Is it too early to brand biomarker discovery as a“Hype”?

It is very interesting to read some of the recent studies and discussions in various scientific publications which revealed the negative aspects of biomarkers. Question is whether these reports or views really conveying the true message or simply trying to create a “negative hype” that the biomarker discoveries are a failure: a conclusion based on discouraging results from a particular technology area? We would like address two different aspects in this blog, first “biomarker hype” and second the current limitations of proteomics technology that may have created this “biomarker hype”.

The diagnostic biomarkers can be used not only for the detection of diseases but also for monitoring the drug effect as well as for defining drug dosage. It is a well-established fact that the use of biomarkers in multiple clinical applications may have significant impact on early diagnosis, clinical monitoring, fine-tune the efficacy of the drug. If the definition of a biomarker is based on the concept of “one biomarker-one disease”, it will be hard to find biomarkers for complex diseases, such as cancer. Our current understanding of clinical diagnostics, infrastructure, and expertise may dictate that the single biomarker approach would be more practical, feasible and reliable. At the same time, we should also consider the fact that major breakthroughs in recent years on gene and protein expression analysis have not only resulted in generating significant scientific discoveries for clinical applications of multiple biomarkers but also the involvement of these biomarkers in disease pathogenesis and progression. On the contrary, we are still spending valuable scientific resources to find perfect single biomarkers for various diseases. A recent study from National Cancer Institute (Cancer Prev. Res. (2011) 4:365) has been cited as a classic example of biomarker hype or the failure of biomarker discovery. According to this report the researchers have tested more than 35 ovarian biomarkers that have been claimed in many scientific publications to be better than CA125, a well established ovarian cancer biomarker. After analyzing hundreds of tissue samples, the researchers found that none of the biomarkers were better than CA125. Now the question is, based on this experimental data, can we truly claim that those biomarkers which did not perform well in this particular experiment are not better than CA125 and hence, are not true biomarkers? Several questions still remains unanswered such as: do we know for sure how many patients who were diagnosed using CA125 biomarker were responded completely or partially or none to the chemo or radiation therapies? If we would have carefully analyzed the CA125 selected patients along with other biomarkers (in combination), wouldn’t it be possible to predict a specific biomarker combination pattern that would have helped us to understand: a) the disease variation among patients and b) the patient response to a given therapy, which will ultimately help us to develop better treatment methods. The biomarkers are not only for the detection of a disease but also to provide clues for the disease state and variation in a particular patient. Expression level of a particular biomarker or a panel of biomarkers can be used to select a specific drug dose or a treatment regimen. Wouldn’t it be reasonable to think that the presence of other biomarkers in addition to CA125 in ovarian cancer patients might have some biological and clinical significance? Otherwise, a test sample from an ovarian cancer patient should not have shown positive to a second biomarker that is not present in healthy controls.

Based on our analysis using data from various clinical trials, we found out that it is very difficult to find biomarkers that have high specificity, in terms of diagnosing a particular cancer in all the patients who were enrolled in a clinical trial. It was also true that in some studies a particular biomarker that was found in majority of cancer patients were absent or merely detected in few patients, though these patients were all diagnosed with the same cancer like the biomarker positive patients. The data from clinical studies needs to be evaluated critically for developing innovative strategies for biomarker discovery and clinical diagnostics assays. All the biomarkers that are experimentally validated need to be carefully evaluated and considered for developing most efficient and clinically useful diagnostic/prognostic assays. Biomarker or proteomics researchers need to develop creative thinking, planning and execution of ideas with a “birds view” not with a “microscopic view” that fails to interpret the long-term impact of research findings beyond the areas of expertise. In other words, for developing an intelligent and smart research plan for high value areas like biomarkers, researchers need to develop broader understanding and knowledge on current and future clinical applications of biomarkers. Constant interaction with clinical and diagnostic researchers in academic and industrial laboratories could be a first step towards achieving this goal. Collaboration with researchers from established pharmaceutical or biotechnology companies will be an advantage to academic researchers to design their strategies and experiments that may lead to the discovery and validation of clinically viable biomarkers.

