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Feature Articles : Apr 15, 2009 ( )
Focused Assays Lead to More Potent Drugs
Label-Free Remains No.1 Cell-Based Trend, but Electrophysiology Is Not Far Behind!--h2>
The demand for new drugs is pushing companies to develop cell-based assays that focus on a specific cellular event, potentially leading to tailored and more potent therapies. These assays also hold promise to predict toxicity, improve extrapolation to humans, and identify optimal leads earlier in the drug-discovery process. The companies highlighted in this article will share recent developments in this field at two upcoming conferences: Informa Life Sciences “Cell-Based Assay” conference to be held in London and CHI’s “Cell and Tissue-based Assays for HTS” meeting to be held in Philadelphia.
There are many factors to consider before and during cell-based assays, says Lisa Minor, Ph.D., principal scientist, drug discovery, Johnson & Johnson. Some of these include whether the assay has the right signal, the correct biological event is measured, and the right cells are used.
Another challenge is cell lines. “What you’re really concerned with is that the cell doesn’t have endogenous expression of your target and that the only event you’re seeing is what you’ve added to the cell.” A recent concern focuses on cell lines that are contaminated with other cell lines, or may not be the right species. Dr. Minor says several companies are developing tools to qualify for species and tissue type.
The biggest trend in cell-based assays, says Dr. Minor, is label-free technology. “You can pick up minor cell changes you can’t see with a microscope, and without having to do multiple assays on the same cells.” She says everyone would like to use primary cells, but they are limited in number and their characteristics are difficult to maintain. Electrophysiology with high throughput is also an emerging technology. Although Dr. Minor says better electrophysiology systems are needed, she is not sure about improvements to other technologies yet. “You don’t realize you need something until somebody shows up with an idea.”
DiscoverRx recently updated a platform developed years ago by its CEO for cell-based assays. “We’ve put some new improvements and twists on it and those are what have made it amenable to measuring protein-protein interaction events in live cells,” explains Keith Olson, Ph.D., vp of R&D.
The PathHunter™ assays incorporate a target protein fused to a b-galactosidase (b-gal) peptide expressed in the cytoplasm. Another portion of b-gal (enzyme activator) is expressed only in the nucleus. When these two interact, active enzyme is formed, generating signal. This can detect protein translocation to the nucleus or nuclear envelope degradation during mitosis.
All the assays run on any chemiluminescence-capable plate reader with a single reagent addition step, do not require special instrumentation or antibody labeling. An advantage of using an enzyme approach is that it amplifies the signal—up to a twenty or thirty signal to background ratio. “One of the challenges of FRET and HRET, the two most common ways to address protein-protein interactions, is that they don’t generate a lot of signal. After you finish building an assay, you may only have a two- to threefold signal to background ratio,” Dr. Olson states.
In addition, the system is tunable. “We’ve been able to modulate the b-gal reporter system so we have essentially no background association of the two components. The only way to generate signal is to have productive protein-protein interactions.”
The company also offers the PathHunter b-Arrestin Assays for over 100 G-protein coupled receptors (GPCRs). These are high-throughput screening assays for monitoring GPCR activation following ligand stimulation, without imaging instrumentation, fluorescent protein tags, or radioactivity. It provides direct measure of b-Arrestin binding to the GPCR of interest during activation, making it useful for de-orphanizing novel GPCRs. Dr. Olson says the company has recently started a program to look at transcription factors and cytoplasmic kinases involved in general cell signaling events.
xCELLigence™, a cell-based assay system codeveloped by ACEA Biosciences and Roche Applied Science, combines microelectronics and cell biology to provide a label-free process to monitor live cells under physiological conditions. A disposable 96-well E plate contains a microelectronic biosensor in each well. “The cells interact with the gold microelectrodes, leading to changes in impedance,” explains Yama Abassi, Ph.D., senior director, cell biology, ACEA. Each change in the cells’ status, i.e., adhesion, cytotoxicity, cell proliferation, cell-cell interactions, or morphological changes, alters the impedance measurements, and therefore, can be quickly detected in real-time.
Eliminating labels brings the cells closer to physiological conditions and avoids costs associated with radioactive dyes. The microelectronic readout is noninvasive, so cells are not destroyed and can be continuously monitored. This, says Dr. Abassi, allows the user to build a kinetic profile of the experiment. “It gives us a quality-control picture of how the cells are behaving inside the wells and then when we treat the cells with a particular compound, we can observe if the response is immediate. This could not be accomplished by standard endpoint assays.”
Recent research efforts have been focused on two areas: morphological profiling and safety pharmacology. “We’ve noticed that, when we grow cells and add compounds, we get specific genetic signature patterns. These are dependent on the mechanism of action of the compound,” Dr. Abassi states.
Safety pharmacology efforts are focused on cardiotoxicity effects. His group uses embryonic stem cells differentiated into cardiomyocytes and monitors the effects of pro-arrhythmic compounds on these cells. This is significant because, according to Dr. Abassi, “almost 30 percent of lead compounds being developed ultimately fail because of some type of cardiotoxicity. It’s a huge problem and people are looking for ways to predict it much earlier in the drug discovery pipeline.”
Label-Free HT Platform
Label-free systems can generate fewer false positives and reduce nonspecific effects that occur in labeled assays, states Debra Gallant, product marketing manager, MDS Analytical Technologies. The company’s CellKey™ platforms (96- and 384-well formats) use a noninvasive, impedance-based approach that measures changes in impedance in response to activation of signaling pathways within cells.
