Scientists Take Aim at Disease-Causing RNAs Using Small-Molecule Drugs

This chasm has left many drug developers interested, instead, in targeting RNA—specifically, using small molecules that could bind to the three-dimensional structure of misbehaving RNAs and prevent their translation into problematic proteins or, if they are noncoding, block their cancer-promoting function within the cell. Compared to other RNA-targeting drugs on the market—such as antisense therapeutics, which inhibit dysfunctional RNAs using synthetic complementary RNAs—small molecules generally have low toxicity profiles and are easier to deliver to where they’re needed, even when the target is a solid tumor or a cell lurking beyond the blood-brain barrier. Indeed, some scientists have speculated that the most elusive targets in cancer biology—the transcription factors c-Myc and STAT3 and the cell-signaling protein KRAS, for example—might be made accessible using this strategy.

Many proteins are basically not druggable today because they don’t have those obvious small-molecule binding sites.

—Michael Gilman, Arrakis Therapeutics

But researchers have had several reasons to be skeptical about finding the right drugs for the job. For starters, RNA—protein-coding messenger RNA (mRNA) in particular—is considered relatively malleable, flipping from one three-dimensional configuration to another in unpredictable ways. What’s more, scientists have long assumed that RNA has limited structural diversity due to its four-nucleotide alphabet, making individual sequences difficult to target with selectivity on the basis of their structure.

That view is now beginning to change. In 2018, biomedical scientist Kevin Weeks at the University of North Carolina at Chapel Hill and colleagues published results from E. coli showing that RNAs with complex structures that are predictable from nucleotide sequence alone are far more common than once thought (Cell, 173:181–195.E18). The team also found that highly structured mRNA showed lower levels of protein expression than flexible, linear mRNAs, suggesting that manipulating RNA structure using small molecules could indeed influence protein production.

Weeks is hoping to harness knowledge of RNA’s three-dimensional structures to develop RNA-targeting therapeutics at Ribometrix, the startup he cofounded in 2014. The company raised $30 million in Series A funding—and many other companies taking the same tack are enjoying similar investor interest. Last summer, for example, Massachusetts-based Skyhawk Therapeutics, which is developing small molecules to target mis-spliced RNA in diseases ranging from cancer to neurological conditions, secured a $60 million upfront payment as part of a deal with pharma firm Celgene, and raised an additional $40 million in equity investment. Other industry heavyweights, including Novartis and Merck, have begun developing their own programs to target RNA with small-molecule drugs.

“The number of targets, the number of diseases that one could go after by targeting RNA, is almost unimaginable,” says Matt Disney, a chemist at the Scripps Research Institute in Jupiter, Florida, and founder of Expansion Therapeutics, a startup headquartered in San Diego, California, that is currently working to develop small-molecule drugs to target mutant mRNAs implicated in a degenerative muscular condition called myotonic dystrophy. “It’s nearly everything.”

Blocking protein translation

While excitement about the therapeutic potential of RNA-targeting small-molecule drugs is relatively recent, several compounds that block protein translation of specific mRNAs have been on the market for a while. Naturally occurring antibiotics such as tetracylines, macrolides, and aminoglycosides, as well as synthetic oxazolidinones, act by disrupting the function of bacterial ribosomes—the sites of mRNA’s translation into protein—which themselves are made of RNA. And bacterial riboswitches, noncoding structures in mRNA molecules that regulate gene expression by binding to metabolites and ions, have been successfully targeted using small molecules such as Merck’s ribocil. The rapid emergence of bacterial resistance to these molecules, however, has stalled progression to the clinic.

See “Riboswitch Flip Kills Bacteria”

These cases of small molecules binding to RNA were dismissed as outliers by many researchers. Then, a couple of years ago, there were two landmark discoveries: scientists at pharmaceutical giants Novartis and, later, Roche—the latter in collaboration with PTC Therapeutics—independently developed similar small-molecule drugs for the treatment of spinal muscular atrophy (SMA), a disease frequently caused by faulty splicing of RNA transcripts of the SMN2 gene. Both compounds, branaplam and risdiplam, respectively, were discovered through high-throughput screening techniques not specifically tuned to search for RNA-targeting molecules. But it was later found that the drugs repair the faulty splicing pattern by stabilizing the pre-mRNA molecule in a way that ensures that exon 7 of the gene, which is usually skipped in SMA sufferers, is retained. The drugs are currently performing well in Phase 2 clinical trials.

Noncoding RNAs are also frequently implicated in the development of disease, offering a suite of possible targets for RNA-targeting small-molecule drugs.

The progress of these two drugs has revitalized researchers’ hopes of finding small molecules to halt the expression of disease-promoting proteins. “If they are approved, it will be a landmark achievement for the RNA-targeting field,” says Disney.

Several companies are now aiming to target mRNA directly with small molecules that could stop protein being made by physically preventing translation at the ribosome or by recruiting a nuclease from within the cell to break down particular transcripts. Successfully targeting mRNAs in these ways “would change the face of drug discovery by enabling proteins to be targeted even before they are fully synthesized,” writes Weeks in an email. Researchers at Ribometrix and Arrakis, for example, tell The Scientist they are on the hunt for small molecules that can bind the mRNA of oncogenic transcription factors such as c-Myc, although successes in drugging such high-profile RNAs have yet to be reported.

But oncology has plenty of other mRNA targets to choose from. For instance, in 2014, researchers at the University of California, San Diego, led by biochemist Thomas Hermann identified a potential therapeutic target on the mRNA coding for thymidylate synthase, an enzyme that confers resistance to some chemotherapy drugs (Biosci Rep, 34:e00168).

