Note: this article first appeared in the Fall 2014 issue of PittPharmacy.

The transmitted light module at the top of the ImageXpress Micro automated high content imaging platform. Automated high content screening (HCS) platforms provide integrated solutions for both the acquisition and analysis of digital images of thousands of cells that have been arrayed into the wells microtiter assay plates.

Discovery of the cellular pathway that causes a disease starts the exciting and challenging process of discovering a drug or new combination of drugs that will correct the pathology. The sheer number of chemical compounds that might have the desired effect is daunting. Millions of compounds could potentially be tested, but testing that many compounds in animals for effect is not possible. Paul Johnston, PhD, has been addressing that vexing problem for the last 20 years.

A biochemist by training, Johnston’s stock-in- trade is developing high throughput and high content screening assays capable of testing hundreds of thousands of chemicals for activity. Each time Johnston develops a new assay, he needs to find a key point in the biochemical pathway where interaction with a test compound yields a measureable desired change. He then develops highly specific and robust cellular assays using robotics to create a system that can screen thousands of compounds per day. Johnston must grow, harvest, and count the cells, uniformly dispense them into wells, and then incubate them with the test compounds. He asserts that “the scientific payoff is better using cells than in non-cell- based experiments, which are easier to perform.

“If you find a hit in a cell-based assay, you generally have a better lead because you already know the compound works in a cell.” “What I hope to see are compounds that I identify moving through the drug development process. Then comes the next batch of questions: Will the compound be absorbed and distributed throughout the body? Will it be toxic? How will the body metabolize it?”

Johnston, a research associate professor in the department of pharmaceutical sciences, has been asking those questions at the University of Pittsburgh since arriving in 2005. Pitt’s Drug Discovery Institute had just won funding through the pilot phase of the National Institutes of Health’s roadmap initiative, which sought to transform biomedical research by overcoming some defined knowledge gaps. Johnston, who previously worked in the pharmaceutical industry, was hired and spent his first few years at Pitt building a high throughput screening center, ending up with one of the most comprehensive facilities of its kind in academia.

Paul Johnston Lab
Paul Johnston (seated) with his lab team, left, front to back graduate students Stan Kochanek, Ashley Fancher, and Tim Pouland. Right, front to back, research associates Yun Hua, David Close, and Daniel Camarco.

“For some approaches, I’d certainly say that we could compete with big pharma,” he says. The lab collaborated with both Pitt researchers and scientists from outside universities, guiding them about which assay formats to select and helping to perfect the assays to the point where they’re ready to begin screening.

“That’s a really important point,” said Johnston. “A lot of investigators in big pharma and academia have been running these assays for years and think they’re ready to go,” but almost universally, the assays have not been fully developed and involve too many chances for statistical variability.

About three years ago, Johnston left the Drug Discovery Institute to set up his own chemical biology lab at the School of Pharmacy, where he continues to collaborate with investigators. Currently, most of his projects focus on cancer therapeutics.

“Many of my colleagues are interested in, and working on, drugs to cure cancer,” he explains. “It’s a major unmet need.”

Cancer in the crosshairs

Johnston recently collaborated with Jennifer R. Grandis, a Pitt Medical School researcher who is focused on head and neck cancer, to develop a high-content imaging assay to identify selective inhibitors of STAT3 pathway activation that is common in head and neck cancer. The STAT3 and STAT1 pathways are
involved in the regulation of cell growth and they work in opposite directions. STAT3’s target genes inhibit cell death (apoptosis), promote cell proliferation, and hinder anti-tumor immune responses. STAT1’s target genes, on the other hand, activate cell cycle arrest, promote apoptosis, and enhance anti tumor immune responses. STAT1’s target genes, on the other hand, activate cell cycle arrest, promote apoptosis, and enhance antitumor immunity. If a compound inhibits STAT3 selectively and does not interfere with STAT1 signaling, the compound would be a potentially effective anticancer drug.

Johnston, in developing his screen, tested several head and neck squamous cell cancer (HNSCC) cell lines as candidates for the assay based on the biochemistry of the STAT3 pathways and the cytokines that activated the pathways. He also developed a counter screen for STAT1 that could be conducted in the same cells. All of the 22 steps in the assay were automated and then validated by testing a 1,280-compound library of pharmacologically active compounds. Johnston’s High Content Screen is now being used to screen chemical libraries to identify new drug leads to treat head and neck cancer.

In July 2014, Johnston and Nathalie Wong from the Chinese University of Hong Kong announced a collaborative research agreement to screen the 50,000-compound Pharmacy School Diversity Library to identify inhibitors of liver cancer cell growth.

AR-TIF2 SlideSpecifically, they are looking for compounds that target higher levels of the GHF-H1 onco- gene, which is associated with more aggressive tumors. Wong is providing the cell lines, and Johnston is developing the high throughput screening assays. Their hope is to flag compounds that might one day be developed into new drugs for liver cancer therapy.

“In the end, we want to provide [researchers] with a list: Here are the top hits from the screen that would selectively kill liver cancer cells that overexpress GHF-H1,” Johnston says.

In an independent prostate cancer project, Johnston’s approach was to optimize a high content biosensor imaging assay that would measure the interaction between the androgen receptor that is critical for the proliferation of prostate tumor cells and a coactivator that helps regulate gene transcription. The assay he developed is significant because it could help identify drugs that would work in the more aggressive forms of prostate cancer that continue to grow even after surgery and relapse from the standard androgen ablation therapies.

Another contract from the National Cancer Institute charged Johnston with developing miniature assays for drug combination screening in several forms of cancer, including melanoma, leukemia, prostate, and ovarian cancer. The idea was the find two drugs that, when combined together, synergistically killed more cancer cells more effectively than they would alone.

Dawn of discovery

Working at the very beginning of the drug development life cycle, Johnston’s
sophisticated high content screens play an important role in the
discovery of new therapeutics. His role is enhanced by the collaborations
he has been able to build with Pitt colleagues, particularly in the
School of Pharmacy, where researchers commonly investigate transporters,
metabolism, drug absorption, and distribution—all factors that influence how well a drug achieves its goal. That ability to collaborate across disciplines is crucial to his program’s success, he notes.

“If I find some good-looking hit and lead compounds, I have chemistry collaborators who put some skin in the game and do some synthesis  around the leads,” Johnston says. “In academia, unless you can establish collaboration with the people who have skills different from yours, you have to teach yourself how to do it. Having my group be in the School of Pharmacy, where I’m side by side with these colleagues, means that it’s a much more efficient process.”

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