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Archive for January 2012


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?

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 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?

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. Strategies for Rational and Personalized Cancer Biomarker Discovery

2. Cancer Theranostics – Potential Applications of Cancer Biomarker Database
3. Are stem cells ready as a next generation drug discovery tool?
4. 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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Scientific and technical areas covered: Drug discovery, Stem cells, Genomics, Proteomics, Biomarkers, Clinical diagnostics, Companion diagnostics, Molecular diagnostics, Personalized medicine (theranostics), Plant and agricultural biotechnology

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Availability of a comprehensive cancer biomarker database may opens up scientific and technical opportunities in developing innovative oncologic theranostics (Rx/Dx), a diagnostic therapy process that leads to the development of successful personalized medicine strategies in cancer treatment.

With the growing trend towards the advancement of personalized medicine concept, there is a need to develop strategies and tools that can be used for individualized diagnosis and treatment. Theranostics based tools, a combination of diagnostics and therapeutics approach, offer promising agents that can be used for the improved diagnosis and treatment of various diseases. In oncologic theranostics, developing innovative personalized cancer treatment rely on the identification of novel cancer biomarkers and diagnostic assays to identify patient’s response to a particular drug, for optimizing personalized drug treatment regimen (drug dose, drug treatment schedule etc) and for monitoring the efficacy of treatment (disease stage, tumor progression, tumor recurrence etc). A best example for this will be the approval of Genentech’s Herceptin® with DakoCytomation’s HercepTest® for breast cancer theranostics. Future development of cancer theranostic tools depends on the discovery and validation of existing or novel cancer biomarkers. A database that contains comprehensive information on discovery phase or clinically validated biomarkers, along with therapeutic drug target information, can be a powerful tool in developing novel theranostic assays as well as for the discovery of new drug targets based on theranostics (Fig.1). A combination of therapeutic drug target and biomarker pathway analysis, in particular companion diagnostics pathways, can pave the path towards developing innovative strategies in cancer theranostics.

Fig. 1: Theranostics and cancer biomarker database in personalized medicine


Biomarkers that have potential applications in cancer theranostics can be broadly classified into:

1. Imaging biomarkers: Drug molecules labeled with imaging tags (e.g. NRI, MRI etc.) and antigen-directed imaging drugs (e.g. radiolabeled antibody drugs) are the very good examples. In this case, a single molecule can be used as a diagnostic and therapeutic agent, EGFR, VEGF and TAG-72 are very good examples where antibodies against these drug targets tagged with imaging markers can be used in theranostics. Imaging biomarkers can also be very useful in targeted surgical treatment of cancer. Labeled antibody based detection of phosphorylated or dephosphorylated will be an attractive theranostics tool in phosphorylation-dependent targeted cancer therapy and diagnosis. Epigenetic biomarkers are another attractive target for developing cancer theranostics. Applications of additional tools such as nanoparticles and gold particles have been demonstrated in theranostics.

2. Diagnostic/prognostic protein biomarkers: Immunohisotchemistry and immunoassays (e.g. ELISA) can be used in theranostic applications. Identification of diagnostic biomarkers that can be used as therapeutic drug targets will have significant impact in theranostics. Development of protein biomarker-directed antibodies (labeled) or small molecules or aptamers can be a potential theranostics tool.

3. Molecular diagnostic markers (genes/SNPs/miRNA/epigenetic): PCR, qPCR, DNA sequencing (including next generation sequencing), and microarray based technologies can be used as theranostics tools. Single step diagnostic therapy, like labeled antibody drug based theraostics, may be a challenging task with molecular diagnostic biomarkers, possible exceptions are siRNA or miRNA based cancer therapies.

4. Cell based biomarkers: Cancer stem cells, circulating tumor cells (CTCs) and tumor-infiltrating immune cells (CD68-positive macrophages/T-cells etc.) can be used in cancer theranostics. The diagnostic and therapeutic significance of these cell based biomarkers have been demonstrated in several published studies.

5. Drug efficacy/response/predictive biomarkers: Biomarkers include proteins, gene, miRNA, SNPs, metabolites etc., which can be successfully used for the development of companion diagnostic assays. These biomarkers can also become a therapeutic drug target for further discovery of theranostics based therapeutic drug targets.

6. Combination therapy response biomarkers: Combination therapy approaches have been demonstrated as an efficient treatment method for various cancers. However, the availability of companion therapy response biomarkers are limited (some of these biomarkers are included in our cancer biomarker database). Wide adoption of combination therapy as a method for cancer treatment may warrants a need for the discovery and validation of new biomarkers associated with combination therapy.

Identification of new biomarkers and availability of large number biomarkers may result in the development of theranostics for most of the cancer types. A cancer biomarker database that contains comprehensive and cumulative information on experimental and clinically validated biomarkers, especially companion diagnostic biomarkers and therapeutically relevant biomarkers, may opens up scientific and technical opportunities in developing innovative oncologic theranostics (Rx/Dx) tools.

Sciclips cancer biomarker database contains more than 8700 cancer biomarkers, which are classified into 1) diagnostic biomarkers 2) disease predictive/risk assessment biomarkers 3) drug efficacy/response biomarkers 4) prognostic biomarkers and 5) cancer companion diagnostics biomarker pathway. The biological and molecular functions, biological process associated, chromosomal location, SNPs and protein-protein interaction networks of each biomarker are listed in this database. This comprehensive information will be useful for the validation of existing biomarkers and for the identification and validation of new biomarkers for cancer theranostics. Please follow the link to see the details of cancer biomarker database: http://www.sciclips.com/sciclips/diagnostic-prognostic-cancer-biomarker-main.do

Related tools:

1. Biomarker protocols
2. Bioprotocols
3. Biomarker News
4. LinkedIn® Theranostics group

Related blogs:

1. Strategies for Rational and Personalized Cancer Biomarker Discovery

2. Cell based reporter assays: misleading approach in drug discovery?
3. Are stem cells ready as a next generation drug discovery tool?
4. Cell Based Reporter Assays vs. Animal Studies in Drug Discovery- Potential Limitations, Risks and Liabilities


Comprehensive Cancer Bomarker Database

Sciclips consultancy

Diagnostic & Prognostic Biomarker Database

Bioprotocol database (open access)

Combination therapy database (open access)

Therapeutic drug target database (open access)

Bioinformatics databases (open access)

Stem cell researchers database

A comprehensive database for stem cell researchers, Stem cell research reagents, Stem cell patents, Stem cell grants, Stem cell clinical trials, Stem cell statistics, Stem cell news and Stem cell open innovation ideas. Please visit http://www.sciclips.com/

Sciclips consultancy

Proteomics researchers database

A comprehensive database for proteomics researchers, Proteomics research reagents, Proteomics patents, Proteomics grants, Proteomics clinical trials, Proteomics statistics, Proteomics news and Proteomics open innovation ideas. Please visit http://www.sciclips.com/

Share or sell research reagents prepared in your laboratory

Drug target database (open access)

Pharmacogenomics protocols online

Proteomics and Mass spectrometry (MS) protocols online

Therapeutic drug targets database (open access)

Drug discovery, Biopharmaceuticals, HTS assays protocols online

Protein display/Protein engineering/Directed evolution protocols online

Molecular biology and Cell biology protocols online

siRNA protocols miRNA protocols online

Video protocols

Cell culture video protocols, Cytotoxicity assay video protocols, Stem cell video protocols, Molecular biology video protocols http://www.sciclips.com/sciclips/video-protocols.do

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Bioprotocols online (open access)

Combination therapy database (open access)

Therapeutic drug targets database (open access)

Bioinformatics databases (open access)

HTS assay protocols online (open access)