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Cell Based Reporter Assays vs. Animal Studies in Drug Discovery- Potential Limitations, Risks and Liabilities
Posted by: sciclips on: January 16, 2012 In: Drug discovery Comment!

This blog critically analyzes the potential scientific limitations, risks and liabilities of cell based reporter assays, including non-natural protein based reporter assays (e.g. circularly permuted firefly luciferase or GFP reporter assays) and stem cell based assays, in drug discovery and development. Scientific arguments have been put forward to suggest that current format of cell based reporter assays may not be ideal for replacing animal studies. This blog also analyzes the potential negative impact of patents and intellectual property (IP) rights in fostering innovations in cell based assays. Finally, this blog warrants imposing accountability, tighter regulations and demanding higher standards in cell based assays that are used in drug discovery and development. There is a notion that cell based assays can be an attractive alternative for animal based drug discovery assays, an argument based on the fact that human cell based assays are more relevant to humans rather than the animal model studies. Convenient assay readout format, including GMP method development, and low cost are the other factors that favor cell based assays over animal based studies. However, cell based assays, including 3-D cell culture assays, do not replicate the complex nature of cellular organization, cell-to-cell communication, coordinated cell signaling pathways, tumor microenvironment etc., like in animal model studies. Even then, cell based assays (especially primary cell based assays) provide valuable information that are very critical for drug discovery and development. On the contrary, several reporter based or genetically modified (GM) cell based assays that are marketed for drug discovery may not only a scientifically non-viable alternative for animal studies but also come with several limitations and potential liabilities, possibly in animal studies. Cell based reporter assays may not be considered as an alternative to animal model studies?

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Cell based reporter assays that are marketed for drug discovery assays are often projected as an alternative to animal based studies, without well-proven scientific basis to support these claims. The assay principle and the methods appear to be scientifically appealing if it is not subjected to in-depth or comprehensive scientific analysis, which is often ignored or not considered as a factor that may have long-term consequences in any drug discovery/development studies. There are several key issues with cell based reporter assays, some of which we have already addressed in earlier blogs. Genetically modified cells used in cell based assays can be different from parental cells, primarily due to genetic and physiological alterations resulted from long-term cell culture conditions and forced expression of reporter gene/proteins, often with human or non-human protein fusion partners. Long term cultures can induce mutations and forced expression of reporter genes/proteins can alter the cell signaling pathways, which may result in false positive/negative results. The genetic and epigenetic impact of random integration of reporter genes, can vary based on the integration site or copy number of integrated reporter genes or heterogeneity (both genetic and epigenetic) of such cells, are often not thoroughly studied or understood in cell based reporter assays. Random integration of reporter genes, both in coding and non-coding regions, can alter or silence certain genes or affect genetic regulatory mechanism, and the resulting cell lines may not be uniform for such genetic or epigenetic changes. In addition, differential expression of microRNA (miRNA) or variations in metabolite pathways, which play significant role in cellular regulation and signaling process, are not well studied in genetically modified cell lines with reporter genes. The above-mentioned genetic and non-genetic factors, some of which have been discovered in recent years, can be major challenges in developing cell based reporter assays. However, if cell based reporter assays are used in drug discovery studies without understanding these critical factors successful drug discovery/development process can be scientifically challenging, cost-prohibitive and time-consuming. It can be even worse if cell based reporter assays are based on non-natural reporter proteins such as circularly permuted firefly luciferase or GFP based reporter protein. Circular permutation is considered to be an elusive molecular evolution process that involves gene duplication or exon shuffling and circularly permuted proteins may not be naturally present in mammalian cells. Very few naturally occurring circularly permuted proteins have been reported in bacteria and in plants (e.g. chloroplast associated bacterial homolog protein). Based on these scientific evidences, the reliability of circularly permuted firefly luciferase based reporter assays needs to be carefully analyzed since in vivo expression of circularly permuted proteins in cells may lead to significant changes in genetic regulatory mechanism (e.g. translational machinery), cellular metabolism, cellular physiology and cell signaling pathways. The question is, can we rely on non-natural reporter protein based cell based assays in drug discovery or development? Though the drugs that are initially selected using such cell based reporter assays do not reach up to human clinical trials, testing such experimental drugs in animals may pose liabilities. The obvious reason is, these drugs are screened and selected using non-natural reporter protein expressing cells, which may be abnormal cells. Since the natural genetic machinery of normal cells may not tolerate such non-natural proteins, forced expression of these proteins in normal cells may result in genetic or epigenetic abnormalities, which may lead to anomalous cell signaling or metabolic pathways. Extensive scientific validation of these cells or cell lines is required, prior to any animal testing, to address the biological relevance of the assay and biological or genetic or toxicity equivalence to normal cells. We do not disregard the fact that there are several reports on the use for circularly permuted proteins for improving enzyme stability or protease resistance etc. But, these proteins were artificially synthesized, expressed and purified using recombinant methods for functional or structural studies. On the contrary, in drug discovery/development applications cell based circularly permuted reporter proteins are being used for analyzing and predicting critical drug-induced outcomes using in vivo studies. The truth is that current cell based reporter assays did not adapt or evolve in parallel with recent scientific discoveries, which may have significant impact on cell-based assay outcomes. Integrated multidisciplinary considerations are needed to develop efficient cell based assays. There are several technologies currently available for analyzing the genetic, proteomic, epigenetic, miRNA or metabolite changes in genetically modified reporter cell lines. At least, some of these analyses need to be done before we use cell based

