Euroscreen S.A.

Our assays are using recombinant cell lines stably transfected with plasmids encoding apoaequorin, the GPCR of interest and if necessary, a recombinant G protein to redirect the coupling of the GPCR toward calcium signaling. After clonal selection the best clones are selected on the basis of pharmacological characterization, high signal to background ratio and stability of expression. Euroscreen has developed this method for performing high throughput functional screening of GPCRs (Aequorin assay).

Aequorin assay has been successfully applied for high throughput screening of agonists, antagonists and allosteric modulators for different families of GPCRs as well as for use in deorphanization programs. It is also very useful for molecular pharmacology, profiling and custom assay development such as for the testing of functional antibodies.  

Aequorin is a photoprotein isolated from luminescent jellyfish (like various Aequorea species e.g. Aequorea victoria). Aequorin is composed of two distinct units, the apoprotein apoaequorin, with an approximate molecular weight of 22 kDa, and the prosthetic group, coelenterazine, responsible for emission of light.

In the presence of molecular oxygen the two components of aequorin assemble spontaneously, forming the functional protein. Four EF-hand type regions have been identified in the structure of Aequorin and at least 3 of them function as binding sites for Ca+2 ions: Ca+2 binding to these EF hands triggers a conformational change of the protein, that leads it to oxidize its prosthetic group, coelenterazine, into excited coelenteramide and CO2.

As the excited coelenteramide relaxes to the ground state, blue light (wavelength = 469 nm) is emitted and can be measured by a luminometer. Cultured cells expressing the aequorin gene can effectively synthesize aequorin in a stable or transient way, however recombinant expression only yields the apoprotein, therefore it is necessary to add coelenterazine into the culture medium of the cells to obtain a functional protein and subsequently use its blue light emission to measure intracellular Ca+2 concentration. Coelenterazine is a hydrophobic molecule, and therefore is easily taken up across the plasma membrane of higher eukaryotes, making aequorin suitable as a Ca+2 reporter in mammalian cells.

The advantages of aequorin-based functional assays

1) Luminescent signal. Aequorin has a number of advantages over other Ca+2 indicators: Coelenterazine alone does not emit light. Moreover the light emitted by the oxidation of coelenterazine does not depend on any optical excitation, so there are no problems associated with auto-luminescence (as for auto-fluorescence) when using aequorin.

2) Wide dynamic range. The sensitivity range of aequorin is well suited for measuring [Ca+2] between 50 nM and 50 µM, corresponding to the calcium concentrations found in vivo. The wide dynamic range, together with the low buffering effect (see below), makes the photoprotein the tool of choice when it is desirable to estimate quantitatively the large [Ca+2] increases that occur in some cell types.

3) High signal-to-noise ratio. As mammalian cells are not naturally endowed with chemiluminescent proteins, the background of aequorin measurements is close to zero. Moreover, the steep relationship between the increases in light emission and Ca+2 concentration (see above) accounts for the very large luminescence peaks observed upon stimulation of cells. For an average level of transfection, a signal to background ratio over 50 is easily obtained both in 96 and 384 well format.

4) Low Ca+2 buffering effect. Thanks to the excellent signal to noise ratio, reliable aequorin measurements can be obtained with moderate levels of expression of the aequorin probe (i.e. <1 µM). In addition, aequorin has a low affinity for Ca+2 ions (Ca+2 association constant = 7.106 M-1). Thus, though in principle all Ca+2 probes perturb Ca+2 homeostasis because they bind Ca+2 and thus act as Ca+2 buffers, this effect is much less relevant for aequorin than for the trappable fluorescent dyes (typically loaded at concentrations of >50 µM, i.e. >100 times higher than recombinant aequorin).

5) Low cost: 10 times less expensive reagents than for a luciferase assay or FLIPR assay.

6) Universal coupling to calcium for most of the receptors through co-expression of an adequate signaling protein. This allows screening of a target without previous knowledge of the specific signaling pathway and/or without the previous knowledge of its natural ligand (i.e. good success for screening of orphan receptors)

7) Short exposure time of cells for agonist testing (around 30 seconds compared to several hours for transcription-based assays). This allows testing of the activity of cytotoxic compounds and avoids metabolism of the tested compounds.

8) Detection of agonists, antagonists and allosteric modulators. The screening of agonist and antagonist or allosteric modulators is performed in series with the same samples allowing to clearly characterize compounds in the primary screening.

9) The assay is homogeneous (no separation step) thus limiting the handling. Using the Aequorin assay method, a single cell preparation can be used for a full day of screening. Stability of the signal during all the day, and from day-to-day is validated before performing the screening. Moreover, the use of frozen cells in such screening is allowing to complete a screening campaign with one unique batch of cells.