The false hopes that are created by current proteomics approaches can be blamed for the “biomarker hype”. More often the term “biomarkers” are confined to or projected as proteomics based biomarkers; mainly by the proteomics researchers. We sometime knowingly or unknowingly tend to ignore the fact that biomarkers can be proteins, peptides, DNA, mRNA, SNP, miRNAs or metabolites. It would be tough to predict whether protein or molecular biomarkers will be more commercially viable and successful; not to forget the fact that molecular biomarkers are more robust and cost effective than protein biomarkers. Even if we consider proteomics is the way for biomarker discovery, current proteomic approaches would need a 360^° make over, especially in sample preparation and analysis methods that are suitable for clinically relevant biomarker discovery platform. Starting from sample preparation to enzyme digestion followed by fractionation are the major limitations of mass spectrometry (MS) based proteomics approach in a clinical set up. The difficulty in sample analysis, sample loss, lack of reproducibility and the high cost in identifying a biomarker are additional limitations. It is even more confusing when proteomics approaches claim absolute quantitation of biomarkers based on the data that have been generated through complex sample preparation that lacks reproducibility. Moreover, the biological significance and relevance of absolute quantitation of biomarkers in diagnostics applications are debatable. Unless, we have a robust sample preparation and analysis technique, MS based proteomics approaches may continue to generate false positives/negatives. Unfortunately, optimization of sample preparation techniques also includes development of new LC and MS instrument platforms that can handle heterogeneous “dirty” samples without specialized sample cleaning steps.

The lack of true collaboration between mass spectrometry scientists, biological and clinical researchers is another limitation in MS based approach. The protein mass spectrometry field is generally dominated by analytical scientists and there is an urgent need for more productive collaborative research, not just a core facility confined consultation, to identify true biomarkers that are biologically and clinically relevant. Analyzing the peak shifts and peptides may not be the only major thrust area, rather, the identification of biological relevance and validation of biomarkers should also be the priorities of proteomics researchers. Proteomics laboratories need to develop strategies, skills and infrastructure for true biomarker discovery. Currently, mass spectrometry based proteomics skills or expertise is confined to very few laboratories or core facilities that have independent mass spectrometer capabilities. This severely cripples innovations in proteomics based biomarker discoveries. Breakthroughs in biomarker discoveries may happen once portable mass spectrometers are available to research laboratories, like PCR machines or ELISA readers. It is noteworthy to point out that emerging areas such as MALDI imaging using tissue arrays and whole protein mass spectrometry analysis look very promising and hopefully these technologies might address the current limitations in proteomics based biomarker discovery.

The current criticism over biomarkers can be purely based on the putative or false biomarkers that were identified and claimed using mass spectrometry based proteomics approaches. Nonetheless, the failure of proteomics technology should not invalidate the biomarkers (proteins, SNPs, miRNA etc) that were discovered and validated through other conventional and modern biological tools, such as tissue arrays and various antibody based technologies, in a pre-clinical or clinical set-up. These technologies will continue to discover new biomarkers that may have the potential for developing novel diagnostic tools.

