More than 50% of all current drugs and nearly 25% of the top 200 best-selling drugs target G-protein-coupled receptors (GPCRs).These heterotrimeric proteins contain seven membrane-spanning segments and consist of α, β, and γ subunits.
To add to the complexity, there are four main subfamilies depending upon their a-subunit structure: Gs, Gi/o, Gq, and G12/13. Thus far, endogenous ligands have been suggested for more than 200 GPCRs. Because there are likely 8001,000 human GPCR genes, discovery of drugs that target orphan GPCRs (whose ligands are unknown) may have enormous economic impact.
As a result, companies are developing enhanced methods or new techniques for high throughput screening of GPCRs. For some, monitoring the responses of living cells provides a sensitive measure for detecting GPCR responses.
Examples include measuring calcium flux, employing green fluorescent protein (GFP) as a reporter along with high throughput imaging, and complementation techniques where read-outs are detected by spontaneous fusion of artificially split proteins. But cell-based screening methodologies are not without their problems such as interference from non-specific ligands or other GPCRs.
Thus, other companies pursue non cell-based screening techniques such as incorporating the exquisite sensitivity of mass spectrometry or improved traditional methods to monitor binding of GTP to GPCRs.
Reversing Order of Addition
Measurement of intracellular calcium levels provides a sensitive means to detect GPCR activities. So-called calcium flux determinations help disclose signaling events of receptors. Euroscreen (www. euroscreen.be) has developed a novel way to screen compounds that interact with calcium-coupled GPCRs. They found that reversing reagents' traditional order of addition presents multiple advantages.
"Overall, our methodology provides a functional, high throughput cell-based screening technology," explains Vincent Dupriez, Ph.D., head of molecular biology and co-inventor of the technique. "The principle is based on a photoprotein called aequorin that originated from jellyfish and luminesces robustly and rapidly after binding by calcium.
"Basically, we load GPCR-expressing cells with the apoaequorin cofactor coelenterazine, dilute the cell suspension in assay buffer, and inject into microtiter plates containing the compounds to be screened. A flash of light is emitted after calcium increases following GPCR activation.
"Traditional tests add reagents opposite to this. But, reversing order of addition provides several important advantages. It provides great flexibility and reduces the screening time and the amount of drugs used."
"Adding cells to compounds offers a unique way to search for natural or surrogate ligands for any calcium coupled-protein," Thierry Christophe, Ph.D., head of screening, reports. "It provides higher throughputs because injectors do not need to be extensively washed, as is the case when injecting compounds. It is also much less expensive in terms of materials and provides better consistency of the results since it eliminates the need to grow adherent cells on assay plates."
The company's high throughput screening proprietary platform is called AequoScreen. It also offers a range of products and services including custom screening and a large collection of recombinant GPCRs as stable cell lines or as ready-to-use-membrane preparations.
The use of living cells to screen for compounds that activate or deactivate GPCRs has the advantage of honing in directly on an in vivo process, according to Carson Loomis, Ph.D., vp of research for Xsira Pharmaceuticals (formerly Norak Biosciences; www.xsira. com).
"We use a technology called Transfluor for detecting and quantifying GPCR desensitization and internalization. This technology is based on agonist-induced translocation of GFP (green fluorescent protein) that is linked to -arrestin2, a marker of receptor activity," Dr. Loomis explains.
"Since desensitization occurs only in the setting of an activated receptor, monitoring arrestin translocation allows us to detect if the GPCR is activated and determine the amount of activation."
Dr. Loomis says that the transfluorescence technology easily lends itself to high throughput screening. "With robotics and automated image analysis software, we can derive measurements from about 100 plates (each with 384 wells) per day."
Imaging approaches for screening GPCRs is a new and evolving methodology with several advantages over traditional methods, indicates Dr. Loomis.
"These techniques measure not only the intensity of the reaction, but, importantly, allow us to monitor movement within live cells. They look at a different biological process than other GPCR read-out assays.
"With Transfluor, there is no limitation by the type of GPCR since the assay will work with both known and orphan receptors. Other advantages are that this technology allows us to look for both agonists and antagonists of therapeutically relevant responses and other read-out assays, such as measurement of calcium release and cyclic AMP levels."
Norak's assay technology has been licensed by AstraZeneca, Merck, Roche, and others. Using this technology, these companies have investigated several GPCR subtypes applying automated imaging methods to screen large compound libraries.
