The National Institutes of Health has awarded Southern Research Institute a $256,384 grant under its Molecular Libraries Roadmap to develop a high-throughput assay that will screen for novel inhibitors of phosphodiesterase isozymes by measuring the levels of cyclic guanosine monophosphate in live cell populations.
Gary Piazza, manager of the cell biology and immunology group at Southern Research Institute and principal investigator on the grant, told Cell-Based Assay News that his group is collaborating with Blueshift Biotechnologies to take advantage of the company’s IsoCyte laser scanning fluorimeter, which has capabilities that make it particularly suitable for the assay.
In particular, IsoCyte is able to measure FRET (Förster resonant energy transfer) by anisotropy — the degree of polarization of emitted light from a molecule after it is excited with polarized light. The anisotropy capability promises to enable faster measurement as well as a much higher signal-to-noise ratio compared to standard FRET.
“This is not your conventional fluorescence assay for high-content screening,” Piazza said. "We will rely on fluorescence polarization in the context of FRET to image and measure intracellular GMP levels using a fluorescence protein construct that serves as a cGMP biosensor and instrumentation from Blueshift Technologies that is capable of measuring anisotropy."
One driver for developing the assay, he said, was that "we have previously shown that certain phosphodiesterases are elevated in tumors and therefore cyclic GMP levels are low. … This provides a unique technology to screen for novel cyclic GMP phosphodiesterase inhibitors that may be elevated in tumor cells and represents a novel strategy for cancer drug discovery."
Chris Shumate, vice president of business development at Blueshift, explained that FRET is traditionally done by measuring the emission ratios of two fluorophores as they interact. “The problem with that,” he said, “is that there has never been a high-content assay based on FRET just because of the cumbersome nature of doing that measurement. Often those two emission spectra overlap so significantly that it’s difficult to deconvolve and get the ratio.”
FRET by anisotropy, however, should “allow any protein-protein interaction to be measured on a cell-by-cell basis in living cells at high throughput,” Shumate said, noting that the system can perform a typical assay in four to eight minutes per well plate.
Blueshift sees the work with SRI as further proof of concept for IsoCyte’s anisotropy capabilities. Shumate said that the company has so far completed a caspase assay using FRET by anisotropy “as well as some model fusion systems.”
After the project is complete, Shumate said the company hopes to develop “a series of kits” for measuring protein-protein interactions in live cells in high throughput.
Evan Cromwell, president of Blueshift, said that the company is also developing assays for the system with the University of California, San Francisco, and Active Motif.
“I think where the development really has to come is in these FRET sensors — developing a fluorescent protein pair, a [cyan fluorescent protein/yellow fluorescent protein] pair that shows very good FRET interaction and change in FRET upon some cellular event,” Cromwell said.
“That’s one of the things we’re working on with Gary, is to develop the FRET sensors that would go with our instrumentation.”
Blueshift and the Piazza lab at SRI have worked on pilot studies to characterize the cGMP biosensor in transient transfected cells [CBA News 09-22-06], but the NIH grant will provide funding to further advance the assay development effort for high-throughput screening, which will involve the development of stable
transfected tumor cell lines.
”Once we develop the assay for high-throughput screening, we plan to submit the assay to the NIH Molecular Libraries Screening Center Network for screening against its large repository of small molecules,” Piazza said. SRI is an NIH-designated Molecular Libraries Screening Center.
Cyclic GMP is of particular interest in cancer research because it regulates tumor cell survival, among other cellular processes. Phosphodiesterases are known to degrade cGMP, so cancer researchers are interested in identifying PDE inhibitors that could prevent the degradation of cGMP and thereby enhance its effects in tumor apoptosis.
“This is not your conventional fluorescence assay for high-content screening.”
“Previous studies that we have conducted suggest that cGMP PDE inhibition is an important off-target effect, which is responsible for the tumor cell growth inhibitory activity of certain highly potent nonsteroidal anti-inflammatory drugs such as sulindac,” Piazza and colleagues explain in the abstract for the NIH grant.
“These studies led us to hypothesize that safer and more selective cancer drugs can be discovered by interrogating a large compound library for cGMP PDE inhibitory activity.”
Piazza explained that one challenge in identifying useful PDE inhibitors for cancer applications has been the sheer number of PDE isozymes and the potential for mutations in tumor cells. "There's a very large family of these isozymes," he said. "There are probably well over 50 or so isozymes that exist in the human genome, but it's not clear which are useful to target for cancer."
The cell-based cGMP imaging assay is "a novel strategy to develop molecular probes that should provide valuable tools to identify those isozymes that are expressed in tumor cells and regulate tumor cell growth or survival," he said.
According to the grant abstract, the SRI team will develop a novel cGMP biosensor based on a chimeric protein composed of cyan- and yellow fluorescent proteins fused with the cGMP binding domain of PDE2 or PDE5.
“Fluorescence resonance energy transfer (FRET) results from a conformational change of the protein upon cGMP binding to cause decreased polarization (anisotropy) of YFP fluorescence, which provides a highly specific analyte to image intracellular cGMP in live cell populations using a laser scanning fluorimeter,” the abstract states.
Aims of the study include generating a stable transfected colon tumor cell line that expresses the cGMP biosensor, developing an image-based assay to measure cGMP levels in transfected cell populations, and configuring the assay for high-throughput screening.
“A key innovation of this cell-based assay is its ability to identify pharmacologically relevant cGMP PDE inhibitors that suppress tumor cell growth without prior knowledge of the specific isozymes involved in carcinogenesis,” the abstract states.
Piazza said that while his group is partnering with Blueshift, it’s likely that the resulting assay will not only run on the IsoCyte.
“That remains to be seen,” he said. “We do have an Evotec [now PerkinElmer] Opera at Southern Research Institute, and we will be comparing that to the Blueshift IsoCyte to see which one has advantages, so it will give us some ability to compare them.”