Recent Biomarker Patents

1. Predictive models for diagnosing and assessing the extent of coronary artery disease

2. In-vivo detection of a biomarker in the gastrointestinal tract

3. Biomarkers for the diagnosis of intra-amniotic infection in pregnant women

4. Method for the detection prostate cancer cells in urine

5. SNP biomarkers assessing the risk of developing chronic obstructive pulmonary disease (COPD)

6. Biomarkers for predicting the efficacy of polyamine-type chemotherapy in breast cancer patients

7. G-protein as a biomarker for the diagnosis of autism spectrum disorders

8. Gene expression biomarkers for the diagnosis and prognosis of infectious diseases

9. FGF-23 as a biomarker to identify mortality risk in asymptomatic chronic kidney disease patients

10. Method of screening for an elevated risk of lung transplant rejection

11. Serum biomarkers for the diagnosis of invasive Candida infection

12. Stool biomarkers for the detection of colorectal cancer and pancreatic cancer

13. Biomarkers for predicting cognitive decline in an Alzheimer's disease patient

14. Biomarkers for predicting the efficacy of bevacizumab treatment in colorectal cancer patients

15. Biomarkers for IgA nephropathy

16. Mitochondrial DNA for monitoring toxicity of a HIV drug treatment

17. CEA binding RNA aptamers for the diagnosis and treatment of cancers

18. RNA biomarkers for the prognosis or early detection of lung cancer

19. Molecular diagnostic methods for Streptococcus pneumonia

20. Molecular diagnostic methods for Clostridium difficile

21. Biomarkers for assessing the risk of neovascular age-related macular degeneration (AMD) in a patient

22. A method for analyzing the C-terminal amino acid sequence by mass spectrometry

23. Molecular diagnostic methods for Neisseria, Chlamydia, and Chlamydophila bacteria

24. Metabolomic biomarkers for the diagnosis of non-alcoholic steatosis (NASH)

25. Biomarkers for determining the status of female fertility

26. Detection of ataxia telangiectasia healthy carriers

27. Novel stem cell cancer markers for the diagnosis and treatment of solid tumor breast cancer

Details: http://www.sciclips.com/sciclips/drug-discovery-news.do

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Recent Drug Discovery Patents

1. eEF2K as a therapeutic drug target for the treatment of Alzheimer's disease
2. Promoting neuronal differentiation of a neural stem cells using COUP-TFI protein inhibitors
3. Herbal extracts for the inhibition of cancer stem cells
4. Integrin alpha 5 agonists for promoting osteoblast differentiation of mesenchymal stem cells
5. A matrix for the delivery of osteogenic human mesenchymal stem cells
6. miR-126 as a target for the inhibition of angiogenesis
7. Treating leukemia with rice prolamin
8. Inhibitors of Elk-1 or MSK-1 for treatment of mood and anxiety disorders
9. PEGylated IGF-1 for the treatment of amyotrophic lateral sclerosis (ALS)
10. Anti-pleiotrophin antibodies for the treatment of cancer
11. Clotting factor-Fc chimeric proteins to treat hemophilia
12. Cell-permeable peptide inhibitors of the JNK for the treatment of various diseases
13. Dp71 as a therapeutic target for inhibiting or stimulating angiogenesis
14. IL-6 and IL6R for the treatment of radiation or chemotherapy induce toxicity, xerostomia
15. Hedgehog inhibitors for the treatment of fibrosis
16. Biomarkers for predicting the efficacy of polyamine-type chemotherapy in breast cancer patients
17. Biomarkers for predicting the efficacy of bevacizumab treatment in colorectal cancer patients
18. Mitochondrial DNA for monitoring toxicity of a HIV drug treatment
Details: http://www.sciclips.com/sciclips/drug-discovery-news.do

Recent Drug Discovery and Biomarker Research Highlights

Drug discovery

1. Induced pluripotent stem cell based human cellular model for Rett syndrome
2. Water drinking could assist overweight children in weight loss
3. Organic cation transporter 2 (OCT2) as potential pharmacological target for mood disorders therapy
4. HDAC6 inhibitors for the treatment of Charcot-Marie-Tooth disease
5. Protein complementation with resonance energy transfer to study conformational changes in response to activation of a GPCR heteromer
6. STAT3 pathway as a therapeutic drug target of prostate cancer stem cells
7. A prodrug approach for improving tuberculosis drug targets
8. Novel thalidomide derivatives for the treatment of sickle cell diseases
9. Novel isoniazid derivatives for the treatment of tuberculosis
10. iPS cells derived neural stem cells for glioma gene therapy
11. Identification galactose-1-phosphate uridyltransferase (GALT) inhibitors for classic galactosemia treatment
12. Potent inhibitors of dihydroorotate dehydrogenase (DHODH) for the treatment of malaria
13. Promising reversible and selective MAO-B inhibitors identified
14. A screening strategy to identify drugs that suppress statin muscle toxicity
15. Fluorescent sensors to study cell signaling molecules with high spatial and temporal resolution
16. Anti-progastrin antibody for the treatment of breast cancer and colorectal cancer
17. Plasminogen activator inhibitor-1derived peptides for the treatment of asthma
18. Parstatin peptide for the treatment of myocardial ischemic injury
Details: http://www.sciclips.com/sciclips/drug-discovery-news.do


