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Cell based reporter and in vitro homogeneous assays that are used in drug/drug target screening are considered to be powerful and convenient tools in primary screening of compounds or drug molecules. Research reagent and pharmaceutical/biotechnology companies are heavily involved in developing new technologies and methods for primary screening using homogeneous or cell based reporter assays. We are skeptical about the actual benefits of currently used cell based reporter or homogeneous assays that are vulnerable to false drug/drug target identification. The logical reasons for our skepticism are explained in following paragraphs.

In vitro homogeneous assays may not identify all potential drug candidates: The concept of homogeneous assays to “add, mix and read”, looks very elegant. One can screen thousands of compounds in a day without involving cumbersome and error-prone multistep methods. However, the question that remains unanswered is: -Are these homogeneous assays just for convenience and not really meant for “true” discovery of potential drug candidates? Is there a huge possibility to miss most promising drug candidates by adopting homogeneous assays in primary screening? Let us take an example and try to analyze whether these questions are relevant or not. Luciferase based assays are the most sensitive methods and are being widely used in various drug discovery assays. We will take kinase assay as an example to demonstrate potential advantages and disadvantages of homogeneous assays. Currently, there are few reagent companies who sell kinase assay products that are based on ATP quantitation (ATP depletion based assay) using luciferase reagent. At a glance, this assay looks very promising. The assay is done by performing a kinase assay. ( kinase + substrate + ATP+ buffer components + test compounds (potential kinase inhibitors)) followed by the detection of ATP using luciferase reagent (luciferase + luciferin). The ATP in the kinase reaction drives a luminescent reaction catalyzed by luciferase. Luciferase uses ATP to convert luciferin into oxyluciferin and light. In the presence of kinase activity, ATP is depleted from the reaction; thus, there will be less ATP to catalyze the luciferase reaction resulting in less emitted light. In screening applications for kinase inhibitors, emission of higher light output is scored as positive hits. A careful analysis of this assay raises several questions. The ATP, luciferase and luciferin are the moving targets in this assay. Possibly, test compounds can affect any one of these moving targets, in addition to the target kinase, since test compounds are not removed during the detection step. . If these happens, it will result in false positives or false negatives. Possibly, the effect of test compounds can be tested using control experiments using compounds alone with luciferase reagents. However, if test compounds are hydrolyzed or modified in presence of kinases and if these modified products or bi-products affect any one of the luciferase detection components, this will drastically affect the outcome. It is not possible to speculate that all existing or future compounds will not affect luciferase assay reagents, including non-ATP based Renilla luciferase assay. At the same time, one can argue that tens of thousands of compounds have already been screened using luciferase based assays and there were no reports on false positives or false negatives. How could we confirm this when the assay cannot differentiate between true or false positives/negatives? It is not possible to speculate that compounds that were negative in these assays are truly “negative” and vice versa. This is the major issue with any reporter (enzymes) based assays such as luciferase or beta-galactosidase or beta-lactamase or GFP etc. There are even kinase assays in the market that utilize multiple enzymes in addition to luciferase (e.g. ADP based kinase assays). These assays are more vulnerable to false results because in addition to luciferase reagents additional enzymes present in this assay can become the target for test compounds. Several reporter enzyme based assays are available for various drug screening assays such as cAMP, proteases, caspases etc. There is huge likelihood of getting false positive hits while using two enzyme systems in which inhibitors may find luciferase instead of kinase. We believe, enzyme based reporter assays may not be suitable for homogeneous primary screening, which may result in selecting wrong compounds for further screening by leaving most promising compounds undetected, unless all test compounds were tested parallel using other more predictive assays such as radioisotope based assay for kinases. The convenience of homogeneous assays in primary screening of thousands of compounds can lead to the selection of a wrong drug candidate, which may or may not end up as a true drug.
There are many questions yet to be answered: a) if we are aware of these limitations why the research community is still using in vitro homogeneous assays for drug discovery research? b) Are we using the homogeneous assays since these assays are simply more convenient, need less resources/reagents and less expensive than multistep approaches. The truth is, these assays are not cost-effective in the long term. Reagent companies are more interested in selling their products or marketing their technologies, with or without worrying about the long-term scientific impact of their products.

