Illuminating Nonspecific Activation
Natasha Thorne, Ph.D., a post-doctoral fellow at the NIH National Human Genome Research Institute, has carried out investigations of drugs targeting many conditions, especially human genetic disorders. Working in the NIH Chemical Genomics Center under the leadership of James Inglese, Ph.D., and Douglas Auld, Ph.D., Dr. Thorne and her colleagues have employed a high-throughput screening assay that uses firefly luciferase and Renilla luciferase to validate the purported nonsense codon suppression activity of compounds.
Part of the NIH Roadmap for Medical Research is directed toward the screening of chemical libraries of low molecular weight compounds in a search for innovative pharmaceuticals. The collection is vast, including more than 300,000 different substances aimed at the entire range of human illnesses. The results of these screening studies will be shared through peer-reviewed publications and the PubChem database.
Since traditional high-throughput screening presents a single concentration, with a subsequent detailed follow-up of promising candidates, many hopeful molecules may be lost due to inauspicious choices of the initial set-up conditions.
In the platform that Dr. Thorne and her coworkers have adopted, however, a quantitative high-throughput approach is put into play, which includes a seven-point titration in 1,536-well plates. Because of the volume and complexity of the data, a bioinformatics program is brought to bear on the analysis.
Firefly luciferase is used as an indicator of target activity, however the compounds being evaluated can interact directly with the luciferase, causing unanticipated and confusing artifacts. In fact, when the team investigated a 70,000 compound library they identified 3% of the molecules as being directly active against the luciferase.
An example of how these concerns were realized comes from studies on PTC124 (from PTC Therapeutics), which is believed to suppress nonsense codons. Duchenne muscular dystrophy and cystic fibrosis are sometimes caused by nonsense mutations that prematurely terminate the proteins specified by these genes. “Our data suggests that the initial discovery of PTC124 may have been biased by its direct effect on the luciferase reporter,” Dr. Thorne said. Nonsense codon suppression is under clinical investigation by other laboratories, but it is too early to rule on its clinical promise.