Biomarkers

1. Multidrug resistance (MDR) gene signatures as prognostic biomarkers of ovarian cancer
2. Urinary metabolomic biomarkers of broccoli and Brussels sprouts consumption
3. Protein standard absolute quantification (PSAQ) method for the measurement of cellular ubiquitin pools
4. Proteins associated with aggressive prostate cancer identified
5. Urinary NGAL a promising early marker for monitoring renal impairment in patients with short-term type 2 diabetes
6. Novel glycan biomarkers of colon cancer
7. REGIV as a biomarker of peritoneal recurrence after curative resection in gastric cancer patients
8. Biomarker panel for the diagnosis of live injury
9. kPCR assay for the detection of KRAS and PIK3CA mutations in colorectal cancer
10. Urinary metabolomic biomarkers for the diagnosis of prostate cancer
11. In vivo imaging of ADLH in cancer stem cells
12. EML4-ALK gene or protein fusion as a biomarker for the diagnosis of non-small cell lung cancer
13. Progastrin as a biomarker for the diagnosis of breast cancer
14. Progastrin as a biomarker for the diagnosis of liver cancer, cirrhosis, and hepatitis C
15. MAGEC2- a biomarker for the differential diagnosis of testicular germ cell tumors
16. PRDM1 protein a potential biomarker of radiation therapy–induced secondary malignancies in pediatric Hodgkin's lymphoma
17. Genetic variations in chromosome 11 to predict schizophrenia risk
18. GRP78 SNP as a predictive biomarker of liver cirrhosis in persistent HBV carriers
Details: http://www.sciclips.com/sciclips/drug-discovery-news.do

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

In recent years, there is a considerable push in adopting stem cell based assays as a drug discovery tool. The major argument behind this initiative is stem cells can differentiate into specific cell types, which can be used for targeted drug screening. Major pharmaceutical companies and public funded academic institutes have been started investing significant amount of effort and money on stem cell based drug discovery with the hope that this approach will ultimately provide real breakthroughs in drug discovery research as well as significant cost savings. There is no doubt that stem cells may offer unique opportunities in drug discovery, but the scientific data to support this notion may not yet well established to the extent that is applicable to drug discovery screening.

A most popular application of stem cells is the use of stem cell derived cardiomyocytes as a screening tool for testing cardiac toxicity of drugs. One can argue that there is enough scientific evidence to prove that stem cell differentiated cells are genetically similar to corresponding normal cells and can be used as a drug screening tool. This may or may not be true based on answers to following questions. a) can stem cells be a better tool in drug screening assays than currently used cell/animal based assays? b) are stem cell derived cell/tissue types are genetically and physiologically identical to the desired natural cell types, for example cardiomyocytes? c) are these proposed stem cell based assays will provide better tools to reduce or eliminate fatal side effects of drugs, e.g. cardiotoxicity? It is important to note that current use of cell based assays (non-stem cell based) failed to reduce or prevent fatal side effects of drugs. Based on this, it is hard to argue that currently proposed stem cell based assays provide any better solution to current problems. We need to understand genetic and physiological make up of stem cell derived cell types before we adopt this technology for making life saving decisions. In any means, our opinion does not imply that stem cell based products are not suitable for developing drug discovery assays. Our goal is to critically analyze the scientific rational behind current approaches.

Currently used confirmatory tests are not sufficient enough to establish the use of stem cells in drug discovery screening.

