SRI's Piazza and Keeton, and Blueshift’s Cromwell Discuss Assay Development for the NIH's MLSCN

The National Institutes of Health’s Molecular Libraries Screening Center Network, part of the NIH’s Roadmap for Biomedical Research, has established a new way for academic and non-profit institutions to operate — namely, as industrial-scale drug screening operations. But it is also serving as a proving ground of sorts for innovative screening technologies.

At the Southern Research Institute’s MLSC, lead investigator Gary Piazza and colleagues are using Blueshift Biotechnologies’ IsoCyte laser scanning system to develop high-throughput cellular assays based on fluorescence resonance energy transfer (FRET) and fluorescence anisotropy detection. If successful, the project could be a major boon for nascent Blueshift, which is still trying to build a foothold for IsoCyte in the academic and pharmaceutical screening markets.

At the Society for Biomolecular Sciences’ annual meeting held this week in Seattle, Cell-Based Assay News caught up with Gary Piazza, SRI associate research biologist Adam Keeton, and Blueshift President Evan Cromwell to discuss the SRI-Blueshift collaboration in more detail.

Gary, what is your involvement with SRI, and how does that work within the scope of the MLSCN project?

GP: I am the program director for the SRI MLSC. We do a number of different assays for the NIH MLSCN – biochemical assays, cell-based assays, molecular assays. We’re developing new capabilities for image-based assays and, as part of this initiative, we’ve been collaborating with Blueshift to develop a novel image-based assay to be able to measure changes in cGMP levels in live cell populations. Cyclic GMP is a molecule involved in signaling and plays a role in a diverse number of physiological and disease processes, such as cell survival, smooth muscle relaxation, memory, and cancer. There are really no assays that are available to quantify fluxes, or dynamics of cGMP in cells. We’re interested in developing a fluorescence-based assay to be able to quantify live cells. It is based on FRET and anisotropy measurements.

There isn’t anything available now to measure changes in intracellular cGMP levels?

GP: No, there certainly is nothing to measure changes in cellular cGMP levels themselves. So this is truly an innovative method that, if we can get it working, would have broad utility.

The Blueshift technology that you’re using is called the IsoCyte. Is that actually an imaging platform?

EC: IsoCyte is a laser-scanning imaging system that collects four channels of information. What we can do is collect two colors of anisotropy, which allows us to then measure FRET pairs for cGMP. We can reference to the anisotropy changes, as well as looking at polarization ratios, and it gives you a very robust way to measure the occurrence of FRET in a live cell.

GP: We’re interested in developing this method for high-throughput screening to identify molecules from the NIH Roadmap small-molecule repository for compounds that might affect cyclic GMP, whether it’s through inhibiting its degradation through, for example, phosphodiesterases; or through activating it via guanylate cyclases.

Why did you select the Blueshift technology for this assay? There are other technologies on the market that could be adapted to this type of assay.

AK: The reason we went with the Blueshift instrument is its specific ability to measure anisotropy, which is a way of measuring FRET that gives a very high signal-to-background or signal-to-noise ratio as compared with conventional and more traditional ways of measuring FRET. These are not thought of as lending themselves to a very robust assay. That’s really the strength of this, that it allows us to measure FRET in a very robust way on a cell-by-cell basis.

GP: This is also a non-invasive, or less invasive, method relative to other types of conventional fluorescence imaging platforms.

EC: Yes, it is a laser-based system, but it is a low-powered laser, so you are not doing any photochemical damage to the cells.

This is designed to be a live-cell, kinetic assay?

GP: Yes, you’re getting spatial information, kinetic information, and an idea of the percentage of cells that are responding to stimuli.

What about the actual assay system, in terms of the FRET dyes and the cellular constructs? Did your lab develop these?

GP: This is through a collaboration with the University of Wurzberg [Germany]. They were the ones that were responsible for engineering the specific constructs we’re using to transfect cells. It’s been a very productive international collaboration, and our intent is to develop stable transfected lines that would express these constructs, using colon tumor cells. We have evidence that cGMP is an important signaling molecule for inducing apoptosis. So the ultimate goal is to identify compounds that would have potential as anti-cancer drugs for colorectal cancer.

Within the MLSC program, you said you’re conducting both HTS biochemical assays as well as cell-based assays. What other cellular assay technologies are you working with besides this one?

GP: We recently developed and ran a cell-based assay to measure Her-2 expression in cancer cells. We also have an assay in our program that would reverse resistance to cancer chemotherapeutic drugs. We have assays to measure yeast life span, as well as a yeast-based assay to measure vesicle trafficking. We also have a novel anti-bacterial assay to look for anti-bacterial compounds. These are all assays that we are running as part of the NIH Roadmap activity.

The data from this work will eventually be deposited into PubChem?

GP: Eventually. I should emphasize that these are the early stages of assay development. The ultimate goal is to develop this into a valid and robust high-throughput screen, so we’re applying for NIH grant support for additional funding for that development. The intent is to develop the assay and then send it to the Roadmap initiative for screening.

Evan, what is the importance of an academic center like SRI, which is heavily involved in a screening project the size and scope of the MLSCN, adopting your technology? Would you like to eventually sell this into pharmaceutical screening labs?

EC: We already have an instrument placed in the Vanderbilt MLSC. But the technology that we’re using to try to help Gary develop this to a high-throughput screen is called FRET by anisotropy, which was developed by David Piston at Vanderbilt. We have a license to that technology, and we’re definitely looking for partners to help develop sensors that can utilize this technology. Gary is the example of a perfect partner for this type of application. A FRET sensor has tremendous potential utility, and it is valuable to have a screening center that is top of the line to help develop this into an assay. Once that occurs, I think there will be a lot of interest in doing this in other laboratories, and at pharmaceutical companies.

What are the next steps in developing this into a high-throughput assay?

GP: Well, we are still in the early stages of assay development, but the next step is to develop stable transfected cell lines to be able to develop this into a robust HTS assay. Then we want to be able to configure it for screening, and then do validations with some compounds that are known to affect cGMP levels in cells. We need to identify positive control compounds and characterize their activity in our cell-based models.