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DiscoverX, PatoBios to Marry Flagship Techs, Co-develop Live-cell Assay for GPCR Screens


Canadian start-up PatoBios and assay provider DiscoverX have forged a scientific collaboration to combine their flagship technologies and develop a high-throughput live-cell assay for GPCR screening, CBA News has learned.

The deal may help PatoBios exploit the high-throughput potential of its patent-pending GPCR screening assay, which has thus far only been used in lower-throughput academic research settings, while possibly giving DiscoverX another cellular drug-discovery assay to add to its growing portfolio in this area.

In separate presentations at IBC's Assays and Cellular Targets conference held late last month in Bellevue, Wash., Brian O'Dowd, co-founder of PatoBios and Richard Eglen, CSO of DiscoverX, disclosed the collaboration.

Although full details of the alliance have not yet been worked out or formally announced, O'Dowd later told CBA News that he expected the companies would eventually be able to co-develop a product for sale to the pharmaceutical and academic drug-discovery markets. Eglen, meanwhile, told CBA News that the collaboration is currently research-based, but that will possibly evolve into a business agreement.

"That's still to be decided," Eglen said. "We have a research collaboration at the moment, and Brian has been using DiscoverX [technology] to develop his technology. We'll see how the data progresses, and how the business stage progresses. We're looking for the level of interest and reaction from pharmas and so on."

Specifically, the collaboration involves PatoBios' "multipurpose original cellular assay," or MOCA, and DiscoverX's ProLabel expression vector, which is based on the patented technique known as enzyme fragment complementation.

"It's very much a research collaboration at the moment, and we're looking for the level of interest and reaction from pharmas and so on."

The MOCA assay stemmed from work that O'Dowd did as a post doc in the lab of Duke University's Robert Lefkowitz, in order to discover agonists and antagonists of known and "orphan" (unknown) GPCRs. O'Dowd moved on to the University of Toronto as a professor of pharmacology, and in early 2004, along with colleague Susan George, founded PatoBios to exploit the technology for drug screening (see CBA News, 6/1/2004).

The basic strategy of the MOCA assay is to engineer a cell line to express a genetically modified receptor on its surface. The receptor contains a nuclear localization sequence that causes the receptor to translocate to the cell's nucleus, and a reporter molecule — usually GFP — that allows the user to track the translocation. As explained on the company's website, "interaction of the modified plasma membrane protein with structurally compatible compounds [drug candidates] prevents the translocation away from the cell surface and is measured as protein retained on the cell surface."

Therefore, PatoBios' assay allows researchers to detect GPCRs that localize at the cell surface by imaging the GFP that was attached to them, and then identify compounds that disrupt this process. However, this approach was not amenable to the type of high-throughput cell-based assay that drug companies might use in a primary assay, according to Eglen and O'Dowd.

"There's a difference when a drug is present, or when it is not present, in the amount of receptor that you can measure at the cell surface," O' Dowd explained. "There are lots of different ways to measure that, but you want something suitable for drug screening, automation, and homogeneous assays. It immediately struck me while looking at the [enzyme fragment complementation] technology that using it with our technology would be a good combination."

Enzyme fragment complementation is a general term for a technology called ProLabel that underlies several of DiscoverX's cell-based assay offerings. EFC is a homogeneous detection technology based on two genetically engineered ß-galactosidase fragments — a large protein fragment termed the enzyme acceptor, and a small peptide fragment termed the enzyme donor. Separately, the fragments are inactive, but in solution, they recombine to form active ß-galactosidase that hydrolyzes a substrate to produce a chemiluminescent or fluorescent signal.

In an assay combining the two technologies, DiscoverX's large enzyme acceptor fragment would essentially be the reporter molecule — thereby replacing GFP and allowing the PatoBios MOCA assay to be detected using a simple luminometer or fluorometer. Researchers would not be able to visually see the receptor translocation to the nucleus, but the throughput would be greatly increased, possibly making the assay amenable to high-throughput, primary, cell-based screens for modulators of known and orphan GPCRs.

