December 1, 2006 (Vol. 26, No. 21)

Screening Technologies and Assay Development for GPCR Receptors

Signal transduction is a key mechanism that brings about several physiological responses in a cell. Aberrant signaling can result due to gene mutations or regulatory protein overexpression. It is presumed that about 400 human diseases are due to signal transduction pathway defects. Thus, signal transduction research is a major area of focus in the academic and industrial world. This article highlights some of the talks that were presented at “Assays and Cellular Targets”, a recently held IBC conference.

Biased Agonists

Erin Whalen, Ph.D., a senior research associate in the lab of Robert J. Lefkowitz, M.D., at Duke University Medical Center (, presented a seminar titled “b-arrestin Signaling: Expanding the Therapeutic Utility of 7-Transmembrane Receptors.” Dr. Whalen’s talk focused on the role of b-arrestin-mediated signal transduction and the therapeutic potential of modulating such signal transduction pathways.

“Since G-proteins were discovered almost all signaling and receptor research has focused on canonical 7-transmembrane/G-protein signaling. GPCR’s play a critical role in many cellular and physiological processes, and hence these receptors are prime targets for drug development. GPCRs can be desensitized and downregulated by the G-protein coupled receptor kinases (GRK) and b-arrestins. However, it has only recently become apparent that the b-arrestins can also act to facilitate signaling,” explained Dr. Whalen. “Further, the idea that we can target GPCR receptors to modulate b-arrestin signaling independent of G protein activation is a new and novel concept.”

Dr. Whalen presented data that described and validated a new class of drug entities known as biased agonists/permissive antagonists. These are drugs that inhibit receptor-mediated activation of G proteins, but can stimulate b-arrestin signaling.

The physiological effects of b-arrestin signaling are yet to be fully characterized, but they have been shown to play a role in anti-apoptosis and improved cardiac function.

She presented proof-of-concept data with a biased agonist SII (a mutant angiotensin II peptide) that activated b-arrestin-mediated ERK phosphorylation via the AT1A cardiac receptor. This translated into improved isolated cardiomyocyte function and additionally slowed the progression of experimental cardiac dysfunction in vivo.

The importance of b-arrestin-mediated ERK phopshorylation was also demonstrated with other physiologically relevant receptors such as V2R, PTH1R, and b2AR. A key point conveyed was that b-arrestin-mediated signaling is distinct from G-protein signaling and GRK isoforms can differentially direct b-arrestin mediated desensitization, internalization, and signaling.

The Market Potential

“b-arrestin-biased agonists describe a new class of drugs and represent a value-added product for current drug screening programs. One can screen pre-existing compound libraries and previously discarded compounds for modulation of b–arrestins as well as GRKs. More than 50 percent of drugs in the market today target GPCRs or 7TMRs. Thus, the market potential for such b-arrestin-biased ligands is very high,” concluded Dr. Whalen.

Alzheimer’s Disease

Susan Catalano, Ph.D., director of discovery biology at Acumen Pharmaceuticals (, presented a seminar titled, “Discovery of Anti-ADDL Therapeutics for Alzheimer’s Disease: The Critical Role of Image-based Assays in Primary Neuronal Cultures.” Acumen’s small molecule research is focused on developing drugs that target amyloid b-derived diffusible ligands (ADDLs).

“The onset of Alzheimer’s disease (AD) starts with the overproduction and/or decreased clearance of Ab1-42 monomers in the brain, resulting in monomer accumulation.

“These accumulated monomers then form oligomeric assemblies known as ADDLs that bind to neuronal receptors and inhibit long term potentiation (LTP),” explained Dr. Catalano.

“LTP is important for memory function. The disruption of LTP by ADDLs initially results in memory loss, and eventually in the dementia that is characteristic of late-stage AD-afflicted patients.”

Dr. Catalano presented historical data that clearly showed elevated levels of ADDLs in AD brain, AD CSF, and transgenic mouse models. She discussed Acumen’s finding that ADDLs bind only to a subset of neurons and that this binding is inhibited by anti-ADDL antibodies.

Acumen has adapted a two-pronged approach to developing anti-ADDL drugs, namely preventing ADDL assembly and inhibition of ADDL binding to neuronal receptors. “There are multiple intervention points and therapeutic targets in the ADDL pathway, such as ADDL-based vaccines, anti-ADDL antibodies, receptor antagonists, signaling antagonists, and assembly blockers,” stated Dr. Catalano.

Data was presented on a few lead compounds that function as ADDL assembly blockers and inhibit ADDL binding as demonstrated by ADDL-binding imaging assays with hippocampal neurons. The combination of primary neuronal cell model plus imaging allows Acumen a significant advantage in assessing how compounds block selective ADDL binding.

“The few drugs that are available on the market only treat the symptoms of Alzheimer’s Disease by acting on neurotransmitter systems in the brain. They can slow the decline in Alzheimer’s Disease patients by only about six months. We believe our approach targets the underlying cause of the disease, and can prevent or even reverse its course. We believe that our image-based approach can produce better drug candidates faster,” Dr. Catalano said.

“It is extremely challenging to perform AD research, and clinical trials are very long and expensive. The use of patients’ ADDL levels as biomarkers in these clinical trials will be invaluable.”

