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).

G_q and G_i 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.

G_q 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 G_s/i protein-coupled receptors is assessed by measuring the level of cAMP, whereas the IP1 accumulation is used as a functional marker of a G_q-coupled target.

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.