ChemiScreen novel GPCR stable cell lines for calcium mobilization assay platforms

In today’s modern era of drug discovery, however, the process of compound selection typically involves screening compounds against a specific target or class of targets. Biochemical and pharmacological parameters are defined, a lead compound or group of compounds is selected for optimization, and with often years of work, a compound eventually becomes a drug. But without the aid of modern targeted therapies, more than half of today’s prescription drugs have emerged as potent and selective GPCR-targeted compounds. The therapeutic relevance of GPCRs was quickly realized and targeted efforts for drug discovery have ensued against specific GPCRs.

Working with a receptor as a drug target was originally fairly straightforward. GPCRs are intrinsic membrane proteins with seven trans-membrane spanning regions, so target purification necessitates retention of the cell membrane to keep the receptor as close to its native, relevant conformation as possible. Compounds are then screened for their ability to bind to the GPCR, typically measured by the displacement of a radio-labeled ligand. This requires identification of a ligand in the first place. This often tedious and difficult process is beyond the scope of this review, but can drastically limit the ability to perform targeted drug development.

As understanding of GPCR function evolved, it became clear that nuances of receptor activation required additional cellular components to accurately predict ligand activity. The receptors are coupled to heterotrimeric G-proteins, which are comprised of   and  subunits and categorized into four main classes based on the  isoform in the complex: Gi/o, Gq, Gs and G12. Depending on the class of  subunit, the G-proteins can effect different signaling 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 signaling. These subtleties necessitated assay development that could quantify the pathway(s) activated upon ligand binding, thus cell-based assays were developed to measure these nuances of signaling.

Cellular levels of second messengers, such as inositol-phosphate and cyclic-AMP, are modulated by GPCR activity. Reagents to measure the levels of these messengers were developed and applied to functional studies of GPCR signaling and ligand/compound characterization. However, the sensitivity, operational complexity and cost of these assays prevented widespread adoption for high throughput screening (HTS). A standardized method for screening GPCRs was needed. For GPCRs coupled to Gq proteins, the pathway effected induces changes in intracellular calcium levels. An elegant solution was found to measure ligand-induced effects of these GPCRs by combining a cell-permeable compound that becomes fluorescent upon calcium binding and an instrument configured to measure the fluorescent signal in real time (e.g. a Fluorometric Imaging Plate Reader - FLIPR). These instruments evolved to read entire plates of cells, satisfying the requirements for high throughput screening. Still, this methodology was applicable only to only a subset of GPCRs.

Chemicon has developed a novel method of receptor expression and G-protein coupling to create a reliable and universal calcium mobilization assay platform for all GPCRs. Fortunately for researchers, nature has given us a valuable tool – a promiscuous G protein that couples to a wide range of GPCRs. The ChemiScreen cell lines were developed by identifying cell lines with endogenously high levels of these promiscuous G-proteins (G15 or G16). These novel cell lines were then coupled with a unique mammalian expression vector capable of producing high levels of surface-expressed GPCRs that are subsequently coupled to the promiscuous G-proteins. 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 particular second messenger effected by the GPCR is difficult if not impossible. By coupling these ‘orphan’ GPCRs to calcium mobilization, researchers can screen for natural or surrogate ligands for these receptors. The high levels of surface expressed GPCRs also serve as an ideal source for membrane preparations and Chemicon offers a variety of these as products for use in conventional ligand-binding studies.

To further enhance calcium flux and increase the signal generated by GPCR ligand binding, two of the three ChemiScreen cell lines express an endogenous store-operated Calcium Release Activated Calcium (CRAC) channel to allow maximum calcium influx. The calcium mobilization readout of each type of GPCR (class A, B, C and Gi/o, Gs, Gq, G12 coupled) in these engineered cell lines has been pharmacologically validated using an array of known small molecule and peptide agonists/antagonists. In case studies, the IC50/EC50 values and rank orders of potency remain the same in FLIPR assays as they are in ‘native’ assay formats, such as cAMP assays, secondary functional assays, reporter gene assays and radioligand-binding assays. The ChemiScreen cell lines are easy to grow and an ideal tool for high throughput calcium mobilization assays with a superior signal-to-noise ratio. To date, more than 40 cell lines and membrane preparations are commercially available with many more in development.