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Feb 15, 2010 (Vol. 30, No. 4)

Refined Tools Aid in Pathway Analysis

Scientists Explore Range of Options to Remove Roadblocks Along the New Product Development Highway

  • JAK-STAT Pathway

    Signal transducers and activators of transcription (STATs) mediate the effects of growth factors and cytokines to regulate the expression of genes involved in cell proliferation, differentiation, inflammation, migration, and apoptosis. One of several sessions that Paul A. Johnston, Ph.D., research associate professor, pharmacology and chemical biology at University of Pittsburgh School of Medicine, presided over at the meeting discussed the role of STAT3 in squamous cell carcinoma of the head and neck.

    “One of my collaborators, Jennifer Grandis, Ph.D., was originally interested in developing a standard STAT3 reporter assay for screening. Reporter assays are easy to develop and fairly easy to run in high throughput, but there are also a lot of challenges associated with them post screen, there is a lot of potential for artifacts especially when you are looking for inhibitors.”

    Drs. Johnston and Grandis settled on a high-content imaging approach. “I knew that there was STAT3 published procedures out there. I thought it was going to be straightforward, but it was not.”

    In order to perform proof-of-principle experiments, the researchers obtained antibodies for STAT1, STAT3, and STAT5. Activated STAT3 is an oncogene that directs tumor cells toward proliferation and survival, induces angiogenesis and alters the tumor microenvironment, and promotes tumor metastases through its effects on cell migration and invasion.

    In sharp contrast, activated STAT1 is considered a tumor suppressor because it is a potent inhibitor of tumor growth, promotes tumor cell apoptosis, and enhances tumor immunity. “We needed antibodies for STAT3 and STAT1 so we could look for something that selectively inhibited STAT3 without inhibiting STAT1,” said Dr. Johnston.

    “We were able to test and identify antibodies that worked and were able to develop a three-colored dual-antibody assay that measures nuclei and both STAT 3 and STAT1 tyrosine phosphorylation. STAT tyrosine phosphorylation triggers STAT dimerization, nuclear translocation, DNA binding, and transcriptional activation.”

    Dr. Johnston cautions that one size doesn’t necessarily fit all. “Many natural product derived anticancer compounds are not the easiest compounds to synthesize. Also, the cellular activities aren’t always restricted to a single target or pathway. I think this is the real value of content imaging.”

  • Small Molecule Discovery

    Click Image To Enlarge +
    Formylated peptides (agonists=red) and phenyl-butazone derivatives (antagonists=blue) docked into a homology model of the N-formyl peptide G protein-coupled receptor

    The whole concept of flow cytometry is quite simple, observed Larry Sklar, Ph.D, principle investigator at the University of New Mexico Center for Molecular Discovery, who explained that “when people run flow cytometers, one sample is one file. 

    “Generally, flow cytometry doesn’t do high throughput—doesn’t do many samples very well, very quickly—so we created a platform to solve that problem. Our technology is high-throughput flow cytometry, which is intrinsically high content because you can make many measurements simultaneously.”

    Eric Prossnitz, Ph.D., professor of cell biology and physiology at the University of New Mexico, spoke extensively about the HyperCyt flow cytometry platform for high-content high-throughput small molecule discovery. “The platform is evolving for 1,536-well plates and direct sample delivery. Recently, we demonstrated HTS applications with primary cells and yeast multiplex model systems for TOR pathway analysis, as well as innovative molecular assays for intracellular trafficking pathways.”

    The platform can be used to make molecular measurements using assemblies of protein-protein or protein-small molecule interactions on particles. “This is a unique technology that is still in development. One of the challenges we have is keeping up with the sheer throughput of the technologies that are already well established. We can work with complex cell suspensions, bacteria, and yeast in suspension. We can look at, not just high content, but also multiple processes and multiple targets simultaneously, because of the way flow cytometry can color code samples and analyses.”

    Dr. Prossnitz seeks molecules that can regulate physiological processes. “Fundamentally, we are interested in understanding complex interactions between estrogen and its multiple receptors. Until a few years ago, there were two known receptors and it was thought that all interactions were mediated by those two receptors. It’s been somewhat challenging to make headway in this space.”

    “Fortunately, we isolated a compound that selectively activates the new receptor GPR30, which was published in 2006. Since then, over 30 or so publications have used this probe to ask questions in almost all areas of physiology and biology; without this probe, it would have been far more difficult to ask these questions.”

    Dr. Sklar noted that the flow cytometry platform is well suited to fill a unique niche in small molecule identification for cell and molecular assays in suspension, especially in complex cell suspensions for primary cells, hematopoietic stem cells, and leukemia.

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