Cell Based Assays - Seeing the light
High-context cell assays have the potential to take the pivotal role in the drug discovery process. So what are the features of today's leading edge cell based assay technologies and where does the future lie?
We live in interesting times. On one hand the completion of sequencing of the human genome presents the drug discovery community with a wealth of new potential targets, biological questions to ask and problems to solve (1); while on the other hand many new and increasingly sophisticated technologies are being developed which allow these new questions to be asked with the reasonable prospect of getting an answer.
Many technologies originally developed for automation of HTS (high density microplates, robotics, automated liquid handling, laboratory information software, etc) have been exported and applied in other disciplines to bring a factory approach to high-throughput biology in genomics (2). This process has now come full circle with the fruits of this work now re-entering the drug discovery process as new targets in search of validation and drug screening assays. While this wealth of new prospects brings the promise of new targets, it also brings uncertainties.
It has been estimated that costs for developing drugs for new targets emerging from the Human Genome Project will be significantly greater than for established targets (3), where costs are already considered to be excessive by many. These new targets will require validation, and for targets outside of those molecular classes and cellular processes currently familiar to the pharmaceutical industry, may also require new assays and assay technologies.
So it is therefore somewhat ironic that those technologies originally designed to remove bottlenecks in drug screening, having been taken outside and applied to genomics, are now contributing to the generation of a new bottleneck in the postgenomic era. As little as one or two years ago all focus on bottlenecks in drug discovery was on processes downstream of primary screening in secondary screening and ADME-Tox (4), and developments in solution and cell-based screening technologies were evaluated against their place and potential in the classical linear drug discovery pathway (5,6).
Today as technologies and biological discoveries advance on parallel fronts, the demands of functional genomics and target validation are clearly joint contenders for the most significant bottleneck in rapidly advancing the development of new drugs.
Where do cell based assays fit into today’s drug discovery process? At one level, particularly for primary drug screening, cellular assays can be viewed solely as a means to an end (7). If a cell assay enables a target to be screened in a more efficient manner than a solution assay, then the cell assay is a viable approach, even if in this context the cell is in fact merely a conveniently pre-packaged collection of assay components.
While this approach may be enabling in certain aspects of HTS, it ignores the fundamental potential of a cell based assay to characterise, analyse and screen a drug target in situ and in context.
Many processes in drug discovery, while all addressing aspects of the same biological continuum, operate independently, often using different assay systems and technologies. While this situation may be a pragmatic result of technological reality, it is not an efficient process. Recent and continuing developments in technology have provided drug discovery with an increasingly powerful armoury of tools to dissect biological processes. Today many of these tools can be used in conjunction with live cells on a variety of instrument platforms that together span a technology continuum across biology and drug discovery from genotype to phenotype to screenotype (8).
Consequently with these tools researchers in different disciplines can begin to use the same sets of tools to perform complex multiparameter assays for target validation and screen development using live cell assays. They also have the ability to transfer the same assay readout into a robust high throughput screening format, and to return screening hits for further characterisation. Consequently, high-context cell assays have the potential to take a pivotal role in the drug discovery process in applying high-quality criteria to inputs from high-quantity activities (Figure 1).
What are the features of leading edge cell based assay technologies that enable this continuum? Today, fluorescence technologies predominate, and many technologies based on reporter gene assays (9), ion channel probes (10) and green fluorescent protein (GFP) (11,12) are in widespread use.
While these basic tools remain valuable, further developments of these and other probes, coupled with advances in instrumentation are extending the capabilities of cell analysis systems for target validation and drug discovery. Today’s fluorescent probes and reporter technologies with the greatest potential for enabling a biology and technology continuum share a number of common characteristics:
Such fluorescence reporters available today and in development by a number of companies, will permit the design of live cell assays with the ability to analyse individual biological responses across cell populations, to track the movement of molecules within cells, to correlate responses with cell cycle position or other inherent cellular parameters, and to analyse two or more independent assay readouts from the same cell. Coupled with appropriate hardware and analysis software these reagents will allow this level of sophistication to be achieved in high throughput with robust assay performance.
In what today may be viewed as the traditional heartland of cell based drug discovery assays, two relatively mature technologies, FLIPR™ (Molecular Devices) and VIPR™ (Aurora Biosciences) are used extensively for screening GPCR (13) and ion channel targets (14) using calcium and voltage sensitive fluorescent probes. These assays are based on measurement by macro imaging and consequently yield data at the cell population level; fully exploiting the potential of cell based assays requires the ability to acquire data at the cellular and sub-cellular level.
The most significant advance in realising the potential of cell assay technologies has been the development of high-resolution, high-throughput imaging systems which are capable of multicolour detection of fluorescence reporters in fixed and live cells. The ArrayScan™ from Cellomics (15) is a first-generation example of microscope imaging adapted to a high-throughput platform for ‘highcontent screening’.
The ArrayScan has been applied to analysis of GPCR internalisation (16) and a number of other applications (17), with the majority of assays being performed on fixed cells. A further development of this platform, the ArrayScan Kinetic, has been designed to extend the capabilities of the system for live cell assays. Further cell based platforms have been developed by Acumen and others (18).
