Living the high (throughput) life
MARK S. LESNEY
HTS looks for rewards from the use of cellular assays.
ILLUSTRATION: GEOFF SMITH
Everyone talks about the marvels of drug design in silico or the utility of cell-free screening using cloned receptors. The hottest news is of some newly synthesized target molecule derived from genomic studies. But sometimes, drug discovery must be tackled as if it were literally as big as lifeby using living cells for a particular high-throughput assay.
According to D. Lansing Taylor and colleagues at Cellomics, Inc. (Pittsburgh), The postgenomics era has ushered in the era of the cell (1).
Cell-based assays are critical to assessing the behavior of a drug in an environment comparable to its presumed site of action. A drugs toxicity, ability to enter a cell, and overall efficacy can be determined under something more like field conditions. And unlike simple biochemical assays, in which the target protein for a drug is already known, cell-based assays keep open the possibility of discovering drugs that interact with hitherto unknown targets.
But even knowing the target protein for a drug is not necessarily enough for an appropriate noncellular assay. Researchers are increasingly realizing that the primary amino acid sequence can rarely predict in situ structure or interactions, especially for drugs targeting membrane-bound or compartmentalized proteins. The final architecture of a receptor is profoundly affected by the cellular environment in which the protein finds itselffrom nearby lipids and accessory proteins to changes induced by pH or coenzyme interactions. Artificial cells or lipid micelles can solve some of these problems, but in many cases there is nothing in the drug discovery process like life to mimic lifethe laboratory equivalent of the clinical trial.
The problem, of course, for high-throughput screening (HTS) of cells, is that life is a bit more delicate and finicky than a simple protein in solution or one bound to a microtiter plate or polymeric beads.
First, in the initial growth stages there is the requisite to maintain sterility, proper temperature, CO2 and O2 levels, and dispersion or adhesion. Then, optimum conditions must be controlled throughout the distribution of cells into the appropriate HTS vehicle. In most cases, this involves localizing the cells in place, usually in specially designed microtiter dishes. The unique requisites of certain cells or the demands of a particular assay typically require the use of plates with special coatings, often poly-d- and poly-l-lysine or collagen.
Any necessary subsequent growth must be allowed for, and appropriate conditions must be maintained during the screening period. There is no point in using sophisticated fluorescent probes to study the effect of your target compounds on growth or apoptosis when your cells are barely clinging to life, either because of a leak in your CO2 system or from covert mycoplasma contamination. These dangers can make cell cultures seem a nightmare compared with using target proteins taken from a vial stored in the 20 °C freezer for instant use.
To make matters worse, in many cases the cell types needed as a particular drug target may not be easily amenable to culture. Highly differentiated cells may have nonobvious, or currently unknown, requirements for surviving in culture. And rarely do they maintain the full gamut of their unique characteristics long-term in vitro. Here, the area of cell differentiation studies, including a significant amount of research using controversial stem cells, is a critical path to future developments.
But even with all of these problems, cells remain an important accessory to the drug discovery process and are likely to become even more so.
How cells die
Quickly identifying potential drugs that kill, debilitate, or transform cells is an important part of the drug discovery process. In the case of cancer cells, the goal is to develop chemotherapeutics that promote differential cell death (i.e., kill cancer cells while letting normal cells live). This search is often a quest to discover the triggers of apoptosisa complex cascade system actively leading to cellular suicide.
A wide variety of chemical probes is used to monitor the difference between living, dying, and dead cells (living cells uniquely take up or exclude an increasingly complex battery of dyes or fluorescent compounds). The most common dye systems, such as methylthiazol tetrazolium (MTT), are based on the conversion of tetrazolium dyes to blue formazan products. Unfortunately, many standard assays for cell viability do not take into account the process-oriented nature of apoptosis.
Because apoptotic or otherwise damaged cells may take a long and variable time to die, the gold-standard assay is clonogenicit consists of taking treated cells, plating them, and determining the number of single-cell-derived colonies that subsequently form. This assay gives the best indication of long-term cell viability after drug treatment, although it is more time-consuming and difficult than instant viability checks. Changes in cell density or metabolic parameters associated with growth can be measuredthe flip side of monitoring cell death. Clonogenic assays are also used to assay toxicity by demonstrating differential survival and growth.
