Researchers from the Scripps Research Institute this week reported on a new computational approach for designing small molecules against RNA using only the target's sequence.
Called Inforna, the strategy was used by the team to create small molecules for microRNA inhibition, in some cases with equal or greater selectivity than oligonucleotides, opening the door to alternative strategies for therapeutically targeting the small, non-coding RNAs.
The work shows that "one can take the products of a genome sequence and use that to directly design small-molecule lead therapeutics," Matthew Disney, a Scripps researcher and the study's senior author, said. Additionally, "it suggests that RNA is druggable with small molecules and maybe [these compounds are] competitive with oligos in terms of selectivity."
Despite the abundance of potential therapeutic RNA targets in the transcriptome, "non-ribosomal RNAs are rarely exploited with small molecules," the scientists wrote in a paper appearing in Nature Chemical Biology.
"People simply don't have an understanding of what types of RNA folds bind small molecules and what types of chemical structures bind to RNA," Disney explained. "If people go to do a straight high-throughput screen of RNA targets, you get much lower hit rates for those targets relative to proteins. There is just a limited understanding of how to drug RNA."
But if this hurdle can be overcome, and the ability to convert RNA sequence into secondary structure accurately merged with information about RNA secondary structural elements that bind small molecules, then the field would have "one very immediate way to leverage all these genome-wide association studies for disease into small molecule therapeutics," he told Gene Silencing News.
To that end, the scientists developed Inforna, which computationally folds RNA sequences and runs them through a database of RNA fold/small-molecule interactions.
For the study appearing in Nature Chemical Biology, the sequences and secondary structures of all known hairpin precursors in the human transcriptome were downloaded from miRBase and their structures were modeled via free energy minimization.
Inforna was then applied to the full set of secondary structures, providing the targetable motifs in each of the RNAs and listing the corresponding small molecules that bind them — a process that covered more than 5.4 million possible interactions.
The lead interactions were then refined using two criteria: the targetable motif must be in a Drosha or Dicer processing site, as these are cleaved to produce precursor miRNAs and mature miRNAs, respectively; and the miRNA in question must have been shown in other studies to be causative of disease.
Overall, the team identified 22 different miRNA precursors, including ones linked to prostate, breast, and ovarian cancers, as well as Parkinson's disease, that could be targeted with small molecules.
Using a statistical method they had previously developed to score RNA motif-small molecule interactions, the researchers measured the fitness of their small-molecule leads for binding its corresponding RNA motif in the precursor miRNA target, finding one targeting the miR-96 precursor was a perfect match, targeting a fold that was not present in any other miRNA precursor.
When tested in a primary cell line, the small molecule lowered the expression level of miR-96 by 90 percent at 40 micromoles. Because the precursors of miR-96, miR-182, and miR-183 are transcribed as a single transcript, the research group looked at the effects of the small molecule candidate on other miRNAs and found that it silenced miR-96 without having much of an effect on the other two miRNAs.
Taken together, these findings showed that the small molecule "was selective in theory, and selective and bioactive in practice," Disney said.
The research team also examined the effects of the small molecule on the downstream targets of miR-96, which has been shown to be upregulated in cancer and linked to oncogenic transformation through its silencing of forkhead box protein O1 (FOXO1).
The small molecule was introduced into breast cancer cells where it boosted FOXO1 levels three-fold, Disney explained. More importantly, the compound induced apoptosis in the cancer cells — an effect that appears to be a result of its effect on the miR-96/FOXO1 pathway based on data showing that the apoptotic effect could be significantly reduced by silencing FOXO1 mRNA expression.
Notably, the scientists also compared the effects of their miR96-targeting small molecule to seed-targeting locked nucleic acids — commonly known as tiny LNAs — and traditional antisense oligonucleotides.
The antisense molecule was found to affect the expression of 12 miRNAs including its target by at least 2.5-fold at 100 nanomoles, according to the paper. The tiny LNA, meanwhile, only "modestly discriminated" between miR-96 and miR-183, silencing its target by about 90 percent and miR-183 by about 50 percent at 50 nanomoles.
"One of the big impediments to getting people interested in targeting RNA with small molecules is the lack of selectivity," Disney said. "I don't know how general it is that small molecules can be at least as selective as oligonucleotides, but this [work] says that, at least for this case, it is possible."
He attributed the small molecule's selectivity for miR-96 to its 100 percent fitness as determined by Inforna, which suggests that it could be possible to predict if other molecules would be equally selective for other miRNA targets — work that he said is currently underway in his lab.