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NIH Dual-Reporter Assay Enhances Precision, Excludes Nonspecific Transcriptional Effects

Researchers at the National Institutes of Health have published the first cell-based assay run at the NIH National Chemical Genomics center — a two-reporter luciferase-based assay to identify upstream inhibitors of the NFκB pathway that appears to offer a number of advantages over previous methods. 
The study, reported in this month’s issue of Assay and Drug Development Technologies, describes a two-reporter luciferase-based assay that uses red and green click-beetle luciferases to identify stabilizers of the IκBα protein in the lymphoma cell line OCI-Ly3.
According to Eric Davis, a senior research fellow at the Metabolism Branch of the National Cancer Institute and lead author of the paper, the dual-reporter format offers several advantages over single-reporter assays and previous two-color assays, including enhanced precision and the exclusion of nonspecific transcriptional effects.
In addition, the assay lends itself well to a high-throughput screening format. The NIH researchers validated their protocol in a 1,536-well format to screen more than 2,600 bioactive compounds and note in the paper that they have used the assay to screen 66,000 compounds in a study “to be published in a future report.”
“We wanted something that would specifically report on the biology of the lymphoma cells that we used, which for our purposes was the green beetle luciferase-IκBα fusion,” Doug Auld, group leader for genomic assay technologies at the NCGC and a co-author on the paper, told Cell-Based Assay News this week.  
“We also wanted something to report on cell uniformity, specifically related to cytotoxicity or nonspecific effects, or even the pipetting itself,” Auld said. “And we wanted something that could report on the number of cells per well, which for our purposes was red beetle luciferase. The ratio of the green and red luciferase reporters allowed us to normalize out many of the artifacts that typically occur during cell-based assays.”
Auld said that the dual-reporter design helped uncover an unexpected number of nonspecific signal activation events that would have gone unnoticed in a single-color assay.
“Although this has been reported before in the literature, we weren’t anticipating as many as we saw here,” he said. “The ratiometric nature of this assay allowed us to flag them immediately.”
Auld noted that a one-reporter system would have yielded a number of false positives, which “would have been a real problem.”
While the use of a second reporter protein is not uncommon in cell-based assays, Davis said that the NIH method is better than other two-color assays that use firefly and Renilla luciferases because the click-beetle luciferases are derived from the same organism and therefore use the same substrate.
“In terms of the logistics and mechanics of doing a high-throughput screen, you’ve cut your workload in half right there,” he said.
In addition, Davis said that the researchers were able to read their signals “almost simultaneously” by using filters, which simplified the reading. By comparison, when firefly and Renilla luciferases are used together, they each use different substrates that must be added separately and read at two different times.
The authors report in their paper that they identified two primary types of IκBα stabilizers using the assay: those with low efficacy and high potency, and those with high efficacy and lower potency.
“Our goal now is to actually look for compounds that affect the IκBα/NFκB pathway and nominate a chemical probe” that would be confirmed in secondary assays, said Auld.

“In terms of the logistics and mechanics of doing a high-throughput screen, you’ve cut your workload in half right there.”

Once they generate these data the researchers plan to post them on the homepage of the Molecular Libraries Screening Center Network, of which Auld’s lab is a part. Auld said these data may be used “as kind of a chemical probe for IκBα activation” so that “if people want to use it to study that pathway, it will be available to them.”    
Auld said that NCGC chemists are currently developing analogs around “a couple of promising” candidates that came out of the screen. “We are currently trying to optimize them, just for potency and solubility,” he said
Browsing the Molecular Library
Auld said that the NCGC works closely with NIH investigators like Davis, as well as external researchers, to guide assay design.
“We try to advise them on how to develop their assays in a way that will work well with low-volume, automated systems,” he said. Davis is “one of the first ones we talked to about this, and his assay was actually the first cell-based assay run at the NCGC, and it performed very well.”
Auld’s lab is a part of the NIH’s Molecular Libraries Screening Center Network, which was established in June 2005 as part of the NIH Roadmap’s “New Pathways to Discovery” initiative. The network is funded by all of the NIH institutes and is co-administered by the National Institute of Mental Health and the National Human Genome Research Institute. 
The MLSCN centers are screening a central repository of 70,000 compounds that is expected to grow to about 300,000 in another year or so, Auld said. All of their data are deposited in PubChem.
“If someone else wants to look at PubChem, and take these compounds and further develop them, that’s great,” said Auld. “IP can be filed on subsequent analogs. But the whole goal of anything that’s done within the molecular libraries initiative, including the probe development I just described, is to make it freely available.”
The compound repository is managed by BioFocus DPI, a subsidiary of Galapagos. Auld said that the company has a contract with the NIH to array these compounds into whatever format the screening centers need and ship the plates to the centers for screening.

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