Other important reasons to assay cell death in HTS assays include investigating the comparative effectiveness of antiviral and antimicrobial compounds and examining the general cytotoxicity of new drugs (no nasty side effects wanted).
How cells live
Assessing drugs for their effect on active cellular metabolism is key to identifying compounds that affect processes rather than trigger an apoptotic shutdown. One new and popular form of metabolic assay involves the use of a reporter gene fused to the gene of interest, whose product the drug must trigger or affect.
An example of this kind of assay adapted to cellular HTS is Packard BioSciences bioluminescence resonance energy transfer (BRET) assay system. BRET was developed from fluorescence resonance energy transfer (FRET) assays, which measure the proximity between interacting fluorophores to determine the degree of interaction. The enzyme Renilla luciferase (Rluc) is used as a donor, and a green fluorescent protein (GFP) is used as an acceptor molecule. Upon addition of a proprietary compound that serves as the substrate for Rluc, the BRET signal is measured by comparing the amount of blue light catalyzed by Rluc to the amount of green light emitted by GFP. The ratio of green to blue increases as the two proteins are brought into proximityRluc donates its energy to GFP rather than using it to fluoresce itself.
Researchers perform assays by fusing the genes of Rluc and GFP to two respective biological partners that are expected to interact in a cell-based assay. Changes in the interaction (and locations) of these proteins caused by ligands or drug test compounds can be evaluated by monitoring changes in the ratio of blue to green light emitted by cells.
Other forms of metabolic cell-based assays include those that determine various kinetic properties. For example, receptor binding is assayed by examining a unique conformational response of the receptor itself or by looking at specific metabolic changes that happen when a compound attaches to its receptor or is taken into the cell via the receptor. Or assays can monitor the changing cellular concentrations of highly specific metabolic products. One example of the latter is Packards Alphascreen, which assays whole-cell cyclic adenosine 5´-monophosphate (cAMP) by using a biotinavidin-based system that can monitor competition between endogenous cAMP and exogenously added biotincAMP. In this way, important aspects of cellular energetics can be monitored.
The whole shebang
According to Taylor and colleagues, the integration and automation of all such metabolic assays are necessary to properly investigate the next stage after the genome, that is, the cellomethe entire complement of molecules and their interactions in a cell.
The development of new bioluminescence proteins with varied active wavelengths (colors) should result in the ability to decipher ever more complex interactions in multiply transformed cellsa critical necessity in assaying multidimensional metabolic processes for more sophisticated drug discovery using HTS. A similar benefit will derive from the development and implementation of new fluorescent probes for metabolism.
The era of the cell will require the development of automated cell analysis instrumentation and searchable databases of the cellome . . . measur[ing] temporal and spatial distributions and activities of targets and cellular constituents in and between cells using multicolor fluorescence assays (1). Taylor and others are working to develop tools to offer the same type of power for cell analysis that is available for genomics. With this in mind, Cellomics is touting a future in which cells on chips will provide the same type of HTS value as DNA microarrays.
No cell is an island?
Recently, some companies have begun to claim that cells aggregated into their logically complete packagea whole organismhave a place in the world of high-throughput drug screening. Genetically engineered test animals that can be produced and used easily en masse are the key.
Automated fluorescent sorting of small model animals has been shown to be feasible by Union Biometrica (Somerville, MA) and may well prove valuable for HTS in the drug discovery process. The complex object parametric analysis system (COPAS) platform, as it is called, uses liquid flowing through a laser beam to quickly assay a stream of wee animals (such as the nematode Caenorhabditis elegans or the fruitfly, Drosophila sp.) to gather information on their size, which is valuable for determining life cycle stages, and fluorescent marker response, which can verify reporter gene status or a treatment response.
