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Tutorials : Sep 15, 2005 (Vol. 25, No. 16)

Casting a New Light on GPCR Targets

IP-One Fluoresence Immunoassay Useful in Investigating the PLC Pathway

The most important class of drug discovery targets today is undoubtedly the class of G-protein coupled receptors (GPCRs). These receptors account for more than 30% of all targets currently under investigation by the pharmaceutical industry, and this percentage is increasing.

GPCRs are transmembrane receptors coupled to an internal cell machinery just underneath the cell membrane, thus carrying extracellular signals to the cytoplasm. When activated by an external stimulus, such as the binding of a ligand or an agonist, they can trigger physiologically important processes.

Equally, of course, they can be activated, or their activity inhibited, by candidate drug molecules, making them highly druggable targets. They are closely associated with mood-related conditions such as obesity, high blood pressure, sleep disorders, and pain perception, as well as tumor growth and immune response.

Drug discovery scientists use high throughput screening (HTS) to simultaneously test hundreds of candidate molecules for their ability to modulate GPCR activation. They want sensitive, fast, and cost-effective assays that can indicate activation of particular GPCRs by measuring their downstream products. This is not always a straightforward task.

Cisbio international (Bagnols-sur-Cze, France) already developed an assay to detect activation of one of the most important GPCR activation cascades, the cAMP pathway. The company also developed a second assay to help investigate another key pathwaythe PLC cascade.


GPCR activation operates mainly through two major pathways (depending on which particular species of G-protein is involved). One, called the cAMP or Gs-Gi pathway, leads to the production or inhibition of a secondary messenger called cAMP (cyclic adenosine monophosphate). Activation of this pathway is relatively easy to detect because cAMP accumulates in the cell and can be assayed directly.

The other pathway, called the phospholipase C (PLC) pathway, is coupled to a different G-protein called Gq. About 50% of all GPCRs work through this pathway, so it is essential to find a suitable assay to detect their activation.

Unfortunately the secondary messengers activated by Gq are short-lived inositol phosphate (IP) compounds which are degraded by specific phosphatases before they can accumulate in the cell, and are thus hard to measure.

At the moment, most GPCR investigators are using a flash fluorescence instrumental method called FLIPR (Molecular Devices; Sunnyvale, CA) to measure Gq activation. This method detects an intracellular calcium burst that is the ultimate result of the receptor-triggered IP cascade.

However, FLIPR requires a dedicated and expensive fluorescence detector. Second, the Ca2+ burst is very remote from the original GPCR activation event, occurring a long way downstream in the cascade.

This means that a substantial number of the "hits" detected by FLIPR are false positives caused by compounds that interfere all along this signal transduction pathway or the final fluorescence detection.

Depending on assay conditions, these false hits can be as high as 1%, which is extremely large considering that typically only 0.1% of a whole library of candidates are true hits. So the hits generated by the primary FLIPR screen have to be subsequently re-screened or confirmed by another technique to eliminate the false positives.

Freezing the Cascade

The IP-One assay addresses these problems. It works by "freezing" the cascade at a particular point and then measuring the accumulated product, as follows.

Gq proteins, when triggered by a receptor, activate the plasma-membrane-bound enzyme phospholipase C-beta (PLC; Figure 1). This enzyme reacts on an phosphoinositol moiety (PIP2) in the membrane to release inositol 1,4,5-trisphosphate (IP3). This molecule is labile, quickly being transformed into two inositol phosphate subproducts, IP2 and IP1.

Part of the IP3 generated binds to specific receptors on the endoplasmic reticulum, which induce opening of calcium release channels. This quickly raises the concentration of Ca2+ ions in the cytosol, causing the burst of ionic calcium mentioned earlier.

IP3 is very challenging to measure directly because of its short life. This transience also means the ionic calcium it releases cannot be a long-lived messenger either.

However, it has been shown that incubating cells with lithium chloride blocks the final step of the cascade, the conversion of IP1 into myo-inositol. This leads to the accumulation of IP1 in the cells, which can then be detected in the resulting cell lysate by the IP-One immunoassay (Figure 2).

Screening for Inverse Agonists

IP-One quantifies the amount of IP1 accumulated in the cell, giving an exact measurement of the level of activation of the cells' Gq receptors. Cells incubated with a compound that acts as a Gq agonist will show increased IP1 accumulation, while cells incubated with a Gq antagonist or inverse agonist will show diminished IP1 accumulation.