GPCR-mediated Pathways

G-proteins are important binding proteins from physiological and a drug discovery standpoint. They mobilize a variety of GPCR-mediated signal transduction pathways. cAMP and Ca2+ mobilization assays were the classical assays to measure GPCR binding and activation. Kinase mobilization, gene transcription, and regulation of signal transduction are some recently developed assays that are being increasingly used in a cellular context to determine not just GPCR binding but also help assess receptor function.

Richard Eglen, Ph.D.,vp and general manager of discovery and research reagents for PerkinElmer’s Life & Analytical Sciences (, and Beth Rayl, Ph.D., scientific and exeution marketing manager at Perkin Elmer, discussed various cellular screening technologies for G protein-coupled receptors.

“Cellular models provide a rich data set. The availability of advanced detection systems, improved reagents, and automated robotic workstations has eased the running of cellular GPCR assays in an HTS mode. It has also enabled several companies to perform GPCR screening at the primary screening stage of their drug discovery program,” commented Dr. Eglen.

PerkinElmer offers a diverse portfolio of cellular screening technologies for GPCR assays that address receptor-ligand, phospho-ERK quantification, cAMP measurements, reporter gene assays, IP3 quantification, GTP binding, and novel Ca2+ mobilization detection assays. Dr. Rayl’s discussion profiled a Flash luminescence aequorin technology coupled to the company’s LumiLux Cellular screening platform and described the Janus™ Cellular Workstation for use in multiple cellular GPCR assays.

The Flash luminescence aequorin assay, developed at Euroscreen (, is a cellular assay that measures changes in intracellular Ca2+ . It is based on the principle that the apo-enzyme ApoAequorin is converted to active enzyme Aequorin in presence of coelenterazine. Upon Ca2+ binding, Aeqourin oxidizes coelenterazine into coelenteramide resulting in flash luminescent light emission, which is measured using LumiLux.

Calcium mobilization data generated using a CHO cell line expressing recombinant aequorin and the G-protein coupled receptor was presented. Results of 384-well format correlated well with the 1,536 cellular assay format run with 1,500 cells per well.

The data also has excellent correspondence to data generated with traditional fluorescent Ca2+ assays. The assay can be run with suspension or adherent cells and can be performed in one day.

“A key differentiator of the flash luminescence technology compared to the glow luminescence technology is that the signal is intense as it is expressed over a short span of time. This high signal intensity allows one to look at partial activators and allosteric inhibitors, thus providing a wider range of potential drug compounds. When compared to traditional fluorescent-based Ca2+ assays, the flash luminescence signal is 20 to 30 times higher, which is a large window to assay for novel compounds that modulate a signaling pathway,’’ explained Dr. Eglen.

Dr. Eglen also described in detail PerkinElmer’s Janus Cellular Workstation that enables complete assay automation. The workstation comes with prevalidated templates and data for Lance™ cAMP, britelite™, steadylite™ HTS, ATPlite™ and the AlphaScreen™ SureFire cellular kinase cellular assays performed on CHO cells engineered with appropriate receptors.

GPCR Assay Development

Veronica Soloveva, Ph.D., group leader for cell-based assay development in the chemical sciences department at Wyeth Research (, discussed the importance of flexibility for HTS assay development for GPCRs. She highlighted the impact of new emerging technologies that allow flexibility in choosing the most robust assay for HTS campaigns. Dr. Soloveva’s group provides assay development support for all of Wyeth’s HTS programs. Among their HTS campaigns 50% are cell-based, and of those 70% are targeted at different GPCRs.

“It is estimated that about 1,000 different genes encode about 10,000 different GPCR-like receptors but only about 1,000 of them have been characterized. The rest are orphan receptors whose function is unknown, and they have no known ligands. GPCRs are a big family of receptors that play a major role in many signal transduction pathways. Most medical responses in humans are effected through the GPCRs. In fact 12 out of the top-20 selling drugs target GPCRs,” commented Dr. Soloveva.

Wyeth adopts a multicriterion approach for their HTS assay development. Sensitivity of detection, homogenous format, scalability to low volume, robust performance, statistical reproduction, and automation compatibility are key. “These criterion help define data acceptance particularly when the assay is noisy,” stated Dr. Soloveva.

Its assays are typically conducted with engineered cell lines. One of the ways to decrease variability during multiple days of HTS campaigns is to use single lot/batch of scaled-up cryopreserved cells. To achieve consistency in plate handling during HTS campaigns Wyeth has integrated all of their plate readers into automated workstations.

Dr. Soloveva described Wyeth’s various assay capabilities for different types of GPCR assays with relevant data and receptor examples. For Gi protein-mediated GPCR assays, Lance, and EFC Hithunter assays were used to measure cAMP responses. For Gq protein mediated GPCR assays, Ca flux and IP1 assays were conducted. Plate type and plate coatings influenced data outcome.

Receptor internalization assays with b-arrestins were also discussed. “b-arrestins causes receptor internalization. GFP-labeled b-arrestin allows one to visualize receptor internalization upon ligand binding in imaging assays and helps bring more quality to the screens.

This technology is useful for orphan receptors, for receptors that are poorly defined or have weak responses, and are a good secondary functional assay. The goal is to run the assay as close to the receptor as possible to measure the most accurate response. Overall Wyeth has been successful with their HTS GPCR screens,” stated Dr. Soloveva.

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