Thymidylate synthase is a self-regulating protein—the first of its kind discovered in humans—which can bind to a small hairpin structure on its own mRNA to inhibit translation. Hermann, who has previously consulted for Arrakis, hopes that targeting this hairpin with a small molecule could inhibit the enzyme’s production, though so far, no effective small molecule drugs to target that hairpin have been found.

With increasing use of large-scale screening and transcriptome sequencing techniques, the number of potential targets for such approaches is rapidly expanding. In addition to focusing on well-known oncogenes whose protein products remain frustratingly undruggable, several companies are investigating how to target the mRNA products of novel disease-associated genes, even if their mode of action is poorly understood, says Gilman. (Arrakis will not disclose the specific targets it’s exploring.) “We don’t actually need to know much about their function to target their mRNA.”

Targeting noncoding RNAs

It’s not just mRNA that has received interest from drug developers. Noncoding RNAs, which regulate translation, RNA splicing, and gene expression, are also frequently implicated in the development of cancer and other diseases, offering a suite of new possibilities for RNA-targeting small-molecule drugs.

For example, in 2015 Disney’s group reported the discovery of a small molecule that can selectively bind to and inhibit miR-544, a microRNA involved in the development of breast cancer (ACS Chem Biol, 10:2267–276). When mutated, this microRNA silences the mammalian target of rapamycin (mTOR) protein, allowing breast cancer cells to survive the low-oxygen environment deep within a tumor. The small molecule was able to reverse breast tumor cells’ adaptive physiological response to hypoxia, so that low oxygen triggered apoptosis in the cells in vitro and in mice. Two years later, Disney and colleagues reported success drugging miR-210, which is involved in the hypoxic response of both breast and prostate tumors (J Am Chem Soc, 139:3446–455). Disney says his team is still working on targeting miR-544 and miR-210, with the hope of eventually bringing small-molecule drugs for these deviant miRNAs to the clinic.

Another promising target is miR-21, an RNA responsible for inhibiting a number of tumor-suppressing phosphatases. This RNA has been linked to breast, ovarian, cervical, colon, lung, liver, brain, esophagus, prostate, pancreatic, and thyroid cancers. In 2016, Gabriele Varani, a biophysical chemist at the University of Washington in Seattle, and colleagues reported an engineered RNA-binding protein that targets the precursor molecule pre-miR-21 and prevents it from being processed into the mature oncogenic miRNA (Nat Chem Biol, 12:717–23). Varani has started a company to help develop the work commercially, though he notes that it’s still at the investment-seeking stage.

While the RNA biology is very compelling and very exciting, it’s also much less established than the biology around some other therapeutic areas with more-traditional targets.

—Thomas Hermann
University of California, San Diego

At least one approved anticancer small-molecule drug has since been found by Disney and colleagues to act on pre-miR-21, preventing the pre-miRNA’s maturation and reversing a metastatic phenotype in breast cancer cell lines. And in February this year, the team announced another success in targeting the precursors of oncogenic miRNAs—this time miR-515, a microRNA that renders breast cancer cells resistant to the anticancer drug Herceptin. They found a small molecule that binds to pre-miR-515 and inhibits its maturation, restoring sensitivity to Herceptin in breast cancer cells in the laboratory (J Am Chem Soc, 141:2960–974).

A different approach is to decommission faulty noncoding RNAs by using small-molecule drugs that recruit the cell’s own RNA-degrading machinery, specifically enzymes known as ribonucleases. In 2018, Disney’s lab reported the discovery of a small molecule that binds to miR-96, a microRNA that promotes tumor development by repressing the pro-apoptotic transcription factor FOXO1. Once bound, the drug attracts the cellular ribonuclease RNAse L to cleave the miRNA. In the laboratory, treatment with the molecule, named Targaprimir-96, triggered cell death in cancerous, but not healthy, breast epithelial cells (J Am Chem Soc, 140:6741–744).

The path forward for RNA-targeting small-molecule drugs

Despite the hype, experts told The Scientist that any new RNA-targeting small-molecule drugs would have to wait for clinical trials, though they differed in their long-term predictions, suggesting it could take as little as 2 and as long as 15 years for these drugs to reach patients. “It’s not going to be transformative overnight,” says David Hong, an oncologist at the University of Texas MD Anderson Cancer Center in Houston who regularly consults for and receives research funding from the pharmaceutical industry. But “I think the technology will continue to get better.”

Even with ample time, it’s not necessarily smooth sailing from here. For starters, the field’s relative immaturity makes it difficult to predict which small molecules will have the desired effect on function when administered in living organisms, rendering drug discovery in this area a particularly lengthy and expensive process.

“While the RNA biology is very compelling and very exciting, it’s also much less established than the biology around some other therapeutic areas with more-traditional targets,” says Hermann. There may be “more surprises once the compound enters the clinic . . . because we know so little about side effects and off-target effects of these compounds.”

The University of Washington’s Varani shares Hermann’s concerns. “I am worried about target validation
—whether targeting some of these RNAs will actually be as beneficial [in patients] as some of the cellular and in vivo data indicates,” he says.

Nevertheless, RNA-targeting small molecules will eventually be a key part of doctors’ medicinal toolkit in oncology as well as in the treatment of many neurological, inflammatory, immune, and infectious diseases, Disney argues. Over the next decade, for “every large pharma, there’s going to be a gap in their pipeline if they’ve not got an RNA-targeting small molecule in it.”

Claire Asher is a freelance science writer living in London, UK.