We will analyze stem cell based cardiomyocytes, which is one of the most “publicized” applications of stem cell based drug assay, as an example to understand whether stem cell derived products can be used in drug discovery screening assays. We would like to raise one question. Are we in a position to predict confidently that these cardiomyocytes, generated from stem cells have genomic stability? This means, stem cell derived cardiomyocytes do not posses genetic and somatic mutations, DNA polymorphism, chromosomal translocations, genomic instability due to polyploidy or anueploidy, miRNA polymorphism, metabolite variations and proteome polymorphism/modifications. These cellular or genetic changes can result from long-term cell culture system, molecules or genetic modifications that are used for the induction of differentiation and induced genetic rearrangements that are needed for the generation of stem cells, especially iPS cells, which may have some tumor cell properties. Mere confirmation of stem cell derived cardiomyocytes using DNA microarrays is not enough to establish the fact that stem cell derived cell types are genetically similar to normal cardiac cells/tissues. DNA microarray based approach does not address or identify unknown genes derived from alternative splicing/transposons, gene modifications and polymorphism, polyploidy or anueploidy, RNA modifications and epigenetic/mitochondrial DNA modifications. Therefore, the use of current DNA microarray based screening may be insufficient to establish the validity of stem cell derived products.

In addition to genes, miRNA, metabolites and proteins play significant role in genetic and physiological regulation in a particular cell type. A single change in post-translational modification in a protein can drastically change the cellular signaling process, which cannot be detected using DNA microarrays. This is also true with miRNAs and metabolites. The currently used stem cell derived products such as cardiomyocytes need to be established as true cardiac cells, both genetically and physiologically. This requires extensive scientific research. Until then, stem cell based assays for screening life threatening side effects such as cardiotoxicity should be carefully considered for drug development applications. There is a need for the development of reliable assays or technologies for the detection of genomic, proteomic, metabolomic, epigenetic and physiological instabilities in stem cell derived cell or tissue types.

Establishing a regulatory system for drug discovery assays will help in developing new drugs with fewer or no fatal side effects.

The development of new drugs based on stem cell derived assays, with very limited in-depth understanding of the system, needs to be reviewed diligently. How can we confirm that genetic or physiological instability of stem cell derived cardiomyocytes does not affect assay outcome, which is very critical? Can companies who develop such products, for example cardiomyocytes, guarantee that drugs developed using their stem cell technology do not have fatal cardiac side effects?. Under current system, anyone can aggressively market their products/technologies as a drug discovery tool based upon incomplete data or information because of the lack of regulation in this area. There are numerous examples where several products are marketed for drug discovery research without disclosing possible product limitations (see our earlier blog on cell based reporter assays). This is a major issue especially with proprietary/patented technologies owned by assay development companies or CROs. A very good example for this is reporter enzyme, homogeneous and cell based assays. Marketing proprietary technologies for all possible applications, without disclosing limitations, needs to be critically evaluated. So many public funded laboratories and facilities may also be using technologies, which were marketed to these customers with or without disclosing or foreseeing product limitations. It is important to note that consumers are protected from undesirable claims on pharmaceutical drugs, diagnostics and consumer products. Establishing an oversight regulatory system will help in checking product performance claims. Generating scientific and technical accountability monitoring system that starts from the bottom (e.g. reagent companies, CROs etc.) to the top (pharmaceutical companies) will help in generating robust pipeline of safer drugs. Any failure in a given drug should be scrutinized from the drug discovery research to development phase. Creating an oversight system is warranted especially for developing and marketing drug screening assays, similar to diagnostics products. This approach may slow-down development and application of drug discovery assays. However, in the long-run this will have significant positive impact on drug discovery research and development. This will help in selecting the right product for specific applications with complete understanding of limitations, which will help in developing alternative strategies. Finally, it is the choice of drug discovery researchers to address the need for a regulatory system for assays that are used in critical areas of drug discovery and development. These critical areas can be drug metabolism (ADME), cardiotoxicity, other organ/tissue specific toxicity, mutagenicity, and immunogenicity.
Link:: http://www.sciclips.com/sciclips/blogMain.do

Cell based reporter assays: misleading approach in drug discovery?

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.
Link:: http://www.sciclips.com/sciclips/blogMain.do