A novel method of receptor expression and G-protein coupling has created a reliable and universal calcium mobilisation assay platform for all GPCRs.

Historically, successful drugs have been discovered without the need to understand the molecular details of their mechanism of action. However, modern drug discovery is significantly more difficult. Target classes are identified, biochemical and pharmacological parameters are defined, lead compounds are selected for optimisation, and, often after years of work, a drug is developed

Interestingly, without the aid of modern drug discovery, more than half of today’s prescription drugs have emerged as potent and selective G-protein coupled receptors (GPCR)-targeted compounds. Chemicon International, Inc, a US-based supplier of immunological reagents, quickly grasped the therapeutic relevance of GPCRs, and there have been several targeted drug discovery efforts against specific GPCRs.

Working with a receptor as a drug target was originally fairly straightforward. As the understanding of the GPCR function evolved, it became clear that receptor activation required additional cellular components to accurately predict ligand activity. The receptors are coupled to heterotrimeric G-proteins, which consist of a, b and g subunits and are divided into four main classes based on the a isoform in the complex: Gi/o, Gq, Gs and Ga12.

Depending on the class of a sub-unit, the G-proteins can affect different signalling pathways, leading to distinct biological events. Thus, for a given GPCR, the pathway activated is dependent on the particular G-proteins coupled to that receptor.

Further studies revealed that ligands and GPCR-targeted compounds could not be classified as simple activators (agonists) and inhibitors (antagonists). Some ligands decrease the basal level of activity for a given GPCR (inverse agonists), while some only partially activate the full potential of receptor signalling. These subtleties required assay development to quantify the pathway(s) activated upon ligand binding, and cell-based assays were developed to measure these nuances of signalling.

Perfect platform

Chemicon’s ChemiScreen™ recombinant GPCR membrane preparations and calcium-enhanced stable cell lines are exciting new reagents designed for high-throughput screening and drug discovery. The products consist of a series of high-quality recombinant GPCRs that have been stably transfected into a range of mammalian cell lines with high levels of endogenous promiscuous G-protein (Ga15 or Ga16), using a proprietary mammalian expression vector.

This methodology gives researchers a single platform to screen a wide range of GPCRs, regardless of the native G-protein coupling status. For many GPCRs, the natural ligand may not be known, and identifying the second messenger affected by the GPCR is difficult. By coupling these ‘orphan’ GPCRs to calcium mobilisation, researchers can screen for natural or surrogate ligands.

It also facilitates an increased level of receptor surface expression and subsequent functional coupling of the receptor to the endogenous promiscuous G-protein, which in turn leads to calcium mobilisation without having to co-transfect promiscuous or chimeric G-proteins. This method has been successfully used to redirect a variety of G-protein coupled pathways to a common and simple readout for fluorometric imaging plate reader (FLIPR) calcium flux assays, for Gi/o, Gs and Ga12 receptors as well as standard Gq-coupled receptors.

Intracellular calcium mobilisation is further enhanced through the endogenous, store-operated calcium release-activated calcium channel, which ensures maximum dose-dependent calcium influx. The ChemiScreen cell lines are optimised and pharmacologically characterised to test the functional response of GPCRs, using a panel of known ligands in order of potency as well as calcium-sensitive fluorescent dyes in FLIPR assays.

The cell lines are easy to grow and an ideal tool for high-throughput calcium mobilisation assays with a superior signal-to-noise ratio. To date, more than 50 cell lines and membrane preparations are commercially available, with many more in development.

Company profile

Chemicon International, Inc, manufactures and distributes over 6000 products to many scientific disciplines.