Intracellular Calcium Assays

The cell-based calcium flux assay is considered one of the most important screening techniques used in pharmaceutical drug discovery. Gq-coupled receptor-mediated increases in intracellular Ca2+ release can be measured by calcium-sensitive fluorescent dyes, bioluminescent indicators, or reporter gene assays. Gq-coupled receptor activation can elevate a transient increase in intracellular calcium to 400 to 1000 nM from a basal cytosolic level of 10 to 100 nM. Most common calcium-sensitive dyes such as Fluo-3 and Fluo-4 (Invitrogen) used in HTS are single wavelength compounds that result in a 100-fold increase in fluorescence when bound to calcium.

The increase in intracellular calcium is measured by FLIPR® technology (Molecular Devices Corporation, Sunnyvale, California). FLIPR was designed to perform homogeneous, kinetic, cell-based fluorometric assays for both adherent and nonadherent cell lines. FLIPR is equipped with an argon ion laser that excites a fluorescent dye. A cooled CCD camera images the entire plate and detects the emitted light. The device has the advantages of delivering compounds to 96- or 384-well plates and integrates data signals over a time interval.5960 Other readers available to measure calcium transients include the ImageTrack (PerkinElmer) and FDSS 6000 (Hamamatsu Photonics, Hamamatsu, Japan). The advantages and disadvantages of each instrument should be carefully evaluated before an investment is made in these expensive instruments.

Typically, cells are seeded in clear-bottom black plates overnight. It is often necessary to seed weakly adherent cells into poly-D-lysine-coated plates to improve adherence and minimize peeling off of cells during reagent addition. The cells expressing the GPCR of interest are loaded with the acetoxymethyl ester of Fluo-4 (or Fluo-3, its analog) followed by repeated washings to remove the extracellular dye. Upon cellular uptake, the acetoxymethyl ester will be cleaved by esterases to liberate Fluo-4. Upon binding of calcium, the fluorescence of Fluo-4 increases, which can be detected at excitation and emission wavelengths of 488 nm and 520 nm, respectively. The increase in fluorescent response is directly correlated with the increase in cytosolic calcium levels. In FLIPR, the emission wavelength is limited to 530. The availability of a next generation of FLIPR, FLIPR TETRA, with tunable wavelength, enables the utilization of dyes that emit in the red region for calcium assays. This new FLIPR is also equipped with liquid handlers for 1536-well plates.61

Typically in an agonist assay, data are recorded for every 1 sec for 1 min, followed by a 5-sec exposure for another 2 min. GPCR-induced calcium responses reach a peak in less than 20 sec and the duration of the response will be finished by 3 min. For an antagonist assay, cells are loaded with compounds followed by the addition of agonists in FLIPR. Some cells are serum sensitive, resulting in fluctua tions of intracellular calcium that may interfere with the results. Similarly in an antagonist assay, if the agonist of choice is present in the serum, it could change the affinity of the agonist. For example, LPA is present in serum in micromolar range. Growing cells in low serum media or media containing heat-inactivated serum would help to achieve full efficacy and affinity of LPA to endothelial differentiation gene receptor (Gopalakrishnan et al., unpublished observations).

Cells containing anionic transporters (e.g., CHO cells) should be loaded with probenecid to prevent pumping out of the dye during and after loading. FLIPR kits such as calcium assay and the calcium 3 assay from Molecular Devices allow for a homogeneous format by eliminating washing steps associated with the Fluo-3/Fluo-4 format, resulting in easier automation and faster throughput.

In the new calcium kits, the background fluorescence is reduced by the addition of a dye that absorbs the excitation light of the fluorescent dye and/or its emission light without affecting the fluorescence readout from the cells. This eliminates background fluorescence, and the signal evoked from the cells can be detected with higher resolution. A researcher should carefully examine the utilities of different dyes for a particular target because these dyes could exhibit altered pharmacologic profiles with a target. For chemokine receptors and other growth factor receptor targets, calcium 3 assay kits resulted in robust signals and are preferred over Fluo-4 and calcium assay kits.

Intracellular calcium assays offer several advantages for HTS, including the affinity and efficacy of compounds. They can also distinguish full agonist, partial agonists, and antagonists in a single assay setup. However, the characterization of the potencies of agonists and antagonists could be complicated due to the transient nature of the functional response. Activation of a Gq protein-coupled receptor revealed transient kinetics and the maximal response was observed in fewer than 20 sec, which could lead to a nonequilibrium condition during assessment of the potency of an antagonist. Therefore, a competitive antagonist could exhibit a pseudo-non-competitive behavior if incubated with the cells before adding agonist. Kaler et al.62 reported significant differences in the potencies of some of the serotonin antagonists in HEK-5HT2A cells and -5HT2C cells in co-addition of antagonists along with agonist versus preincubation of antagonists with the cells using an automated flow-through fluorescence analysis system (similar to FLIPR technology in many aspects). A fast binding antagonist displays similar affinity in both addition formats; a slow binding antagonist exhibits a lower potency in co-addition compared to pre-addition. Furthermore, spiperone exhibited a classical competitive behavior with serotonin receptors in a co-addition procedure versus a noncompetitive behavior in a preincu-bation format. Therefore, a detailed experimental setup is needed for mechanistic evaluation of agonist-antagonist interactions in intracellular calcium assays.

