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Feature Articles : Jun 15, 2010 ( )
Characterizing Proteins to Bolster Pipelines
Basic Science and Chemistry Play Key Roles in Developing New Therapeutics!--h2>
In a world that is striving for better, cheaper, and faster means of discovering the next medical magic bullet, many researchers are going back to the drawing board. “Column technology for bioseparations stood still while biological sciences advanced,” said Dorothy Phillips, Ph.D., director of strategic marketing in chemistry commercial operations at Waters. “Scientists discovered that the chromatographic peaks were too wide or broad to give the required resolution, resulting in a focus on developing or updating the column chemistries.”
Furthermore, the push for translational medicine has come at a cost to basic science, noted Rich Vaillancourt, associate professor, department of pharmacology and toxicology at the University of Arizona College of Pharmacy. “What we need is a balance between basic science and translational research.”
On the other hand, proteins were a largely untouched frontier in the past because the technology wasn’t available to tap into that potential. “Reversible phosphorylation is the most common regulator of cellular events, which is why kinases are so prominent in drug discovery research,” said Rick Wiese, Ph.D., manager of bioscience R&D at Millipore. “Approximately 30 percent of cellular proteins are regulated by reversible phosphorylation, which gives researchers a pretty big target.”
As the push to move more compounds to the clinic picks up speed, conferences reflect the tension between developing technology and basic science, as well as the priority issues between developing drugs for higher incidence diseases versus niche indications. At the recent BIT “Life Sciences Conference” in Beijing and CHI’s “World Biomarker Conference” in Philadelphia, scientists shared some of their latest discoveries—and challenges—in coaxing answers from proteins to bolster the drug discovery pipeline.
Multiplex Biomarker Panels
In his talk at the CHI meeting. Dr. Wiese spoke about the importance of detecting phosphorylation and other post-translational modifications (PTMs) in order to understand cellular communication and treat many disease states. He noted that current intracellular protein assays such as Western blotting are limited due to a lack of specificity and low throughput. In addition, they are not quantitative, nor are they easily multiplexed.
“Traditional Luminex xMAP cell-signaling assays are multiplexed, but they are qualitative, limited in plex size, and require multiple wells to study multiple PTM sites on a protein. Further complications ensue when there is competition between two proteins in the same well you are trying to interrogate.”
Dr. Wiese pointed out that Milliplex MAP EpiQuant assays are quantitative and can measure multiple PTM sites and total protein in the same well with picomolar sensitivity. “We did a quantitative time-course expression analysis of 105 intracellular phosphotyrosine targets and 42 cytokines/chemokines in stimulated A549 cells. The experiment required less than three 96-well plates, thereby saving sample, reagents, laboratory time, and resources.”
By providing large, analytically validated panels of cell-signaling targets, EpiQuant technology enables researchers to create a kit that best meets their needs and quantitatively measure multiple phosphorylation sites, as well as total protein in the same well, said Dr. Wiese. “In addition, screening large panels can be of benefit because discoveries are often made accidentally; something pops that you weren’t expecting.”
Miniaturization and Microfluidics
Microfluidic lab-on-a-chip represents another area of fairly rapid expansion in technology. Raj Singh, Ph.D., director of biology R&D at Caliper Life Sciences, gave some examples of high-sensitivity LabChip® applications for quality control and characterization of monoclonal antibodies and other proteins in his talk in Beijing.
“Miniaturization using the microfluidics platform has many advantages, including speed and robustness,” said Dr. Singh. “However, it is possible to go too small; if you go too small, you cannot scale up. We are finding that microfluidics, as opposed to nanofluidics, gives the robustness, repeatability, and speed, as well as scalability that scientists require.”
Microfluidic chips form the key components of Caliper’s LabChip systems, which also include a LabChip instrument and experiment-specific reagents and software. Dr. Singh said that the chips contain a network of miniaturized, microfabricated channels through which fluids and chemicals are moved to perform experiments.
The instrument and software control the movement of fluids via pressure or voltage, and an integrated optical system detects the results of the particular experiment. “Because we have great flexibility in channel design and can exert split-second computer control over fluid flow, we have the ability to create chips for a multitude of applications,” said Dr. Singh.
Selling a platform is not enough, he noted. “Generic small molecules are easy to make and characterize; biosimilars, which is where protein characterization lives, are much harder to make, and biosimilar has evolved to biobetter. What we have done is take all the components of the assay—for example, glycan profiling—and put it on a plate and integrate it with our LabChip GXII reader. Just add compounds, follow the protocol, and you have your results. It comes out the same every time. Repeatable and robust—we think this is important in cell culture.”
Waters’ Dr. Phillips provided an overview of UPLC technology in Beijing and how it can be used to characterize proteins. “Advances in HPLC for biomolecules were few and far between until 2004, which is when Waters introduced its UPLC technology that allows scientists to achieve significant increases in resolution, speed, and sensitivity.”
