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.”