GPCRs coupled to Gas or Gai/o proteins can be linked to PLCp to generate an increase in intracellular calcium when coexpressed with chimeric Gaqi/o5 or Gaqs5 proteins. These chimeric proteins can be generated by replacement of five C-terminal amino acids of Gaq with the corresponding Gai/o or Gas residues.63 The receptors do not activate all the chimeric G proteins equally and therefore it is important to test multiple chimeras to determine the best system for the target of interest. Alternatively, receptors can be coexpressed with certain promiscuous G proteins (i.e., Ga15,

Ga16) that are capable of interacting with a variety of Gs-, Gi/o-, and Gq-coupled receptors and signaling through calcium release.64

The promiscuous Ga16 G-protein and the chimeric G proteins are broadly used tools for setting up robust assays for HTS.63-65 Many orphan receptors have been deorphanized through a Ga16 approach.66 The ability of Ga16 to function as a universal adapter protein for GPCRs by coupling to PLCp allows a large number of receptors to generate an increase in intracellular calcium independent of the second messenger pathway normally modulated by the receptor. Therefore, the receptor of interest and the other endogenous receptors present in the parental cell line could couple to Ga16.

One difficulty with using a cell line containing Ga16 for an HTS campaign is this artificial coupling of other nontarget endogenous receptors through Ga16. All the compounds that interact with nontarget receptors in cells will be identified as positives in the primary screening. Thus, testing all the positives in the parental cell line with Ga16 is critical for eliminating the false positives. Again, not all GPCRs can be forced to couple to Ga15 or Ga16. For example, it has been reported that receptors such as dopamine D3, angiotensin ATII, somatostatin SST1, muscarinic acetylcholine M1, tachykinin NK2, melatonin MT1C, CCR1 and CCR2, and adrenergic a1C and a1D do not couple efficiently to Ga16.68-70 Liu et al. reported the application of a Ga16/z chimera to Gi-coupled receptors for higher sensitivity and promiscuity compared to Ga16. The Ga16/z chimera was constructed by replacing 25 or 44 amino acids of the C-terminal domain of Ga16 with those of Gaz. These chimeras were found to be more sensitive for Gi-coupled receptors, although their ability to link Gs proteins to PLCp and calcium was diminished.71 Again, researchers should carefully consider the chimeric/promiscuous Ga16 approach because it may alter the pharmacology profiles of agonists and antagonists.

Recent reports demonstrate that the magnitudes of agonist responses can be improved dramatically by performing the assay at elevated temperature instead of at room temperature. Wong et al. reported the enhanced responses of many neuronal receptors such as a1A and a2A adrenergic, histamine H1, serotonin 5HT1A and 5HT2A, and dopamine D2 and D3 at higher temperatures when coexpressed with Ga15 to make it amenable for calcium assay format.72

Other instruments beside FLIPR have fully integrated robotic capabilities with 96- or 384-well injectors for measuring kinetic readouts for calcium assays including the FDSS of Hamamatsu and the ImageTrack of PerkinElmer. During the last decade, the sizes of corporate screening libraries have increased tremendously, in essence forcing researchers to miniaturize their assays from 96- to 384- to 1536- or 3456-well plates.

Scientists at Abbott reported a microarrayed compound screening format (||ARCS) for identifying agonists for a GPCR.73 In this format, 8,640 discrete compounds are spotted and dried onto a polystyrene sheet that occupies the same footprint as a 96-well plate. The cells expressing the receptor of interest were preloaded with Fluo-4, cast into a 1% agarose gel, placed above the compound sheets, and imaged successively using a CCD system. In addition to the ||ARCS format, gene reporter assays can be employed to increase the throughputs of calcium assays, and p-lactamase assays have been miniaturized to 3456-well plates. One advantage of gene reporter assays over calcium fluorophores is their ability to detect weak calcium signals from GPCRs either because the signal is amplified or the reporter gene construct can be activated through the PKC pathway. Indeed, Gq-mediated activation of PLCp leads to generation of two types of second messengers: inositol-triphosphate (IP3) will stimulate calcium release, whereas diacylglycerol will stimulate PKC activity which activates NFAT transcription through calcineurin.

Intracellular calcium can also be measured using a bioluminescent indicator known as aequorin. The aequorin complex consists of the 22,000-dalton apoaequorin protein, molecular oxygen, and a coelenterazine luminophore. When calcium binds to this complex, the coelenterazine is oxidized to coelenteramide resulting in the release of carbon dioxide and blue light at 468 nm. The utilization of aequorin for GPCRs has been characterized in detail by Ungrin et al.74 Euroscreen has patented aequorin-based cell lines for GPCR screening (AequoScreen); the receptor of interest is expressed in a cell line coexpressed with apoaequorin. The promiscuity of Ga16 is well utilized in the aequorin-based assays to improve the coupling to calcium for both adherent and nonadherent cells. Typically, cells are loaded with coelenterazine for at least 3 h. During the incubation step, coelenterazine enters the cells and conjugates with apoaequorin to form aequorin, which is the active form of the enzyme. Upon receptor activation, the light is emitted for 20 to 30 sec. Conventional luminometers or instruments such as the Lumax (CyBio AG, Jena, Germany) and FDSS 6000 can be used to measure the luminescent signal. This functional screening assay has been tested with several GPCRs. The potency of agonists is similar to those obtained from radioligand binding and/or other functional assays. Unlike fluorescent calcium indicators, Ca2+-bound aequorin can be detected without illuminating the sample, thereby eliminating the interference due to autofluorescence from the screening compounds.

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