Cell based reporter assays may not be ideal for drug discovery: Cell based reporter assays that are used in drug/drug target screening are more vulnerable to false identification of drug molecules. In a reporter enzyme based assays, it is not possible to confirm that the positive readout is really due to a) the test compound or b) hydrolyzed products of test compounds or c) induction of alternative signaling pathways by test compounds, which trigger the activation of reporter enzymes. For example, fusion protein based stable cell lines are used in GPCR assays. In a primary screen, a compound may be selected based on its effect on GPCR activation. It is very hard to confirm whether the GPCR activation is not due to the effect of the compound on fusion protein, rather than the actual GPCR protein? The GPCR protein fused with a reporter protein does not behave or have the structural/surface integrity of the same un-tagged GPCR protein. A compound or protein that can recognize the untagged-GPCR in cells may not always recognize GPCR protein fused with another reporter protein and vice versa. In addition to this, expression of foreign proteins, like GPCR fusion proteins, can induce genetic and physiological changes within a designer stable cell line, which is different from normal cell lines. If so, how could we differentiate the effect of a drug on GCPR activation through normal cell signaling pathway, not through new proteins or metabolites that are induced in genetically modified stable cell lines? One should also take account of the genetic modifications that can occur during continuous culture of stable cell line selection procedure. The drug discovery screening assays using a drug target fused with a reporter enzyme or protein may not always mimic the effect of a drug on that particular wild type drug target (without fusion protein). Successful examples can be cited, but, these are not universally applicable. Cellular regulation is highly conserved and tightly controlled, otherwise a single mutation in a protein or a defect in protein-protein interaction would not have led to the onset of human diseases. The fundamental question we need to ask whether a drug molecule selected as a “positive drug candidate” based on its effect on a drug target protein fused with another reporter enzyme will have similar effect or binding characteristics on wild type drug target protein (non-fusion). Researchers have to give more emphasis on the physiological and genetic effects of fusion proteins in cells and how do these changes affect cell signaling pathways, which in turn affect the efficacy of the screening assay?

Unlike in vitro homogeneous assays, it is nearly impossible to have real negative control in cell based assays (considering the effect of cellular components on hydrolysis or degradation of test compounds). This makes even harder to identify potential false positives or negatives in these assays. There are several cell based reporter assays in the market for drug discovery research and these assays should be carefully analyzed for long-term benefits in identifying true drug candidates that are safer to use. On the other hand, cell based assays (without reporter enzymes) can be a powerful tool in analyzing the global effect of any given drugs. If we can analyze total changes within a cell, at protein or nucleic acid or metabolite level, due to the effect of a particular drug, possibly we can understand more about the effect of drugs at physiological or cell signaling level that can pin point some clues to the potential side effects. Innovative technologies that can thoroughly analyze these changes in cell based assays and in vivo animal model studies will have great potential in future drug discovery research. For example, it is possible to develop techniques for measuring cAMP using non-invasive and non-reporter enzyme based methods. If we can measure glucose molecules real-time in human blood using non-invasive methods, similar methods can also be feasible in measuring metabolites such as cAMP, phosphoinositides, P450 actiavtion/inhibition etc. in a cell based system. Along with the activation of cAMP, if we can identify molecular and physiological changes that are induced by test compounds will provide critical insights into the possible side effects of drugs. Such innovative approaches in drug discovery research will give us hope in developing safer drugs in future.

Are there any solutions? Obviously, one can ask that we have questioned the use of assays that are being widely accepted by the drug discovery research community. Are we right? If so, what are our solutions? From our point of view the questions we raised are relevant to current drug discovery approaches. Some existing solutions: a) instead of homogeneous or cell based reporter assays (exceptions can be based on targets), researchers may try to adopt techniques or methods that involve two or more step procedures for compound screening. It is true that this approach is not convenient like homogeneous assays, but multiple step methods can be automated like homogeneous assays. For example, radioisotope based kinase assays are widely used to confirm the kinase activity. However, this assay is not amendable for high throughput format because of safety and waste disposal issues. Peptide or solution array (glass slide or 96/384 wells) based assays coupled with antibody or phosphospecific detection reagent can identify “true” potential drug candidates than homogeneous assays. In these assays test compounds and other components are removed prior to the addition of detection reagents. Moreover, these are direct assays, phosphospecific, rather than currently used indirect kinase assays. Also, innovative technologies need to be developed for the detection of phosphate groups without using antibodies.

b) We need to develop new methods or modify existing methods for the applications in drug discovery. A very good example is ADME/Tox assays. Mass spectrometry (MS) assays are widely used with accuracy and reliability. These technologies can be adopted for various drug discovery assays rather than using reporter enzyme based assays. Mass spectrometry based assays can detect direct effects of drugs without manipulating cells or in vitro conditions. Similar MS assays can be developed for several drug discovery screening assays, which can quantitatively identify true changes in metabolites or proteins due to the drug treatment. HTS assay methods needs to be developed for most of the MS based drug discovery assays, which include cAMP, phosphoinositides, lipids, proteases, caspases etc. Multistep antibody based assays, not homogeneous assays, or antibody arrays are also very powerful tools in addition to MS assays. Non-specificity and cost are the major hurdles in using antibody based HTS assays.