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reporter assays in drug discovery/development studies. In contrast, emerging technologies like non-invasive imaging of metabolic changes in tissues or whole animals using Raman spectroscopy look very promising in drug discovery research. Innovations in similar non-invasive tissue or animal/human based technologies, which do not involve genetic modification of cells or tissues, may open up new avenues in developing alternative technologies to cell based reporter assays. The utility of stem cell based drug discovery assays can be misleading? Stem cells are often projected as an alternative cell based system for drug toxicity, cardiotoxicitiy etc. We have addressed the limitations of stem cell based assays in drug discovery in our earlier blogs. Here we would like to address whether currently used or proposed stem cell based assays can be an alternative to animal model studies. Our answer would be no. Like any genetically modified cells, stem cells are also prone to genetic, epigenetic and physiological changes. Without understanding the physiological and genetic/epigenetic consequences of stem cell based assays, including stem cell based reporter assays, any testing of drugs selected using such assays may have adverse consequences in animal studies. However, stem cells can be a potential alternative to animal studies, provided that stem cell based drug discovery assays are developed with sound scientific principles where critical and fundamental questions are addressed and solved; with careful consideration of long-term impact. It should also be noted that normal cell based assays may not be an ideal tool for predicting adverse drug reactions (ADR), which may require animal studies. However, stem cell based assays coupled with cell/tissue-specific differentiation may offer unique tools for predicting ADR, which can be characterized though genetic, epigenetic, proteomic or metabolomic analysis. In order to achieve these goals, the scientific knowledge generated from basic discoveries needs to be tested and validated scientifically for developing technically competent and commercially viable drug discovery tools that can be used for developing safer and cost-effective drugs. Any efforts in developing commercially fast-track stem cell based assays based on speculative or nascent assumptions, projected from indirect experimental results, will be scientifically misleading and commercially unsuccessful in the long-term. Intellectual property (IP) rights can be a road block to innovations in cell based assays that can replace animal studies? The patent scenario in cell based assays can be a real road block to further innovations in this area. The major limitations are the availability of cell based assays to the customers because of the intellectual property (IP) rights, a significant factor that affects the development of improved or superior products. Exceptions are cross licensing, which itself is a very complicated process heavily associated with brand name, profitability and market share. Customers are often left with limited choices of using certain assays, which are patented with scientifically questionable broader claims and applications. Often, Claims and Experiments results/observations in patents can be scientifically unrelated. With respect to product end-user license, the use of reagents is also restricted to specific cell based assay products, even though similar superior reagents may be available in the market. The IP rights may also lead to extended licensing agreement that can generate royalty from drugs discovered though certain cell based assays, including stem cell based assays. The question is, if a drug fails due to adverse drug reactions (ADR) in patients, whether the manufactures of cell based reporter assays are also responsible for the liabilities similar to the drug manufacturer? Irrespective of these IP related issues, cell based assays that are marketed without considering the scientific merits and potential long-term risks and liabilities in animal or human studies may have to be cautiously used in drug discovery/development. Also, the use of certain cell based reporter assays, like the non-natural reporter proteins, in publically funded drug discovery projects needs to be reevaluated because the scientific validity of these assays are not clearly demonstrated. Such accountability may lead to the generation of patents with scientifically valid claims that are established through strong scientific and experimental design and analysis. This approach may ultimately help in developing innovative cell based drug discovery/development assay products or similar technologies that can replace, at least part, extensive animal studies. What are the solutions?