“But as the biotherapeutics technology advances, the separations problems become more complex. For example, the challenges that we face include thoroughly characterizing biotherapeutics—among them proteins, peptides, and monoclonal antibodies—with about 40 percent of the current biomolecules drug pipeline devoted to monoclonal antibodies. Small molecule—less than 800 dalton molecular weight—analysis is more straightforward than that for large molecules, which require multiple analytical techniques including LC/MS and two-dimensional LC.”
Dr. Phillips explained that working with large molecules presents its own set of problems. “They are made using a biological process rather than organic reactions. Hence, it is critical that any change in the process be well controlled and understood so you create the same biotherapeutic with the same efficacy and safety every time.”
Waters introduced a new Acquity UPLC methodology for size-exclusion chromatography in March specifically to address the challenges of separating the monoclonal antibody monomers from their aggregates (e.g., dimers and trimers of the parent molecule). “Regulatory agencies such as the FDA require that you monitor and measure the percent of the aggregate present in the drug product,” explained Dr. Phillips. “Improving the level of confidence that you are making the same biotherapeutic every time comes from the use of separations techniques such as Waters UPLC technology.”
Finding the solutions to this problem requires new thinking in terms of the biochemistry of the molecule and chromatography, noted Dr. Phillips. “Thinking in terms of protein technology has brought us back to column chemistry.”
Characterizing MEKK3 Regulators
The regulation of serine/threonine protein kinase pathways that function in stress-related signal transduction pathways is the research focus of Dr. Vaillancourt’s laboratory at the University of Arizona. “Many yeast proteins have mammalian homologs, and MEKK3 was identified in mammalian cells due to similarities with a well-characterized yeast protein. However, the function of MEKK3 is poorly understood in mammalian cells.”
Dr. Vaillancourt noted that MEKK3 has never been purified. “It represents more of a genomics approach, where you have a piece of DNA you can isolate from looking at a sequence in the human genome. What does the protein encoded by this DNA do? In a lot of ways, that’s how this project evolved.”
For his lab, this work is connected to MAP kinase signaling. “All signaling of cells come through these pathways, but the puzzle is finding out what route is used by a hormone, toxicant, or any kind of stimulated growth factor. To get a better idea of how it functions, we took a biochemical approach. Proteins are generally regulated by phosphorylation. If you can figure out how they are phosphorylated, you can identify how they function.”
Another phosphorylation he identified was by a different kinase, called PIM, which has been implicated in cancer and as functioning in viral infections. “So this suggests that MEKK3 can also be implicated in pathways for cancer and in viral infections. This allows us to suggest that MEKK3 could be a target for therapies, and that blocking it can be used as a therapeutic. We have the tools to identify molecular targets, but not the chemicals to develop therapeutics, which is an issue.”
Novel Dx Define Metastic Process
Breast cancer is among the leading causes of cancer-related deaths in women, and in the vast majority of cases, death occurs as a result of metastatic disease, noted Massimo Cristofanilli, M.D., professor and chairman, department of medical oncology at the Fox Chase Cancer Center.
“What we discovered and published in the New England Journal of Medicine in 2004 was that the detection of circulating tumor cells (CTCs) was the single most powerful prognostic factor in metastatic disease. In practice, the number of CTCs before treatment is an independent predictor of progression-free survival and overall survival in patients with metastatic breast cancer.”
Dr. Cristofanilli commented that, while the prognostic value of CTCs was confirmed in subsequent studies, the most recent molecular evaluation of biomarkers in CTCs suggests that cancer cells in the peripheral blood of metastatic patients express a different phenotype than those in the primary tumor. In fact, research now shows a discordance between CTCs and tumor cells with respect to expression of biomarkers assessed for treatment planning (e.g., ER and Her-2).
“This result seems to confirm data from preclinical work indicating the existence of a population of cancer cells, known as cancer stem cells, that may have a more aggressive behavior and different phenotype,” said Dr. Cristofanilli. “Studies have shown that cancer stem cells can survive standard therapy. We believe that improved diagnostics can help us to better understand the dynamics of the disease and evaluate changes in protein expression that can make a difference in prognosis and treatment planning.”
Dr. Cristofanilli presented on some of the novel technologies that are advancing the field. “The CTC chip developed by investigators from Massachusetts General Hospital is highly innovative in that it allows researchers to capture and collect cells for molecular analysis. This technology may represent a step forward in our effort to find better technologies capable of better isolation and molecular profiling of these cells.
“Such an innovation can create a new paradigm for more personalized medicine. In fact, we believe that the data generated suggests that molecular characterization of CTCs can provide additional information for the development of personalized therapies in breast cancer. By allowing real-time evaluation of gene and protein expression, we will be able to select effective therapies.”
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