c) Non-invasive methods will be the most powerful future tool in drug discovery research. These assays do not need manipulation of cells or the use of multiple regents for the detection. Recent developments in Raman spectroscopy and related areas will open up new avenues for non-invasive methods for the detection of metabolites, nucleic acids and proteins within a cell or tissue or organ or even in whole animals. These assays can be accurate, predictive and cost-effective. Reagent and instrument companies should invest their innovative minds to develop such non-invasive methods for drug discovery research. Academic and industrial researchers should come up with innovative non-invasive drug screening assays that can help in generating safer drugs in most efficient way.

End Note: It is not our intention to invalidate all innovative technologies that have been developed by very creative and talented scientists. These technologies have helped in finding new ways to screen drugs molecules that led to several successful drugs in the market. However, it is also true that currently used homogeneous or cell based reporter assays (that are used in drug/drug target screening) do not identify all potential drug candidates. Adoption of these assays by more and more researchers/laboratories worldwide will have negative impact on new drug discoveries. The selection of drug screening assays should be made based on long-term scientific impact of these assays. It is also the responsibility of research reagent companies, who develop and market these kinds of products, to analyze the “true” drug discovery value of their products. Based on recent reports on life threatening side effects of so many valuable drugs it is the right time to argue about existing pre-clinical and clinical drug screening assays and approaches. For example, antidiabetic drugs can cause cancer, heart attacks and so forth. It is really a night mare when we think that a drug for a particular disease can cause several other diseases and side effects. On the basis of these observations, we need to accept the fact that there are issues with current pre-clinical and clinical drug screening assays or methods. In other words, current drug screening assays do not provide sufficient information on potential life threatening side effects of drugs. It is possible to develop novel cell based or animal based drug screening technologies and tools that can predict fatal side effects, possibly by identifying cell based biomarkers (proteins, metabolites, miRNA etc.). We believe radical changes are needed in pre-clinical or clinical drug screening assays. Both industrial and academic researchers need to come up with innovative ideas to address these problems in current drug discovery approaches. Recent HTS initiatives by NIH are excellent avenues for addressing true drug discovery value of existing assays. Regulatory agencies like FDA might need to revisit the relevance and applications of existing pre-clinical and clinical assays in context to potential side effects of drugs.

Related blogs:

1. Cell Based Reporter Assays vs. Animal Studies in Drug Discovery- Potential Limitations, Risks and Liabilities

2. Strategies for Rational and Personalized Cancer Biomarker Discovery

3. Cancer Theranostics – Potential Applications of Cancer Biomarker Database

4. Are stem cells ready as a next generation drug discovery tool?

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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There are several technologies developed for studying protein-protein interactions and several products available in the market using some of these technologies. These technologies include tandem affinity purification and protein microarray based methods/kits for functional proteomics and system biology applications. These technologies look very promising and elegant. However, careful scientific analysis of these technologies raises some concerns and questions. These concerns and questions can be debatable, but, these are important issues to be discussed.

Multiprotein complex interaction studies: Tandem affinity purification (TAP) or similar techniques are mainly used for studying multiple protein-protein interactions in protein complexes or protein network analysis. TAP has significant applications in functional proteomics, systems biology and drug discovery studies. The methods used in TAP can be broadly classified into two categories: 1) protein fusion tag based methods and 2) Immunoprecipitation approaches. Several fusion tags such as Strep-tag, calmodulin binding peptide (CBP), histidine-tag, GST, MBP, HaloTag® etc. have been used for protein-protein interaction studies, including TAP. The use of fusion tags, especially large fusion tags such as GST, MBP or HaloTag®, raises some questions regarding the validity of these large fusion tags in analyzing protein-protein or protein-nucleic acid interactions (e.g. chromatin immunoprecipitation). If a large fusion partner is attached to a bait protein (the protein which is used to find its partner/s), this fusion protein will be significantly different from the natural protein. If this is true, can naturally interacting proteins recognize this new fusion protein? Take an example where calmodulin is fused GST (26KDa) and is used to identify calmodulin binding partners. The natural calmodulin interacts with several proteins at different stages of various cellular events. If the natural calmodulin is fused with a protein like GST, calmodulin-GST fusion protein will not be similar to the natural calmodulin. If this is true, do calmodulin interacting proteins can recognize GST-calmodulin? Probably not all proteins that interact with calmodulin, because protein-protein interactions within a cell are tightly regulated and conserved. The fusion protein will never have the natural three dimensional structural integrity or surface charge, energy state, charge state and so forth. We can argue that, there are several published reports on identifying interacting partners using this technology. How do we know that these proteins are really interacting proteins, except model proteins? Does this mean that the current technology will not determine all proteins that interact with natural calmodulin? A single mutation in calmodulin gene or a modification of a single amino acid in calmodulin (e.g. oxidation) can drastically affect the function of calmodulin. We can argue that fusion partners did not affect the structural integrity of protein partner, may be using a linker. If this is true, how can we explain the reports that some proteins loose it’s function or aggregates once you remove the fusion tag or some protein are functionally inactive if it is fused with a fusion tag or some proteins are folded correctly if is fused with fusion tag (e.g. GST,GFP, MBP,HaloTag® etc). These reports clearly demonstrate that a fusion tag can significantly affect the three dimensional integrity of proteins. A systematic and comparative analysis of fusion proteins vs. non-tagged proteins are needed to confirm the application of large fusion tags in protein-protein interaction studies. This analysis should not be done with model systems; rather it should be done with complex samples. Until we confirm this, antibody or small fusion tag (FLAG/FLASH/Step-tag/histidine tag) based technologies may be more suitable than large fusion tags. Non-tag approaches coupled with antibody based technologies (immunoprecipitation) will be the ideal method. The fusion tags were discovered for helping in recombinant protein purification; any applications beyond this should be critically evaluated based on strong scientific principles.

