February 15, 2009 (Vol. 29, No. 4)

Jean-Luc Tardieu, Ph.D.

Advanced Tools to Investigate All Compound Classes of GPCR Activation

Among targets of interest for the pharmaceutical industry, GPCRs account for more than 30% of all drug screenings. The complexity of the signaling-network pathway induced by a ligand binding to the receptor has stimulated the development of new assay tools to trigger GPCR activation.

The diversity of functional GPCR intracellular responses is first due to the G protein. The first step of this pathway leads to G-protein coupling, which activates regulation of the production of a specific second messenger that can accumulate within the cells (Figure 1).

Second messengers’ accumulation assays allow the detection and characterization of all compound classes, full and partial agonist or antagonist, inverse agonist, and allosteric modulators. For some pharmacological classes such as inverse agonist detection or slow-acting compounds, accumulation assays provide unique and powerful tools from hit selection to lead optimization.

Regardless of the G coupling, second messengers’ accumulation assays can assess all compound classes (Figure 2).

Gq and Gi subtypes activate adenylate cyclase (AC), which modulates the production of cAMP. In the presence of a phosphodiesterase inhibitor (e.g., IBMX) cAMP accumulates and can be assessed with an appropriate cell-based assay.

Gq subtype triggers a transient release of Ca2+ from endoplasmic reticulum by D-myoinositol 1,4,5-trisphosphate (IP3) produced by activated phospholipase C (Figure 1).

The short lifetime of IP3 (30 seconds) makes this detection challenging for measuring GPCR responses, as IP3 rapidly enters the metabolic inositol phosphate cascade. It has been known for decades that lithium chloride (LiCl) leads to D-myo-inositol 1-phosphate (IP1) accumulation upon GPCR activation by inhibiting inositol monophosphatase, the final enzyme of the IP3 metabolic cascade.

Cisbio Bioassays, a member of the IBA Group, has developed a GPCR platform to measure the second messengers’ accumulation. This platform is based on the company’s HTRF® technology. The activation of Gs/i protein-coupled receptors is assessed by measuring the level of cAMP, whereas the IP1 accumulation is used as a functional marker of a Gq-coupled target.

Figure 1. GPCR Gq stimulation activates PLC and triggers the inositol cascade.

Figure 2A. Inverse agonist

Figure 2B. Antagonist stimulation

Compound Activation

The use of an accumulation second messenger assay to screen a library of potential GPCR drug candidates would overcome the kinetic action of the compound. The accumulation process allows the detection of either rapid- or slow-acting compounds (Figure 3).

The ability of an accumulation assay to detect a slow-acting agonist can be illustrated with GSK’s Salmeterol™, a long-lasting agonist of beta 2-adrenergic receptors. Agonists of beta 2-adrenergic receptors used for the treatment of asthma can be classified as rapid- or long-acting compounds. Rapid-acting agonists such as GSK’s Salbutamol™ are recommended for the treatment of symptomatic relief of asthma, whereas Salmeterol, a long-acting agonist, is more commonly used prophylactically in the treatment of the disease.

Figure 3. Beta-2-adrenoceptors are Gs-coupled receptors whose activation leads to increased cAMP production within cells.

Slow-Acting Agonist Activity

In a recent presentation, scientists from a large pharmaceutical company summarized studies conducted in their laboratory using the IP-One assay with Gq and Galpha16-coupled receptors.

One of the key findings of the study was the identification, using IP-One, of slow-acting agonist activity not detected by calcium mobilization.

High-throughput screening of this class of compounds may present technological limitations when seeking agonists with the calcium flux, the most commonly used existing HTS method for Gq-protein coupled receptor cell-based functional assays.

Calcium mobilization is a proven and widely used assay. The method can, however, require access to dedicated instrumentation and is limited by its extremely short readout time. These obstacles can increase screening turn-around and can lead to false negatives, particularly when looking for slow-acting agonists. The results presented show evidence of two slow-acting agonists with IP-One, but not with the calcium flux.

Characterization of Inverse Agonists

This article has shown that with second messengers, all compound classes can be investigated. In addition to the ones cited, this also includes inverse agonists.

A study presented at the Miptec Conference in Basel, Switzerland, late last year showed the characterization of an inverse agonist by using the IP-One Terbium assay. The identification of an inverse agonist effect on a constitutively active receptor is challenging with the calcium mobilization assay.  However, due to its terbium cryptate technology, IP-One Tb’s properties include high light absorption, sensitivity, enhanced signal-to-noise ratio, low false-positive rate, and full reader compatibility, and these accumulation assays provide efficient sensitivity and assay windows to properly perform a screen in HTS conditions (Figure 4).

Figure 4. Agonist and inverse agonist dose response by using IP-One Terbium.

Jean-Luc Tardieu, Ph.D. ([email protected]), is HTRF product manager at Cisbio Bioassays. Web: www.htrf.com.

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