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Synthesis

Probing Drug-RNA Interactions

Drug Discovery: Reactions make it possible to show if a drug candidate hits its RNA target

by Stu Borman
August 12, 2013 | A version of this story appeared in Volume 91, Issue 32

BONDING & CAPTURE
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Credit: Adapted From Angew. Chem. Int. Ed.
In the Scripps technique, a small molecule (purple) that targets a cytosine-uridine-guanine (CUG) repeat sequence in disease-related RNA is derivatized with a reactive group (blue) and a biotin affinity tag (green). After binding, the drug covalently bonds to RNA. The combination is isolated via streptavidin (yellow) binding to biotin, and the RNA is then identified.
This scheme shows how a small molecule can attach itself to RNA.
Credit: Adapted From Angew. Chem. Int. Ed.
In the Scripps technique, a small molecule (purple) that targets a cytosine-uridine-guanine (CUG) repeat sequence in disease-related RNA is derivatized with a reactive group (blue) and a biotin affinity tag (green). After binding, the drug covalently bonds to RNA. The combination is isolated via streptavidin (yellow) binding to biotin, and the RNA is then identified.

Drug researchers, always on the lookout for new targets, are increasingly setting their sights on ribonucleic acids (RNAs). These biological molecules control many essential cellular functions and are associated with some diseases.

But proving that drug candidates hit the intended RNA targets has turned out to be difficult. RNA, in fact, has earned a reputation as being “undruggable” with small molecules because small-molecule/RNA interactions are difficult to detect and characterize. Now, however, two techniques make it possible for the first time to detect such interactions in cells.

In one approach, postdoc Lirui Guan and chemistry professor Matthew D. Disney of Scripps Research Institute Florida added a chlorambucil reactive group and a biotin affinity-purification tag to 2H-4, a small molecule (Angew. Chem. Int. Ed. 2013, DOI: 10.1002/anie.201301639). It targets a repeating cytosine-uridine-guanine pattern in mRNA associated with myotonic dystrophy, a common type of muscular dystrophy. In live cells, 2H-4/chlorambucil/biotin bound to and then covalently bonded to the target sequence. The biotin made it possible to use beads coated with streptavidin, which binds biotin, to isolate and characterize the resulting small-molecule/RNA complex, enabling the scientists to verify that the molecule had hit the desired RNA target.

As a bonus, the covalent bonding interaction boosted 2H-4’s ability to repair cellular defects associated with the disease 2,500 times relative to the efficacy of unmodified 2H-4.

Guan and Disney demonstrated their technique on a repeat sequence in an RNA molecule folded over in the shape of a hairpin. It’s not clear if the technique will work for RNAs folded into more elaborate three-dimensional shapes, but Disney says he believes his team will be able to demonstrate that. “The approach is likely general and could be applied to a wide range of disease-associated RNAs,” he says. Follow-up plans also include enhancing the selectivity of the small-molecule/RNA reactions and identifying RNA target interactions in animal models of disease.

Chemical biologist Victoria J. DeRose and coworkers at the University of Oregon have devised a complementary method that adds a reactive azide group to an RNA-binding small molecule. They demonstrated the approach with the platinum-based cancer drug picoplatin. The azide modification enables the team to use a click chemistry reaction—in this case a cycloaddition between an azide and an alkyne—to attach an alkyne-containing fluorescent marker to the azide-derivatized drug after it has bound to its RNA target (J. Am. Chem. Soc. 2013, DOI: 10.1021/ja402453k). They are then able to detect the drug-bound RNA complex by monitoring its fluorescence both in vitro and in treated cells.

DeRose and coworkers note that variations on this approach could eventually be developed to isolate, purify, and identify drug-bound RNA for sequencing, proteomics, or structure-function studies. Efforts to use click chemistry “to isolate and quantify Pt-bound species are currently under way in our laboratory,” the researchers say.

“RNA is still an underutilized target because not much is known about how small molecules interact with various RNA secondary and tertiary structure motifs,” says Christine S. Chow of Wayne State University, who is a specialist in small-molecule/nucleic acid interactions. “Probing small-molecule interactions with cellular RNA targets has not been achieved before with designed and selected compounds that target specific RNA structural motifs,” she notes.

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