DiscoverX Deal Could Help PatoBios
Avoid Patent-Infringement Issues

PatoBios' development of a potentially commercially viable GPCR screening assay based on the movement of GFP-tagged proteins through cells underscores the favorable intellectual property position Danish biotech BioImage has in the cell-based assay world.

BioImage holds several US and European patents, most specifically US Patent Nos. 6,518,021, "Method for extracting quantitative information relating to an influence on a cellular response;" and 6,172,188, "Fluorescent proteins," which rather broadly protect translocation assays — or what BioImage calls "Redistribution" assays — that involve monitoring the movement of GFP-tagged moieties around a cell for the purposes of commercial drug discovery.

For instance, Molecular Devices sub-licenses IP from BioImage that allows its customers to use its popular Transfluor assay, which MDCC acquired earlier this year from Xsira Pharmaceuticals.

The multipurpose original cellular assay devised by PatoBios co-founder Brian O'Dowd cells for the use of GFP as a way to monitor the movement of cellular receptors to the nucleus of cells. PatoBios may have encountered a pricey IP blockade in its quest to commercialize the assay.

But if PatoBios can successfully commercialize the assay with current scientific collaborator DiscoverX, it may be able to avoid the whole situation, since such an assay would replace the GFP molecule used in MOCA with the ß-galactosidase-based enzyme fragment complementation strategy that underlies DiscoverX's assay technology.

According to DiscoverX's Eglen, meshing the technologies may provide several benefits over the GFP-based method (as well as avoid possible patent issues that could arise with a GFP-based assay; see sidebar for more).

"You don't need to do imaging, so you can do the whole assay in a simple microtiter plate," he told CBA News. "The other thing is that you are putting a very small fusion tag on the receptor — GFP is about 26 kilodaltons and the DiscoverX tag is five kilodaltons — so the smaller the tag, the smaller the effect on the pharmacology. And we get gains in sensitivity. We have an enzymatic detection step, whereas GFP is simply fluorescent, so you get both sensitivity and better pharmacology."

Much of this may be putting the cart before the horse, however: Since the MOCA assay is not yet widely accepted in industry, researchers may still want to see the actual translocation event occur by using imaging with a fluorescent tag.

"I think in the early days of the evaluation, they will probably want to see both, to be quite honest," Eglen said. "The key thing is to show how widely applicable this is to a variety of receptors, including orphans, and also to see how the pharmacology of this method compares to traditional pharmacological methods — as well as maybe correlating the DiscoverX system to the imaging-based method."

As an example, O'Dowd found himself defending the MOCA assay during his presentation at the Assays and Cellular Targets meeting. After his talk, an audience member was dubious that O'Dowd's slides actually showed that the receptor tagged with a nuclear localization sequence and GFP was actually localizing in the nucleus — and he had a point, as many of the slides showed GFP-labeled molecules hanging around in many other parts of the cell.

According to O'Dowd, he and his colleagues have published several peer-reviewed papers on the underlying concepts of the assay, and have one pending in a coming issue of Journal of Biological Chemistry that contains much of the data O'Dowd presented at his talk.

"Fifty percent of what I showed [at the talk] is in that paper, and it went through an extensive peer-review process," O'Dowd said. "With any new technology, you're always going to have people who will question it — which is good — but we have thoroughly investigated this.

"You can talk about 'Does it go to the nucleus?' or whatever, but is there a difference when the drug is present, or when it is not present, in the amount of receptor that you can measure at the cell surface?" O'Dowd asked.

At the very least, Eglen is convinced. "When the receptor is liganded, clearly it will stay at the cell surface," he said. "Brian has detected that by GFP, by HA tagging, and by our system.

"How the receptor traffics inside the cell when the ligand is not there maybe still needs to be worked out," he added. "Brian's work does suggest that it travels to the nucleus, but it's a very complicated process. I think the key to the technology is to understand whether agonists have a different effect from antagonists, so it's more the mechanistic level. At the assay level … your real goal is to measure it at the cell surface. Where it goes elsewhere is another question."

— Ben Butkus ([email protected])

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