Protein array based protein-protein interaction studies: The use of large fusion tag proteins for oriented immobilization of proteins have been claimed as a better method for studying protein-protein interactions using protein microarrays (e.g. GST, MBP, HaloTag® fusion proteins). These claims have to be re-visited based on proven scientific principles. If we immobilize a protein onto a surface using a large fusion tag, how does this approach guarantees that the immobilized protein can identify its interacting proteins similar to the non-fusion protein inside a cell? Proteins are dynamic in nature and there are several sequential physical and biological factors involved in complex protein-protein interactions within a cell. Immobilization of a protein onto a confined position, as in protein microarrays, will limits its ability to interact with real protein partners. Immobilized proteins on any surface may not mimic protein’s function in real cellular context. As we mentioned earlier, the fusion protein will never have natural three dimensional structural integrity or surface for interactions etc. like the “wild type” non-tagged protein. Immobilizing a fusion protein will further jeopardize structural and functional integrity of the protein. It is true that there are several reports and successful products in the market demonstrating that immobilized fusion proteins can be used for enzymatic functional screening. In other words, immobilized proteins are functionally active. Studying the enzymatic function of a protein may not be an issue using protein arrays. The functionality of immobilized proteins can be validated using specific assays. This may not be the case with protein-protein interaction studies or complex interaction analysis. There is no control system to test these interactions, other than using a model system using single or multiple proteins. Any assumptions made on current protein array technologies using large fusion tags should be critically evaluated. Protein arrays may not be a viable tool for studying protein-protein interactions in critical areas like drug discovery and clinical diagnostics, exceptions are antibody arrays. Probably, we will never be able to identify real protein partners using protein arrays or any methods that involves protein immobilization onto a surface.

We should think of alternative technologies to unravel complex or simple protein-protein interaction networks within a cell. A technology that does not require manipulation of proteins has to be considered in identifying true protein-protein interactions. Antibody based technologies are an immediate solution to this, but it comes with several technical challenges such as non-specificity. Emerging non-invasive technologies such as Rama spectroscopy or similar technologies (IR/laser etc) may come-up as powerful tools in future. Innovative technologies that can address true biological interactions have to be developed based on strong scientific principles. Development or use of technologies or products that lack true scientific context may jeopardize scientific innovations; loss of time and money are not to mention.

Innovation Read the article (Opinion) from SciClips published in Future Medicinal Chemistry (September 2010, Vol. 2, No. 9, Pages 1411-1415)

First paragraph

“In 2003, Henry Chesbrough, a professor and executive director at the Center for Open Innovation at University of California Berkeley (UC Berkeley), first coined the term ‘open innovation’, in his book Open Innovation: The New Imperative for Creating and Profiting from Technology [1]. According to him: “Open innovation is a paradigm that assumes that firms can and should use external ideas as well as internal ideas, and internal and external paths to market, as the firms look to advance their technology”[1]. In recent years, tremendous growth has occurred in internet-based information and research networking among companies and customers, resulting in a massive distribution of knowledge in the public domain. Companies are taking this opportunity to connect directly with the customers in order to understand their needs and tap into ideas for product development….”
Read the full article: http://www.future-science.com/doi/pdfplus/10.4155/fmc.10.230


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