Comprehensive Clone Set
OriGene Technologies (www. origene.com) suggests that there are an estimated 340400 pharmaceutically relevant GPCRs in the human genome. "Identifying and isolating so many possible targets to evaluate is a huge hurdle for many companies," notes Xuan Liu, Ph.D., director of marketing.
"Some companies may have 50 or more, but the majority cannot devote so much time and effort to getting more. That's why we developed a nearly comprehensive set of about 350 full-length GPCR clones that scientists can use for their high throughput screening."
Dr. Liu also indicates that not all clones are created equally. "Scientists who clone GPCR genes using PCR often have difficulty determining if changes are artifacts of the PCR process or are naturally occurring. That's why our company uses high throughput cloning and expression profiling to isolate and catalog this collection, which is a subset as of our larger 20,000 full-length human cDNA clones."
The GPCR Cloneset genes are cloned into a nonproprietary CMV vector and provided as bacteria in plates for processing by researchers. This feature allows further investigation of gene functions and developing of transfected or transgenic cells for drug screens. The vector also contains a prokaryotic transcriptional promoter for use in a cell-free system.
Most HTS technologies traditionally rely on using cloned genes to create cell lines that express the protein target of interest. But there are several problems with this approach. Genes generated from cDNA libraries in functional assays may be patented and create infringement issues. Additionally, rarity or variability of genes further complicates such strategies.
To circumvent these difficulties Athersys (www.athersys.com) applies its proprietary technology (RAGE) to functionally express GPCRs in desired cells without ever using clones or isolated gene sequences.
According to Alain Stricker-Krongrad, Ph.D., director of pharmacology, "Our methodology randomly activates genes throughout the genome resulting in a library in which every cell expresses a single unique gene. Thus, we now have cell lines that express GPCRs or other proteins of choice.
"An important advantage of doing things this way is that the level of expression is regulated and driven by the vector. Also, it allows one to hone in on one receptor and its specific signaling mechanism. This is much more physiologic. On the other hand with recombinant DNA there is a risk of over-expression of the receptor that will artificially skew results."
To provide for high throughput, intracellular calcium mobilization and ligand-binding assays can be done in a 384-well format. "We are able to accurately detect agonists, partial agonists, and antagonists in library screens (>80,000 compounds)," Dr. Stricker-Krongrad says. "So, we feel this methodology provides a robust and sensitive functional HTS format."
Enzyme Fragment Complementation
DiscoveRx (www.discoverx. com) uses an enzyme fragment complementation strategy for high throughput cell-based GPCR screening. The technology relies on dividing the enzyme, -galactosidase (-gal), into two inactive fragments, ED and EA.
One of these (the alpha fragment, termed ED) is chemically conjugated to a second messenger. When both -gal fragments encounter each other they combine to form active enzyme, which hydrolyzes a substrate and emits a chemiluminescent or fluorescent signal.
"One of the major advantages of this type of assay is that it is nonradioactive and homogeneous," Richard M. Eglen, Ph.D., CSO, notes. "So, washing and separation steps are not required. The reagents are simply added and the signal read on common plate readers. This allows using automated fluid-handling systems for primary screening of ligands that modulate GPCR activation."
An added benefit of chemiluminescent signals, in particular, is that they can provide a large signal to background ratio, according to Dr. Eglen.
"The assay format is easily adapted to different fluid volumes needed in assays that are miniaturized for high density microtiter plates. Furthermore, the interference potential of compounds in screening libraries is very low, resulting in few false positives and high confirmed hit rates."
Dr. Eglen suggests that the technology also allows GPCR secondary messenger analyses. "In this case, we measure changes in intracellular cyclic AMP for Gs and Gi coupled GPCR receptors. The Gi coupled receptors are often quite difficult to assay, and an assay of this type provides a marked advantage.
"Using a different format, based on fluorescent polarization, DiscoveRx has also developed assays for IP3, a second messenger generated by Gq coupled GPCRs. Together, these assay formats are valuable for screening for novel ligands interacting at known and orphan receptors, particularly as one often needs to measure changes in constitutive activity of these receptors."
Affinity Selection Screening
One downside to cell-based screening methods for GPCR lead discovery is interference by nonspecific ligands and other GPCRs. To overcome this handicap, NeoGenesis Pharmaceuticals (www.neogenesis.com) developed an ultra-HTS in vitro method called the Automated Ligand Identification System, or ALIS, that combines affinity selection and mass spectrometry.
