GPCRs are a tempting target for cell-based assays. Clay Scott, Ph.D., associate director for lead generation at AstraZeneca, discussed his company’s pursuit of assays that will identify the ligands that bind to this family of transmembrane receptors. GPCRs play a critical role in the detection of molecular signals from outside the cell that activate signal-transduction pathways and cellular responses.
Dr. Scott and his colleagues compared optical- and impedance-based biosensors for their ability to detect ligand binding to the GPCRs. According to Dr. Scott, impedance-based assays measure changes in electrical impedance (roughly equivalent to resistance) relative to a voltage applied to a cell monolayer.
Optically based methods, on the other hand, quantify the shift in wavelength of reflected light that occurs due to the refractive properties of the biomass. As the morphology and mass redistribution of the cell changes in response to GPCR-ligand driven dynamics, these events can be detected by the two technologies. However, the AstraZeneca team investigates questions of the suitability these approaches for drug and pharma discovery.
In comparisons of GPCR agonists and antagonists, Dr. Scott noted that, in general, similar values were obtained with optical- or impedance-based assays. Both platforms provide sensitive, label-free precise measurement of these agents’ effects on cellular-response. However, using the impedance-based CellKey assay system (MDS Analytical Technologies), novel data was generated, including temporal responses that distinguish Gi, Gq, and Gs signaling.
Johannes Pschorr, Ph.D., European application scientist for MDS Analytical Technologies, also discussed the CellKey platform. “The system measures impedance changes occurring in response to stimulation or activation of signaling pathways within the cell. A monolayer of cells is seeded into a custom microplate that contains electrodes patterned at the bottom of each well. The CellKey system applies small voltages across a range of frequencies, measuring changes due to transcellular and extracellular current, reported kinetically for each well.”
Dr. Pschorr’s team is confident that these physiological changes occur as a direct result of signaling-pathway activation. The platform offers the sensitivity to measure functional activity of endogenous targets in live cells, as opposed to being limited to detecting overexpressed recombinant expression systems, he said. Additionally, he reported that the platform has the ability to measure any cell type—adherent and nonadherent cell lines and primary cells.
Analyzing receptors that are expressed with the appropriate accessory proteins gives researchers a picture of receptor function in a physiologically relevant environment. This flexibility allows researchers to conveniently screen a wide variety of targets with a single instrument platform, which helps standardize data to support lead compound selection.
One of the significant challenges Dr. Pschorr faces is the complexity of GPCR pathways and their interdependencies. “When screening for receptor modulators, we need to use methods that are as close to the actual cellular response as possible. Trying to interpret results from cells that are not physiologically relevant can lead to less-than-optimal lines of investigation to pursue.”
According to Dr. Pschorr, combining the ability to measure a whole live-cell assay with the ability to measure multiple signal pathways within the cell provides a more informative data result. He predicted that the measurement of integrated response of whole cells rather than measurement of one specific point in one specific pathway will lead to a more thorough understanding of complex receptor activity and compound mechanisms of action.
“The ability to do so in a label-free manner also provides an advantage, both in enabling the universality of the measurement and in allowing researchers to have a robust system to easily weed out nonspecific effects due to labels of the currently employed technologies.”
Human Embryonic Stem Cells
“Human stem cells can be used as a tool in small molecule screening,” said Paul Andrews, Ph.D., senior scientist in the drug discovery unit at the University of Dundee, “but care must be exercised.”
Dr. Andrews is the head of a program that exploits stem cells to serve as physiologically relevant cell models of disease. As Dr. Andrews explained, embryonic stem cells have an unlimited capacity for differentiation, whereas adult stem cells are more circumscribed in this respect. A third option is induced pluripotent stem cells, which are derived from reprogrammed adult somatic cells, and are similar to embryonic stem cells. Being activated by small molecule chemical signals, they offer an opportunity to understand and manipulate developmental events. This is only sometimes true (e.g., in the case of retinoic acid) but the majority of other cases involve protein ligands (e.g., the BMPs, Wnts, and Activins).
Small molecules are useful because they may act as surrogates for the protein ligand by stimulating the pathways or inhibiting a negative regulator. In some cases a series of steps may require a toolbox of molecules to bring about the final differentiated state.
A host of challenges are posed by human stem cells if they are to become an integral part of drug discovery. These include the requirements of feeder-free technology and a need to maintain long-term stability of cultures. At present, the understanding of cellular commitment, the steps required for appropriate differentiation, and avoidance of cell death and heterogeneity are all significant hurdles, according to Dr. Andrews. Nonetheless, stem cells constitute a powerful model for the study of disease states and predictive toxicology.
The stem cell technology program includes several other institutions under a £9.5 million multicenter umbrella pursuing cell delivery, high-content analysis, building of libraries, and development of gene-reporter systems. Cellartis supplies more than one billion cells per week, generated through large-scale automated production. The cells are plated in either 96- or 384-well plates and tested against a large collection of compounds. Included are small, drug-like molecules targeting ATP sites on protein kinases, marketed drugs, bioactives, and epigenetic modifiers.
After processing, the cells are imaged by immunofluorescence and analyzed to quantify a particular marker (e.g., fluorescence due to the activation of reporter genes or the levels of a protein detected by antibody staining).
Positive hits in the initial screening are followed up through potency testing, kinase profiling, mechanism of action studies, toxicity, and gene-expression profiles. One of the targets Dr. Andrews and his team have focused on is the bone morphogenetic proteins, or BMPs, which are members of the TGFβ superfamily of extracellular signaling molecules. They observed strong effects on Oct3/4, one of the master regulatory transcription factor proteins that are essential to maintain pluripotency. Focusing on the ability of the BMP4 to drive differentiation, they identified a number of small molecular antagonists that block this effect and maintain pluripotency.
“We sought to identify both the Oct3/4 upregulating and downregulating agents as early differentiation-promoting factors,” Dr. Andrews said. By screening their kinase libraries and bioactive libraries they identified a number of candidates. Three compounds are currently undergoing follow-up work to determine their mode of action and have shown good efficacy in replacing the endogenous BMP inhibitor Noggin in neuro-ectoderm induction protocols.
Other current and future activities include identification of early differentiation factors, and an induced pluripotent stem cell drug discovery program. “In the future we will need effective mode-of-action studies to reveal new pathways and targets,” Dr. Andrews concluded.
The approaches profiled in this article are among the most promising possibilities for accelerating the drug discovery process and saving time and financial resources. There is a pressing need for new technologies as drug development has lagged, in many cases due to the failure to recognize fatally flawed compounds early in the discovery process. New cell-based assay strategies may hold the key to breaking one component of the discovery roadblock.