RNAi turns poppies off morphine

For the first time, scientists have reported that RNA interference can be used to redirect the opium poppy away from generating morphine to manufacturing precursors of potential new drugs. "This approach makes it possible to force accumulation of other interesting molecules which could be of value in the pharmaceutical industry," study coauthor Philip Larkin at Commonwealth Scientific and Industrial Research Organization (CSIRO) Plant Industry in Canberra, Australia, told The Scientist. His group's research appears in the December issue of Nature Biotechnology. The enzymology of the entire morphine pathway is largely elucidated, but virtually nothing is known of what regulates the accumulation of morphine and its intermediates in poppies. Larkin and colleagues employed RNA interference to silence codeinone reductase (COR), the penultimate enzyme in morphine biosynthesis, and shed light on pathway controls. The researchers incorporated regions of complementary DNA from all seven members of the multigene family encoding COR into a chimeric hairpin RNA binary vector. Larkin said the hope was the resulting poppies would accumulate thebaine, the compound preceding COR in the pathway and substrate for commercial analgesics buprenorphine and oxycodone. "The surprising thing here is that when COR was silenced, the alkaloid which accumulates was not the immediate precursor to the silenced enzyme," Larkin said. Opium poppies usually demonstrate an alkaloid profile of morphine, codeine, oripavine, and thebaine. The 18 transgenic poppies produced by the Australian group instead displayed the nonnarcotic alkaloid S-reticuline, which lies seven enzyme steps upstream of COR, and its methylated derivatives codamine, laudanine, and laudanosine. "This is truly surprising and shows the network is a lot more complex than people assumed—definitely not like in bacteria, when you knock out a gene and the next intermediate would be accumulated," said Meinhart Zenk of Martin Luther University in Halle, Germany, who did not participate in this study. Reticuline is a precursor for potential anticancer and antimalarial pharmaceuticals. It normally makes up less than 2 percent of total alkaloids in the opium poppy. "It's really hard to accumulate some of these intermediate materials. This hairpin RNA silencing method really represents an elegant and cost-effective way to put these precursor molecules out there for people to explore," said George Stefano, director of the Old Westbury Neuroscience Institute in New York, who did not participate in this study. "We don't yet know the biomedical significance of these intermediates. But we're finding reticuline active in the nervous system and elsewhere. You now have this abundant source to explore reticuline, make derivatives, see if you can induce something with greater activity," Stefano told The Scientist. Larkin and colleagues suggest the unexpected reticuline phenotype may have been caused by a buildup of morphine substrates codeinone and neopinione that triggered negative feedback on one or more earlier enzymes or transport steps of the morphine branch. Alternatively, the substrate feedback may have inhibited transcription of genes encoding earlier enzymes or transporters. Loss of COR enzyme from a larger interdependent enzyme complex may also have disabled the other enzyme reactions normally associated with the complex. "Scientists in this field, like ourselves, will be impatient to test some of the hypotheses of how the regulation is occurring—for example, does codeinone inhibit the enzyme reticuline oxidase," Larkin said. "We are already busy silencing other steps to gain more insights into the regulation and compartmentalization of the various steps of morphine biosynthesis." Zenk did not feel reticuline was of much use pharmacologically, "but this hairpin RNA technology showed how very valuable it could be in unraveling these tricky regulation steps. If they can, they could really accumulate intermediates that are valuable," he told The Scientist. In the September 23 issue of Nature, Larkin and colleagues had previously revealed how the conventionally bred top1 mutant opium poppy predominantly produced morphine precursors thebaine and oripavine. While oripavine did have its uses, Larkin said his team aims to generate through RNAi a thebaine-only poppy.

RNAi turns poppies off morphine

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