by Catherine Shaffer
G protein coupled receptors (GPCRs) comprise one of the largest and most important families of druggable targets in the body. Currently, about forty percent of all drugs are targeted at a GPCR. The distinguishing feature of these large, cell-surface proteins is a seven-transmembrane domain. The binding of a ligand on one side of the membrane induces a conformational change on the other side, passing a signal through the wall of the cell. This basic mechanism is involved in a huge number of biomolecular systems. GPCRs have gained significance as targets for a great many diseases, from mood disorders, to inflammation, to cancer. However, the hydrophobic transmembrane domains make GPCRs difficult to assay in vitro. New assay technologies have made GPCRs more accessible, opening up a world of possibilities for drug discovery screening.
Cell-based assays
Most GPCR assays are based on the immunoassay concept, whether it is a traditional in vitro sandwich assay, or a high throughput cell-based assay. Multispan, Inc. is a leader in cell-based assay technology exclusively focused on GPCRs. Their proprietary technology (HEx™) allows robust expression of GPCRs in mammalian cells. Their GPCR expression technology produces optimized surface expression in mammalian cells, especially in certain types of GPCRs that are notoriously difficult to express, such as metabotropic glutamate, neuropeptide, and free fatty acid receptors. These versatile cell lines are adaptable to virtually any assay platform or application, as long as it requires expression of the GPCR in mammalian cells. In addition to first-line high-throughput screening in drug discovery research, HEx has been used to study secondary activities and indications for drugs already on the market. Says Dr. Helena Mancebo, president and CEO of Multispan, "Our strategy is to work with the industry leaders in providing GPCR detection technologies such as MDS (formerly Molecular Devices), Cisbio Bioassays, Promega, and our customers together to provide cell-based assays that are most suited for the biology of each GPCR target."
As an example of the applicability of the HEx cell system, Multispan has teamed up with Cisbio Bioassays, to validate this system using Cisbio Bioassay’s IP-One assay, which targets the inositol phosphate (IP) cascade. Ligand binding to a GPCR stimulates Gq, which induces phospholipase C activation, triggering the IP cascade. Because IP-One accumulates in the cell, it can be used to monitor this signal in the cell. The IP-One assay uses Cisbio Bioassays’ HTRF® technology to generate a long-lasting fluorescence signal when ligand binding triggers downstream activation of IP-One. HTRF combines the concept of fluorescence resonance energy transfer (FRET) with time-resolved fluorometry (TRF). HTRF uses the unique properties of complexes of lanthanides (Europium and Terbium), which captures excitation energy and emits at 620 nm, while also transferring energy to an allophycocyanin dye (XL665) or a near infrared acceptor (d2) for emission at 665 nanometers. The main advantage of this assay is that it overcomes the problem of background fluorescence from biological samples, yielding a more sensitive assay.
Collaborations and partnerships between technology vendors has become the rule in the rapidly growing GPCR assay sector. In fact, these synergies are crucial in developing the next generation of technologies for the lab or clinic. In addition to borrowing Multispan's GPCR-expressing mammalian cell lines, Cisbio Bioassays has also benefitted from a partnership with Covalys Biosciences AG, using their SNAP- and CLIP- tag system for covalently bonding needed fluorophores to protein molecules to develop its Tag-lite platform. Increasing the library of available fluorescent ligands opens the HTRF assay technology to greater numbers of GPCR assays. Says François Degorce, head of marketing at Cisbio Bioassays, "Right now we are in the process of producing more ligands. We have been very successful with vasopressin, dopamine, and CXCR4 receptors. We are progressively moving on to more complicated ligands."
In addition to studying receptor-ligand interactions, Tag-lite has also been used to reveal interactions between GPCRs at the cell surface. In a paper published in Nature Methods, scientists from Cisbio Bioassays, in collaboration with the Institut de Génomique Fonctionnelle (Institute of Functional Genomics, or IGF) in Montpellier, France, produced evidence of oligomeric structure of class A and class B GPCRs, and dimers of heterodimers among GABA receptors.1
Growing the GPCR product portfolio
Responding to market trends and customer demand, PerkinElmer
has developed the industry's broadest offering of technologies for GPCR research in both basic research and drug discovery. Products are tailored for high throughput screening as well as lead compound profiling applications, with an extensive selection of membranes expressing GPCRs and radioligands for binding studies. More than 200 cell lines are validated for HTS, covering a range of cell signaling pathways including cAMP accumulation, changes in intracellular calcium, and receptor-coupled kinase pathways leading to ERK phosphorylation in the Alphascreen® Surefire® assay.
In addition to its biological product ranges, PerkinElmer supplies instrumentation and liquid handling platforms to support its assays, such as the Envision® multimodal plate reader and the Janus® automated liquid handling system. Confocal imaging technologies include the benchtop Operetta screening platform and the Opera confocal microplate imaging reader. Martina Bielefeld-Sevigny, PhD, vice president and general manager, drug discovery and research reagents, bio-discovery, PerkinElmer, Inc,, explains: "We continue expanding our GPCR in several ways. Firstly by continuously adding validated cell lines stably expressing the GPCR in both cultured and ready-to-use cryopreserved cells. In parallel, PerkinElmer continues to develop new technologeis to add to cellular products that address evolving GPCR trends ... emerging applications, such as label-free approaches to GPCR activation, are under investigation."
Allosteric profiling
Functional assay of GPCR signal transduction activity is a very hot field right now, but it's not the only interesting type of study that can be done on these receptors. Of course it is also possible to do nonfunctional binding assays, as on an antibody microarray chip, for example. But another growing area of interest is allosteric binding compounds. Allosteric binding (as opposed to orthosteric binding) occurs at a separate site from the native ligand binding site. Allosteric compounds can modify GPCR activity, without physically blocking the binding site. Understanding of the significance of allosteric binding to human medicine is in its infancy, but it adds a layer of complexity to a system that is already nicely druggable. Millipore, which in the past five years has branched out far beyond its traditional catalog of laboratory filters, has recently announced its new Allosteric Profiler service for screening GPCRs against these potential modifiers. Says Blaine Armbruster, PhD, product manager for GPCR drug discovery for Millipore, "Essentially they're attractive because they don't do anything on their own. They can enhance the body's normal responses and so forth ... the native ligand is sort of an on/off switch. Using an analogy, it [the native ligand] turns your heating system on and off. The allosteric compound would turn it up or down, like a rheostat."
Another benefit of allosteric compounds as potential drug modifiers of GPCR activity is that they tend to bind to less conserved regions of the GPCR. This means that the ultimate drug compound can be much more selective. Ligand binding domains tend to be highly conserved, so a drug intended for one specific GPCR could end up blitzing half of the cell receptors in the body, causing a lot of unwanted side effects, to say the least.
The Allosteric Profiler assay is a calcium-based flux assay. Says Armbruster, "We have a panel of receptors, over 150 or so. Basically you add compounds to plates ... and look for shifts in dose response, which would indicate either positive or negative allosteric activity."
Breakthroughs in assay methods for GPCRs have opened up exciting new possibilities for drug discovery and development, and especially for high throughput screening. The wide-open field has even attracted players previously known for hardware and consumables, such as Millipore and PerkinElmer, to the cell-based assay arena. This is good news for researchers, who have an array of new choices for their investigations.
References:
1Maurel D., et al., “Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization.” Nat Methods. 2008 June; 5(6): 561–567.