize the coupling of GPCRs to G12/13. The GPCR under the control of a modified serum-responsive element (SRE) is transfected into a cell lacking Gq/11 and the agonist-induced activation of GPCR is measured using a luciferase assay.57 A longer incubation period is generally required to produce transcriptional regulation of the gene, which will help the detection of weak agonists in the screen. On the other hand, longer incubation times may lead to unmasking the inherent toxicity of the compounds. A potential interaction with other steps in the signaling cascade can also result in the generation of false positives in the screen.

9.5.2.4 cAMP Biosensor

A cAMP biosensor HTS platform known as ACT: One® is available from Atto Biosciences (Rockville, Maryland) to detect cAMP in live cells. The GPCR of

TABLE 9.2A

Functional GPCR Assays in High Throughput Screening

Compound

CTP Binding

Radioactive

Homogeneous

Interference

[35S]-GTPyS

Y

Y/N

Medium

Eu-GTP

N

N

Low

cAMP

Y

Y

Y

FlashPlate

Y

Y

Medium

BioTrack

Y

Y

Medium

Fluorescence polarization

N

Y

Low-high

TRF

N

N

Low

AlphaScreen

N

Y

Medium

HTRF

N

Y

Low-medium

HitHunter

N

Y

Low

ECL

N

Y

Medium

(3-Lactamase reporter

N

Y

Low-high

Luciferase reporter

N

Y

Low-medium

cAMP sensor

N

Y

Medium

Cells or

Throughput Membranes? Deorphanizing

Medium

M

N

Low

M

N

Y

C

or M

Y

Medium

C

+ M

N

Medium

C

+ M

N

High

C

+ M

N

High

C

+ M

N

Medium

C

+ M

N

High

C

+ M

N

High

C

+ M

N

High

C

+ M

N

High

N

Y

Medium-high

N

Y

Low

N

TABLE 9.2B

Functional GPCR Assays in High Throughput Screening

Compound

Ca2+ Assay

Radioactive

Homogeneous

Interference

Fluorophore

N

Y/N

Medium

(3-Lactamase reporter

N

Y

Low-high

Luciferase reporter

N

Y

Low-medium

Aequorin

N

Y

Low

IP3 assay

Y

Y

Y

AlphaScreen

N

Y

Medium

HitHunter

N

Y

Low

Amersham

Y

N

Medium

Desensitization

Y

Y

Y

Transfluor

N

Y

High

BRET

N

Y

Medium

CypHer

N

Y/N

High

Other

Y

Y

Y

Microphysiometer

N

N

Low

Melanophore

N

Y

Medium

Cells or

Medium

C

Y

High

C

Y

High

C

Y

Medium

C

Y

Y

C or M

Y

Medium

C

N

High

C

N

Low

C

N

Y

C or M

Y

Low

C

Y

Medium

C

N

Medium

C

N

Y

C or M

Y

Low

C

Y

High

C

O c era

Membranes? Deorphanizing Íd

t/l interest is stably expressed in a cell line coexpressing cyclic nucleotide gated (CNG) channels that act as cAMP sensors targeted to plasma membranes and colocalized with AC to permit sensitive detection of a local cAMP rise. A change in cAMP level can be detected using either calcium-sensitive or membrane potential dyes.58 This platform is applicable to Gs- and Gi-coupled receptors. This technology allows screening of receptors not coupled by chimeric and promiscuous G proteins.

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