The most recently developed cell analysis platform, the IN Cell analyser (19,20), from Amersham Biosciences, is a line scanning confocal cell imaging platform capable of high-speed simultaneous threecolour fluorescence image acquisition and analysis from live cells in 96 and 384 well microplates.
These high-throughput cell imaging systems have the potential to address a broad coverage of the drug discovery process from functional genomics to secondary screening and beyond (Figure 2).
Development of intrinsic and extrinsic fluorescent probes for cellular assays is a rapidly advancing area, and is being pursued in many biosciences companies and academic laboratories. One of the largest areas of continuing development is in applications of GFP, and other more recently isolated new fluorescent proteins (NFPs) (21) being developed by Clontech and others, which will undoubtedly allow the development of increasingly advanced live cell assays.
Recent agreements and collaborations between Aurora, Amersham Biosciences and the Danish biotech BioImage A/S, have resulted in the development of a range of GFP assays to analyse key intracellular signalling pathways by following the redistribution of GFP fusion proteins in live cells in real time (22) (Figure 3).
These types of assays will see libraries of cell lines engineered to report on key cellular processes become standard robust tools for drug discovery (23). The combination of GFP with high-throughput subcellular imaging offers a powerful and generic approach to monitoring intracellular events, and has recently been used to great effect in GPCR assays by Norak (24).
As high-throughput imaging instrumentation continues to advance, further developments of intrinsic biosensors (25,26) based on GFP and NFPs offer the potential to monitor protein:protein interactions directly in real time and space. The ability to follow cellular protein connections in situ is a key area for further development of target validation and screening assays and a number of approaches show promise (27), including methods based on enzyme reassembly or complementation such as the DHFR system developed by Odyssey (28).
Advances in intrinsic biosensors are paralleled by developments in extrinsic probes and reporters (29). Reporter gene assays continue to be refined and made compatible with non-destructive assays. Aurora’s GeneBLAzer™ system was the first to allow gene expression control to be analysed in living cells using different detection platforms, including FACS isolation of responsive cells (30).
More recently, Amersham Biosciences has developed a live cell reporter gene system based on its core Cyanine dye technology. The nitroreductase gene reporter system (31) uses CytoCy substrates in which enzyme activation is independent of the chromophore. This allows fluorescent substrates to be designed in a range of colours to allow matching to different instrumentation and multiplexing with other cellular reporters, including GFP. Further development of Cyanine dyes as cellular probes include CypHer™5 (32), a pH sensitive analogue of Cy™5 which allows imaging of internalisation of GPCRs and other cell surface receptors in multiplexed assays with GFP.
A next generation of extrinsic cellular sensors based on site-specific labelling of recombinant proteins (33) or protein domain ligation (34) have the potential to provide tools for directly visualising cellular events that, for reasons of steric hindrance, are refractory to the use of large fluorescent protein labels such as GFP. These techniques will also permit the use of new fluors, and new combinations of fluors, and allow a wider range of fluorescence detection techniques, including timeresolved, lifetime and polarisation to be applied to cellular assays.
Coupled with advances in imaging hardware such sensors may also come into play in dissecting cellular processes at even finer detail through single molecule detection (35). Further scope for multiplexed analyses is provided by new classes of inorganic fluors based on nano-crystals and quantum dots (36) which offer extremely photostable narrow bandwidth labels.
Of course, fluorescence is not the only method of monitoring cellular events and as technologies advance other modes of detection will be employed alongside fluorescence assays, including electronic methods based on electrode arrays such as those produced by Panasonic (37), and cell-transistors (38). Fully exploiting the potential of cell assays will also require the implementation of other complementary technologies which allow precise engineering of cell phenotypes on a transient or permanent basis by modulating protein expression in target cells (39,40).
It is self evident that cellular assays are only as powerful as the available probes and reporters. Full integration of increasingly sophisticated cell analysis tools with enabling hardware will allow advancement of target validation and screening from ‘high-content’ to ‘high-context’, and provide a technology continuum to address biological questions from phenotype to screenotype. Making and using these tools to unravel the interacting networks (41,42) of biological systems using cell assays offers an ongoing challenge. We will continue to live in interesting times. DDW
The author would like to thank Angela Williams, Cell Biology Department, Amersham Biosciences, for providing the data in Figure 3.
Dr Nick Thomas is a Staff Scientist in the Cellular Sciences research group of Amersham Biosciences based in Cardiff, UK. He is currently working on a number of projects developing novel fluorescent probes and reporters for use in live cells for target validation and drug screening.
1 Bumol, TF and Watanabe, AM. Genetic Information, Genomic Technologies and the Future of Drug Discovery. Journal of the American Medical Association 2001 285:551-555.
2 Brent, R. Genomic Biology. Cell 2000 100:169-183.
3 Lehman Bros. The Fruits of Genomics, 2001.
4 Dove, R. Drug screening – beyond the bottleneck. Nature Biotechnology 1999 17:859- 863.
5 Hertzberg, RP and Pope, AJ. High-throughput screening:new technology for the 21st century. Current Opinion in Chemical Biology 2000 4:445-451.