The activity ranking of these agonists and antagonists, when measured by IP-One, has been shown to be identical to a ranking measured by a gold standard laboratory method such as affinity chromatography detection of total radiolabelled inositol phosphates (Table).

The assay is proving especially helpful in investigating a particular class of drug candidates called inverse agonists. These compounds are able to inhibit the so-called "constitutive activity" shown by certain Gq-coupled GPCRsactivity that occurs normally without the receptor needing to be triggered by any agonist.

The pharmaceutical industry has of late become extremely interested in finding such compounds. But constitutive activity often occurs at such a low level that it is challenging to detect using calcium sensors. In many cases, though, IP-One is sensitive enough to measure inverse agonist activity.


The immunoassay is based on Cisbio's homogeneous time-resolved fluorescence (HTRF) technology. It necessitated the design of two custom reagents. One is an IP1 analogue, synthesized by grafting a linker to IP1 so that it could be coupled to a fluorescence acceptor, or to a protein carrier for animal immunization.

The second reagent is based on a monoclonal antibody (Mab), extremely specific to IP1, raised after mice immunization with the IP1 derivative. Importantly, this Mab shows no cross-reactivity with either IP2 or IP3, or any other phosphoinositol-related compounds, including myo-inositol.

For the assay, this Mab is conjugated with the fluorescence donor europium cryptate. This Mab-coupled donor molecule binds to acceptor-coupled IP1 analogue molecules with very high affinity and specificity, triggering fluorescent emission through the fluorescence resonance energy transfer (FRET) mechanism.

The assay itself is a classical competitive immunoassay, based on HTRF. The presence of free IP1 in the cell lysate will reduce the fluorescence signal by preventing some of the IP1-coupled reagent from binding with the Mab-coupled donor. Thus, the amount of signal detected is directly related to the amount of free IP1 in the cell. The signal-to-noise ratio is estimated at about 10, sufficient for the precision of the method, while the assay's IC50 using IP1 standard lies around 500 nM.

The TR-FRET readout is known to give better sensitivity and resistance to interferences than other technologies such as fluorescence polarization or fluorescence intensity. The HTRF method also has the advantage that it is not affected by the innate optical properties of the candidate compounds being screened.

This is achieved by rationing the FRET signal against the innate fluorescence of the donor molecule, which thus acts as an internal calibration standard. This is very important in high throughput screening, in which thousands of compounds with different optical properties are being tested at once.

Global Platform for GPCR Investigation

The IP1 quantification takes only one hour, and requires only an off-the-shelf TR-FRET detector available from several suppliers. Using robotic instrumentation, a single instrument can perform 100,000 assays per day (Figure 3).

A significant advantage of IP-One is its close relationship to Cisbio's existing cAMP assay. The two assays run on the same industry-standard hardware and follow exactly the same methodology and procedure. This offers GPCR investigators a platform to examine both of the principal GPCR pathways, covering in practice about 90% of all GPCRs.

Additionally, Cisbio has recently developed an improved dye chemistry that boosts the performance available from its HTRF methodology. For some years the acceptor molecule of choice for HTRF has been XL665, a chemically stabilized version of the alginate pigment APC.

This acceptor has good quantum yield, high stability in the face of a variety of solvents and buffers, suffers very few interferences, and has well-documented immunochemical properties.

However, XL665 is a large and complex protein of over 100,000 Dalton, almost as large as the Mab to which it is coupled when used in HTRF. This can cause occasional problems, particularly steric hindrance.

The IP-One assay introduces this new proprietary acceptor, a small-molecule organic dye called d2. It has the same photophysical properties as XL665, plus a straightforward immunochemistry and negligible compound interference.

Tested in a kinase assay format against a library of almost 15,000 compounds, the results from d2 showed over 95% correlation with those from XL665. When coupled to Cisbio's IP1-analogue reagent, it enhances both performance and stability of the IP-One assay. Cisbio's cAMP assays are also to be upgraded to use the new acceptor.

In summary, IP-One is a powerful addition to the GPCR researcher's armory. With continuing development of even more specific Mabs and improving signal-noise ratios, it can make a substantial contribution to drug discovery.