A monolayer of cells (adherent, suspension, or primary) is seeded onto a microplate that has electrodes on the bottom of the wells. A small voltage is applied through the electrodes: low frequency passes between the cells, high frequency goes through cell membranes. Impedance can be altered by cell adhesion, morphology, volume, and changes in the interaction between cells.
“This platform is really an enabling tool for measuring endogenous receptors of primary cell types. The resulting data is biorelevant. The industry has recognized that it needs drug discovery assays that are more physiologically relevant in order to help it prioritize its lead compounds sooner and more effectively,” adds Gallant.
Another key feature to this system is onboard fluidics, which enables the simultaneous measurement of cell response while adding a compound. CellKey response profiles assist in understanding the underlying pathways activated upon compound or ligand addition, for example, differentiating between different classes of GPCRs. The profiles can also be used in the later stages of lead identification to study effects of different inhibitors and to gain more detailed information on the mechanism of action of lead compounds. Additional applications being developed for the CellKey system include ion-channel analysis and the use of additional primary cell types.
Brighter TR-FRET Assays
Through a recent partnership with Lumiphore, Cisbio Bioassays now incorporates Lumi4™-Tb cryptate for its HTRF® platform for GPCR screening. This molecule’s structure is a lanthanide tightly embedded in a surrounding macrocyle and provides enhanced brightness and greater stability. “This is important in kinase and GPCR research,” explains Francois Degorce, head of marketing, “where you have to use serum or a complex medium.” Other features include enhanced assay performance in terms of sensitivity, assay window, and robustness.
Two new assays are using Lumi4-Tb: the IP-One Tb and cAMP Tb. Originally launched in 2005, the IP-One was the first cell-based HT system to detect inositol phosphate (IP1), which closely correlates with Gq-coupled activity. By replacing Europium cryptate with Lumi4-Tb, the new assay offers increased sensitivity and enhanced signal-to-noise ratio.
Another advantage to this molecule is that it is compatible with a variety of acceptors, including near infrared and green acceptors like fluorecein. This enables multiplexing assays, while maintaining high throughput. “You can multiplex IP-1 with a cytokine assay to investigate whether a unique GPCR can signal through both pathways. You can also multiplex several cytokines at once.” This assay is called HTplex™ and allows simultaneous codetection of IP-1 and cAMP in one well.
Researchers at St. Jude Children’s Research Hospital are combining cell-based assays, high-content screening, and primary cells to discover new compounds that increase understanding of the pathophysiology of pediatric disease. Taosheng Chen, Ph.D., director of chemical biology and therapeutics, says there are specific challenges with pediatric diseases. “We don’t usually have a validated target to start with, so this must be done first. This is different from big pharma, where every target is fully validated.”
His group uses ACEA Biosciences’ label-free screening and a time-lapse microscope (IncuCyte™) that provides real-time growth of cells. Dr. Chen claims his group started using primary cells to do small-scale, proof-of-principal cell-based assays. The primary cells provide an advantage in that they are physiologically relevant, but do pose some limitations. These include: difficulty in culturing, variability, low fluorescence, and minimal availability.
The hospital has an on-site library of 525,000 commercial compounds, with ongoing efforts to develop its own library of unique compounds more specific to its targets. Dr. Chen explains that when they need to validate a target they use a similar approach as big pharma. He adds that in oncology, however, the target and pathway are often unknown; only the phenotype to achieve is known.
In addition, pediatric diseases have unique targets, such as fusion transcription factors. These are associated with chromosome translocations and common in pediatric oncology. “When these transcription factors are knocked down, you don’t necessarily kill the cells. This indicates there is another mutation involved, which has to be identified,” adds Dr. Chen.
Cancer Drug Combinations
In order to assess the effects of combining two anticancer drugs, researchers at Amgen (www.amgen.com) have enhanced a commercially available cell-based HT assay system. Sharon Zhao, Ph.D., senior scientist, lead discovery, says her group adjusted the automation to fit their assays and plate layout, made it high throughput, and developed their own data analysis calculations.
“We test two compounds mixed together to see if they work synergistically or if there is some other effect. This helps provide information on what two drugs for which patient will work best.” Dr. Zhao reports that the assay monitors how well the drugs kill cancer cells.
This assay can be applied during early- stage drug discovery to see if two drugs can work better together, and during later-stage drug development for clinical compounds. The platform can test 30–50 cancer cell lines (versus about 10—the standard for academia) and more combinations. Primary cells are difficult for this application because they are not easily expandable, so her group uses standard cancer lines like CHO.
Some of the challenges of cell-based assays in oncology include relating in vitro data to in vivo predictions, cell specificity, and off-target effects. Dr. Zhao says they are focusing on expanding the system and trying more cell lines and combinations. “We’re trying to collect information more thoroughly about the cell lines, like expression and mutations. We’re trying to see whether we can correlate this data with other data like gene expression profiling or mutation characteristics. These connections will make our data more useful and more predictable.”
There is no doubt that cell-based assays provide invaluable information for drug discovery. The trends now are disease-relevant and primary cells to create a physiological environment to obtain optimal leads earlier. In addition, there is a push to develop more ways to screen functional activity of endogenous receptors forward in the discovery process instead of downstream after HTS. Researchers are hoping that these assays may help bridge the gap between in vitro assays and in vivo predictions, which remains a big challenge.
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