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Some Chemical Rx Compounds May Affect Signal Modulation of Luciferase Assays, NIH Finds

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This article has been corrected to reflect the fact that researchers would see an activation in an analogous system "if there was genuine readthrough going on, and not reporter stabilization." The original version of the story said they would not observe an activation.

Some chemical compounds may trigger nonspecific but highly reproducible signal modulation in certain luciferase assays, according to a study conducted by the National Institutes of Health’s Chemical Genomics Center.

As a result, scientists should consider potential interactions between reporter enzymes and chemical compounds comprising a screening library, and contemplate running control assays before interpreting data from high-throughput screens, NCGC officials told CBA News this week.

In the study, the NCGC researchers found that some compounds that inhibit firefly luciferase don't dim the enzyme's glow, but actually make it appear brighter. That has implications for those using luciferase as a reporter during drug development, since increased luciferase activity is often associated with enhanced target protein activity.

“Luciferase assays are highly sensitive and very useful assays,” said Douglas Auld, group leader for genomic assay technologies at the NCGC and lead author of the study, which appears online this week in Proceedings of the National Academy of Sciences. “What we are suggesting is that when you run these assays, be careful ... to look at reporter-specific effects.”

“[B]ioluminescent reporters are very powerful reporters, the limitations of which, in regards to chemical libraries, one needs to be very aware,” added James Inglese, deputy director of the NCGC and the corresponding author on the paper.

The NCGC researchers trace the results of their findings to 2005, when they began profiling the Molecular Libraries Small Molecule Repository for luciferase activity, and found that luciferase was an important reporter enzyme for a number of cell-based assays.

Three years later, the team published a paper in the Journal of Medicinal Chemistry that described whether the 70,000 compounds in the library inhibit firefly luciferase.

That study identified “several classes of inhibitors” and found that the structure of some of them resembled that of luciferin, a substrate of luciferase. “They were similar structures, and, sure enough, when we did the enzymology, they competed with the luciferin substrate,” Auld said.

In a subsequent 2008 study published in ACS Chemical Biology, the NCGC team demonstrated that 3,5-diaryl-oxadiazoles appear to cause an activation phenotype in cell-based reporter gene assays that used wild-type firefly luciferase, due to the interaction of the compound with luciferase and the resulting stabilization of the enzyme.

One year earlier, researchers from drug maker PTC Therapeutics published a study in Nature showing that a 3,5-diaryl-oxadiazole called {3-[-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid}, or PTC124, increased the frequency of readthrough of a premature nonsense mutation in a firefly luciferase gene.

This finding led the researchers from South Plainfield, NJ-based PTC Therapeutics to design a luciferase-based assay looking at readthroughs. To do this they put a premature stop codon in the middle of a luciferase gene and looked for compounds that would increase luciferase expression by reading through the stop codon.

In this initial characterization of PTC124, firefly luciferase activity was found to increase in the presence of PTC124, although mRNA levels remained unchanged.

The PTC Therapeutics scientists did not investigate the possibility of luciferase enzyme stabilization, although their data would be consistent with this effect of PTC124, according to the NCGC investigators.

In the NCGC’s PNAS paper, Auld said the researchers designed the luciferase gene with a stop codon in the middle of the gene, “as [the PTC Therapeutics researchers] had done.” Auld’s team was able to reproduce PTC Therapeutics’ results with PTC124, which they synthesized in-house, as well as with related analogs.

To further test the idea of enzyme stabilization, the NCGC team designed another bioluminescent reporter system, which used Renilla reniformis luciferase.

“In this case, we actually made separate cell lines that expressed Renilla variants with a stop codon in the middle of its gene,” said Auld. The scientists knew that 3,5-diaryl-oxadiazoles such as PTC124 did not inhibit Renilla luciferase, and they reasoned that “if we made an analogous system, and if there was genuine readthrough going on and not reporter stabilization, we would see activation in that system,” Auld said.

The researchers then tested this Renilla system with PTC124 and “other analogues” and “did not see any activation of the system, and any inhibition of the enzyme,” Auld said.

The NCGC team also found that PTC124 is a potent inhibitor of purified firefly luciferase, but is inactive against Renilla luciferase. They also demonstrated that incubating purified firefly luciferase with PTC124 protects the enzyme against degradation by trypsin.

“The real summary of the story is that PTC Therapeutics has done further work around the series of compounds,” said Auld, who is not associated with the company.

The firm now has animal model data and clinical trial data suggesting that PTC124 is an effective therapy for cystic fibrosis and Duchenne muscular dystrophy. Results of a trial of the compound in cystic fibrosis patients were published in the Aug. 30, 2008, issue of The Lancet.

“So it is possible, as we said in the PNAS paper, that the compound both inhibits the firefly luciferase and has genuine readthrough activity,” said Auld. “This is a story that has not completely played out, but this is what we would conclude.

“We still think that the structure of PTC124 and the way it was optimized was highly biased by the system that was used,” Auld added.

He said that because his team used an alternative Renilla reporter system to parse out readthrough activity from enzyme stabilization, it was difficult to distinguish the structure that was stabilizing the reporter from structures leading to the genuine biological activity that the PTC Therapeutics researchers were trying to figure out.

“The message is that when you run these types of assays, it is really a requirement to look at alternative reporter systems early on during the optimization of the compound,” said Auld. “And we wonder if there could be better compounds, perhaps related to PTC124 or other structures that do not inhibit the reporter or affect the reporter, which might also have readthrough activity.”

The only bioluminescent reporters that the NCGC team tested in the PNAS paper were firefly and Renilla luciferase. However, because they “wanted to further validate the Renilla luciferase system,” the scientists screened a library of about 1,200 compounds against a purified Renilla enzyme and identified inhibitors.

“We found that some of those compounds showed the same reporter stabilization phenomenon that was seen with firefly luciferase,” said Auld.

“It is to be expected that there will be chemical inhibitors of almost any [class of] enzyme,” Inglese added. “It is just something that I think will be more common than an exception, and the idea, of course, is to get on top of it and know the pharmacology of your reporter, so that you can use it in the best possible way.

“We think that any luciferase will have substances in chemical libraries that will interact with it,” he said. “You have to just know what they are.

“One thing we are doing here at the NCGC is trying to develop better tools and understanding at the very early stage of the drug-discovery process, particularly making sure that people know how to use these tools properly,” said Auld.

He said the work described in the PNAS paper is actually “a twist on the dual-glow system commonly used in cell-based assays, where you have the firefly and Renilla luciferase expressed in the same cell.”

In the past, this has been used as a way to normalize to non-specific effects such as cytotoxicity. “We would suggest that another use of this system would be to look at reporter-specific effects, because they are not likely to be inhibited by the same compounds,” Auld said.

If something is affecting the assay because it is directly affecting the reporter, that activity should go away if the reporter is switched, he added. If it is genuinely active, that effect should be maintained.

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