August 1, 2006 (Vol. 26, No. 14)
The Paradigm of One Gene, One Target, One Drug Is Being Widely Revised
At Cambridge Healthtech Institute’s recent “Intelligent Drug Discovery and Development” conference, presenters talked about the ever-evolving role of the cell-based assay in drug development.
“In the past, the drug discovery model was pretty straightforward: one gene, one target, one drug. This model has failed,” said D. Lansing Taylor, CEO of Cellumen (www.cellumen.com). “A couple of issues relate to this failure, but the bottom line is that the cell is a system, an integrated, interacting network of genes, proteins, and other cellular constituents that produce functions. We need to analyze the systems’ response to drug treatments, not just one target or pathway.”
Taylor’s presentation highlighted a hand-picked selection of the most recent publications from the past year that offer novel technological advances in the cell-based screening arena.
The key to understanding the genome is understanding how cells work using the gene products, Taylor added. “You can manipulate certain elements within the cell and then make systems cell biology measurements. Using the role of p53 in about half of all cancers, we can manipulate the levels of either p53 or HDM2 and then do a readout of 10-15 different parameters, profiling for apoptosis, metabolic changes, multiple organelle functions, as well as other specific biochemical changes.”
And this information is important for moving compounds down the product pipeline. One needs to take the same approach in cytotoxicity profiling. “Greater than 30% of failure is due to toxicity,” said Taylor. “Another 30-40% is lost because the compound just doesn’t work.”
Taylor also thinks that function will be the ultimate driver of pharmaceutical research, and cells will play a critical role since they are the first systems-level organization. “Using multiplexed fluorescence is a key development,” Taylor said.
Dual Stable Cell Line Alternatives
Promega’s (www.promega.com) presentation featured bioluminescent cell-based assays, specifically the dual luciferase assay it has developed. Bioluminescent cell-based assays are well-suited for high-throughput screening because of their high sensitivity, wide dynamic range, and minimal interference by the compounds. “Our goal is to make things as simple for the scientist as possible,” said Erika Hawkins, senior investigator at Promega. “To that end, we constructed dual stable cell lines to reduce false positives resulting from compound-induced cytotoxicity in antagonist screens.”
In agonist screening, single-reporter, uncorrected data cannot distinguish between specific down regulation and cytotoxicity, as both responses exhibit a similar decrease in luminescence. In Promega’s Dual-Glo Assay, however, the use of dual reporters can discriminate these responses.
“Detection systems need to be optimized for a rapid half-life, which means decreasing the time to get a good response, and increasing the window of opportunity for a good response,” Hawkins said. “Using dual stable cell lines reduces off-target response and gives us data that are easy to see and track.”
Promega constructs stable dual luciferase cell lines using two plasmids. One plasmid expresses firefly luciferase genes under the control of a response element (e.g., CRE or NFAT) and a hygro-mycin selectable marker that has been stably transfected into master cell lines. The second plasmid expresses target GPCR (e.g., dopamine receptor D1 or muscarinic 3 receptor M3R) and a Renilla luciferase-neomycin selectable marker fusion. Using destabilized luciferases resulted in faster signal responses and shorter assay times and thus a general reduction in opportunity for cytotoxicity from library compoundsACEA Biosciences (www.aceabio.com), presented label-free technology and applications for cell-based assays. The core of ACEA’s RT-CES system is the microelectronic cell sensor arrays integrated into the bottom of microtiter plates. “For cell-based assays, cells are grown in the individual, sensor-containing wells of the microtiter plate. The electronic sensors provide continuous, quantitative information concerning the biological status of the cells present in the well.”
Changes to the biological status of the cells are measured automatically and in real time by the RT-CES system. There are no labeling steps with expensive reagents, and the entire process is noninvasive and harmless to the cells. “For cell-based assays, we are dealing with a living thing, a live cell,” said Xu. “Without knowing the cell status before the treatment and without knowing the kinetics of live cell response to the exposure of chemical compounds, it will be hard to get a consistent result between experiments.”
The actual variable being measured by the RT-CES system is derived from the change in electrical impedance as the living cells interact with the biocompatible microelectrode surface in the microplate well. Using a proprietary algorithm, the signal is converted to a specific parameter called the Cell Index, a measure of what the cells are actually doing over time growing, spreading, changing shape, dying, responding to specific stimuli, and so on.
A range of cell-based assays can be performed on the RT-CES system, including cell proliferation, cytotoxicity profiling, ADCE, cell adhesion, and functional analysis of receptors, such as GPCR, growth factor receptors, and death receptors.
Xu emphasized the importance of label-free capability. “This is a QC issue with cell-based assays. You need to know that your cell quality is good. I know how important it is for scientists to have the tools they need to do their work.”
Zhong Zhong, vp of drug discovery technologies at Cell and Molecular Technologies (www.cmt-inc.net), demonstrated that division-arrested cryopreserved cells performed with better consistency than actively cultured cells in screening applications. These ready-to-use reagents can be thawed, plated, and used in screening to improve consistency, convenience and cost efficiency.
“Cell-based assays are becoming increasingly popular for small molecule discovery in high-throughput screening,” said Zhong. “Advanced HTS technologies require standardized, consistent cell culture as reagents in order to generate reliable results. Division arrest technology, which is a scalable process, allows the use of frozen assay-ready cells for screening as if they are ordinary assay reagents like enzymes or membrane preparations.”
The division-arrest technology effectively uncouples the laborious, time-consuming process of cell production from the robotic screening process and gives users the confidence to focus on assay development.
Invitrogen (www.invitrogen.com) has a portfolio of cell-based assays. Richard Somberg, Ph.D., research area manager, quantitative biology, presented findings made with CellSensor Technology, a growing product line of tools for pathway profiling.
Cell Lines can be used to screen for perturbagens in over 15-20 endogenous signaling pathways and in specific protein targets involved in these pathways. High-throughput compatibility of these cell lines enables their use in both primary and secondary screens. The CellSensor cell lines are created through stable integration of a response element upstream of beta-lactamase. “It is the combination of response element and cellular background,” Dr. Somberg said, “that determines what signal is transduced.”
And as with any proven technology, there are multiple points of entry. “Customers can design their own vectors, use our off-the-shelf vectors, or we can build vectors and or cell lines to suit customer specifications,” Dr. Somberg added.
Using CellSensor cell lines, a researcher can discover what compounds affect a particular pathway. “The goal,” Dr. Somberg said, “is to build a series of cell sensors so that customers can take their leads to the next level. We have over 30 cell lines ready to screen, and if a customer has a cell model or tissue or specific cell line, they can build their own cell lines using this technology.”
“One important aspectand this is where people run into trouble is that there are sometimes interesting results in the cell model that you can’t always predict from the biochemical profile of a particular compound.”