6 Sundberg, SA. Highthroughput and ultra-highthroughput screening: solution and cell-based approaches. Current Opinion in Biotechnology 2000 11:47-53.
7 Moore, K and Rees, S. Cell- Based Versus Isolated Target Screening: How Lucky Do You Feel? Journal of Biomolecular Screening 2001 6(2):69-74.
8 Screenotype: cell engineered with biological reporter compatible with analysing candidate drug action with a target of interest.
9 Gonzalez, JE and Negulescu, PA. Intracellular detection assays for high-throughput screening. Current Opinion in Biotechnology 1998 9(6):624- 31.
10 Gonzalez, JR et al. Cellbased assays and instrumentation for screening ion-channel targets. Drug Discovery Today 1999 4(9):431-439.
11 Kain, SR. Green fluorescent protein (GFP): applications in cell-based assays for drug discovery. Drug Discovery Today 1999 4(7):304-312.
12 Kendall, JM and Badminton, MN. Aequorea victoria bioluminescence moves into an exciting new era. Trends in Biotechnology 1998 16(5):216- 24.
13 Jurewicz, AJ et al. Fluorometric imaging plate reader: using the orexin receptor to screen 145,000 compounds. Genetic Engineering News 1999 19:44-46.
16 Conway, BR et al. Quantification of G-protein coupled receptor internalisation using G-protein coupled receptor-green fluorescent protein conjugates with the ArrayScan highcontent screening system. Journal of Biomolecular Screening 1999 4:75-86.
17 Giulano, KA et al. Highcontent screening: a new approach to easing key bottlenecks in the drug discovery process. Journal of Biomolecular Screening 1997 2:249-259.
18 Trask, OJ and Large, TH. Automated Imaging: Applications to drug discovery. Current Drug Discovery September 25-29.
19 Hansen, R et al. Measurement of fixed and real time cellular assays using the LEADseekerTM Cell Analysis System, poster presented at 6th SBS Conference Vancouver 2000.
20 www.apbiotech.com/ application/Drug_screening/pu blic/lcas/default.html
21 Terskikh A, et al. “Fluorescent Timer”: Protein that Changes Colour with Time. Science 2000 290:1585-1588.
22 Goodyer, ID et al. Screening of signalling events in live cells using novel GFP redistribution assays, poster presented at 7th SBS meeting, Baltimore 2001.
23 Pagliaro, L and Praestegard, M. Transfected Cell Lines as Tools for High Throughput Screening: A Call for Standards. Journal of Biomolecular Screening, 2001 6(3):133-136.
24 www.norakbio.com/ Transfluor.html
25 Harpur, AG et al. Imaging FRET between spectrally similar GFP molecules in single cells. Nature Biotechnology 2001 19:167-169.
26 Ozawa, T et al. A Fluorescent Indicator for Detecting Protein-Protein Interactions In Vivo Based on Protein Splicing. Analytical Chemistry, 2000 72:5151-5157.
27 Mendelsohn, AR and Brent, R. Protein Interaction Methods – Toward an Endgame. Science 1999 284:1948-1950.
28 Remy, I and Michnick, SW. Visualisation of biochemical networks in living cells. Proceedings of the National Academy of Sciences, 2001 98(14) 7678-7683.
29 Durick, K and Negulescu, P. Cellular biosensors for drug discovery. Biosensors & Bioelectronics 2001 16:587- 592.
30 Whitney, M et al. A genomewide functional assay of signal transduction in living mammalian cells. Nature Biotechnology 1998 16:1329-1333.
31 Ismail, RI et al. Nitroreductase – A new live cell gene reporter system, poster presented at 7th SBS meeting, Baltimore 2001.
32 Cooper, ME et al. pH Sensitive Cyanine Dyes for Biological Applications, poster presented at 7th SBS meeting, Baltimore 2001.
33 Nakanishi, J et al. Imaging of Conformational Changes of Proteins with a New Environment-Sensitive Fluorescent Probe Designed for Site-Specific Labeling of Recombinant Proteins in Live Cells. Analytical Chemistry 2001 73(13) 2920-2928.
34 Cotton, GJ and Muir, TW. Generation of a dual-labeled fluorescence biosensor for Crk-II phosphorylation using solid-phase expressed protein ligation. Chemistry & Biology 2000 7(4):253-261.
35 Byassee, TA et al. Probing Single Molecules in Single Living Cells. Analytical Chemistry 2001 72(22) 5606- 5611.
36 Mitchell, P. Turning the spotlight on cellular imaging. Nature Biotechnology 2001 19:1013-1017.
38 Offenhausser, A and Knoll, W. Cell-transistor hybrid systems and their potential applications. Trends in Biotechnology 2001 19(2):62- 66.
39 Harrington, JJ et al. Creation of genome-wide protein expression libraries using random activation of gene expression. Nature Biotechnology 2001 19:440- 445.
40 Elbashir, SM et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001 411:494-498.
41 Weng, G et al. Complexity in Biological Signalling Systems. Science 1999 284:92-96.
42 Bailey, JE. Lessons from metabolic engineering for functional genomics and drug discovery. Nature Biotechnology 1999 17:616- 618.