Proper sampling technique and automation could transform this technology into an adjunct to ordinary cellular HTS. This could provide the obvious added benefits (compared with simple cellular assays) of assessing such factors as developmental effects, including complex differentiation, hormonal gradients, and organcell communication on the behavior of test compounds. Significantly, the COPAS system can sort organisms and deposit them in individual microtiter plate wells for subsequent growth and reproduction. This is especially important in developing reporter gene populations for subsequent analysis, because the take on reporter gene incorporation is notoriously low for whole organisms in most systems. Such an enhanced population would be required to simulate the transformed cell lines normally used for HTS studies using reporters.
At an even more organized level, evolutionarily speaking, Mermaid Pharmaceuticals GmbH (Hamburg, Germany) and Gene Tools, LLC (Corvallis, OR), have joined forces to work on developing knockout and transformed zebrafishthe aquarium stapleas a high-throughput target discovery technology.
Life in the future
Whether or not whole organism or organismo screening comes into its own, the use of living cells will inevitably remain a prominent part of the discovery process, from target identification to initial screening, from safety and efficacy testing to validation. As far as our current understanding goes, and for all of our improved computer models, the living cell still behaves as if it were greater than the sum of its partsnot because of any peculiar élan vital, but because of its complexity and our ignorance. In an ever-increasing number of instances, even as we learn more about the genome, in vitro chemistry can still only augment biology, not replace it. The rewards of a cell-based approach to drug discovery remain there for the taking.
|Breaching the brain
|However effective a drug may be in in vitro target binding studies, it is no good if it cannot be delivered to its site of action. Thus, a key challenge in screening for drugs that affect the central nervous system, for example, is finding compounds capable of getting from the bloodstream into the brain across one of the most effective physiological walls knownthe bloodbrain barrier. This protective layer of cells is composed of tightly spaced brain capillary endothelial cells surrounded by astrocytes, pericytes, and microglia. Freshly isolated brain endothelial cells have all of the unreliability associated with primary tissue cultures.
The standard substitute for assay purposes is the well-established and consistent MadinDarby canine kidney (MDCK) epithelial cell line, which forms a tight monolayer similar to that of brain endothelial cells. When cultured on a porous membrane, the MDCK line, which also shows physiological similarity to brain cells in several key enzymes, can adequately mimic the bloodbrain barrier for drug screening purposes. The drug candidate is placed in a chamber on one side of a porous membrane covered with the attached mono layer of cells and then assayed for migration into the chamber opposite using any number of analytical techniques, including HPLC, LC/MS, or, for easy automation, fluorescent techniques.
Typically, compounds targeted to the central nervous systemsuch as diazepam, haloperidol, and saquinavirshow equivalent permeability across the MDCK barrier as seen in vivo. Similarly, Caco-2 cell monolayers, which are used to test permeability, are a model system for studying oral bioavailability across the gutblood barrier.
Adapting these types of permeability assays to HTS requires the use of radiation-opaque membranes as microporous inserts on which to grow the cells. Blocking the cross-transfer of fluorescence and luminescence allows data acquisition uniquely from one chamber (bottom or top) by a standard plate readera necessity for automation.
- High Throughput Screening: The Next Generation; Dixon, G. K., Major, J. S., Rice, M. J., Eds.; BIOS Scientific Publishers: Oxford, U.K., 2000.
- Cortese, J. D. At the speed of light: A look at the high-throughput fluorescence laboratory. The Scientist 2000, 14 (14), 18. Available at www.the-scientist.com/yr2000/jul/profile1_000710.html (accessed April 2001).
- Krauledat, P. B.; Hansen, P. W. Automated testing of model organisms. Genetic Eng. News 2001, 21 (4), 32.
- The Web site of Biosignal Packard, a Packard Biosciences company, provides detailed information on BRET, Alphascreen, and related technologies. www.biosignalpackard.com (accessed April 2001).
Mark S. Lesney is a senior editor of Modern Drug Discovery. Send your comments or questions regarding this article to firstname.lastname@example.org or the Editorial Office by fax at 202-776-8166 or by post at 1155 16th Street, NW; Washington, DC 20036.
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