"We begin this process by cloning, expressing, and affinity purifying (using a 6X histidine or other tag) the GPCR of interest. We use a proprietary method to solubilize the receptor so that it has a purity and activity suitable for subsequent affinity-based screening.
"Next, we perform the ALIS screen of the receptor with our mass-encoded libraries that contain more than 5 million small molecules synthesized in house. These compounds are screened as pools of 2,500 compounds per well in ALIS."
ALIS incorporates an automated process for microscale size exclusion chromatography that rapidly separates any GPCR-ligand complexes from unbound compounds. These captured compounds are identified by LC-MS.
Finally, NeoGenesis employs ALIS to determine affinity of the compound hits. "The exquisite sensitivity of mass spectrometry enables such experiments to be done using only low amounts of a purified biomolecule receptor," Dr. Nash says.
Another advantage of the system, according to Dr. Nash, is that, "By relying on affinity, we can simultaneously identify multiple drug candidates that modulate a GPCR target by binding to known orthosteric sites as well as those that bind to novel allosteric sites.
"Drug leads that bind to GPCRs in allosteric modes may provide improved novel therapeutic benefits compared to orthosteric ligands. For example, allosteric ligands may be more sub-type selective, or may cooperate with endogenous ligands to provide tissue selectivity and safer, saturable pharmacological profiles."
NeoGenesis recently used its ALIS System for simultaneously determining the affinities of individual compounds from a chemical mixture. This advance enables rapid optimization of the initial hits' binding affinities using mixture-based chemical synthesis.
GTP Binding Assays
Although GPCRs are a diverse set of receptors, they all control the activity of their respective enzymes by catalyzing the GDP-GTP exchange on heterotrimeric G proteins (Gag). Thus, assessing GTP levels provides a snapshot of changes in GPCR activity.
PerkinElmer Life and Analytical Sciences (www. perkinelmer com) has developed a homogeneous assay for measuring the binding of GTP to GPCRs using wheat germ agglutinin (WGA)-coated red-shifted Image FlashPlates.
Wheat germ agglutinin is a plant lectin that binds to carbohydrate residues and thus can capture glycosylated GPCRs. The radioisotopically based assay uses so-called scintillation proximity to quantitatively detect and quantify agonist induced activation of [35S]-GTPgS binding to isolated GPCR membrane preparations.
"These assays have the advantage of needing no filtration or washing steps that are required for similar methodologies," reports Patricia Kasila, HTS applications scientist at PerkinElmer.
"So, they are both fast and simple. The 384-well format allows up to 50,000 points per eight-hour day. Another advantage is that there is less interference from fluorescent compounds and plates can be read on an imager (ViewLux) that visualizes the entire plate instead of one well at a time. So, the imager based-approach allows for higher throughput."
Kasila also reports the growing demand for non-radioactive assays of GTP activity. "Our Delfia GTP binding assay contains GTP labeled with a Europium (Eu) chelate. The GTP-Eu is incubated with cell membrane preparations and when a ligand binds to the GPCR, so also does GTP-Eu bind to the GPCR. After washing away the unbound GTP-Eu, the signal is measured via time-resolved fluorometry, a well-established technology that exploits the unique fluorescence properties of such chelates. More than 40,000 samples can be processed in an 8-hour day."
Peakdale Molecular (www. peakdale.co.uk) expanded its Peakexplorer screening libraries, with the launch of further GPCR-focused collections. The new libraries combine the company's expertise in proprietary chemistry with De Novo Pharmaceuticals' (www.denovopharma.com) in silico design. These highly focused libraries total almost 3,500 novel druglike compounds and are now available for screening to broaden the exploration of GPCR-relevant chemical space.
"We have now launched three sets of compounds under our Peakexplorer brand," says Gareth Jenkins, Peakdale Molcelar's business development manager.
"The first two, Peakexplorer G1 and G2, were based on six chemotypes combining known GPCR ligand features with novel Peakdale chemistry. The third, Peakexplorer G3 has just been made available and illustrates a further eight chemotypes.
"All of the compounds are druglike, fitting well wih Lipinski's rule-of-five. We also look at calculated polar surface area and rotatable bond counts as indicators of good human gut absorption and, for CNS research, for transport across the blood-brain barrier."