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The primary goal of this blog is not to find or suggest a solution, rather to address the issues that are not often discussed; and to create scientific awareness and facilitate scientific consensus to develop innovative cell based assay tools. We believe imposing accountability, tighter regulations and demanding higher standards in cell based assays may lead to the discovery of innovative cell based tools that can ultimately replace animal model studies, at least in part. Related blogs: 1. Cancer Theranostics Potential Applications of Cancer Biomarker Database 2. Are stem cells ready as a next generation drug discovery tool? 3. Cell based reporter assays: misleading approach in drug discovery? Related tools: 1. Drug discovery protocols 2. Bioprotocols 3. Drug discovery reagents 4. Drug discovery news 5. Comprehensive cancer biomarker database with companion diagnostics pathway
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Tags: adverse drug reactions, animal model studies, cell based reporter assays, circularly permuted firefly luciferase, circularly permuted GFP, drug discovery, Luciferase assays, luciferase reporter assays

4 Responses to "Cell Based Reporter Assays vs. Animal Studies in Drug DiscoveryPotential Limitations, Risks and Liabilities"

1 | rnc123 January 16, 2012 at 10:46 pm It is also interesting to point out that transfection reagents used in creating cell based reporters can also have an impact genetic and protein functions in cells. Citations: 1. Transcriptional effects of transfection: the potential for misinterpretation of gene expression data generated from transiently transfected cells: http://www.ncbi.nlm.nih.gov/pubmed/19594446 2. Artefactual effects of lipid-based cell transfection reagents on AbetaPP processing and Abeta production: http://www.ncbi.nlm.nih.gov/pubmed/16911962 Reply

2 | Sciclips January 18, 2012 at 3:21 am

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We are trying to identify and create a list of technologies or products related to drug discovery/development assays, which do not involve genetic manipulation of cells or reporter genes/proteins or multienzyme dependent readout. The assays include both in vitro and in vivo GPCR, general receptors, cAMP, protease, nuclear hormone receptors, ion channel proteins, protein kinases, PI3-Kinases etc. Please help us in identifying these technologies or products by sharing your information with us at contact@sciclips.com. Reply

3 | sciclips January 24, 2012 at 7:36 pm Additional comments on this topic can be seen at Linkedin Nuclear Receptor Resource group (open group): http://www.linkedin.com/groupItem?view=&gid=2630066&type=member&item=90563288& commentID=65657723#commentID_65657723 Reply

4 | Alan Collis January 25, 2012 at 3:38 pm A well considered and articulate analysis. A question at the core of strategy is how quantitatively predictive is this cell assay to the next, or higher level of evaluation? It may receive scant attention in the fast and furious pursuit of a candidate. Reproducibility in an assay is important, yet predictivity or truth telling quality is what underpins a teams prospects of success. Alan Collis: http://www.linkedin.com/pub/alan-collis/37/a94/54b Reply

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