To the untrained eye, the loops, kinks and folds in the single strand of RNA that make up the coronavirus genome look like a tangle of spaghetti or tangled yarn. But for researchers like Amanda Hargrove, a chemistry professor at Duke University, the complex shapes that RNA takes when it folds itself could have untapped therapeutic potential in the fight against COVID-19.
In a study that will appear in Science Advances Nov. 26, Hargrove and colleagues identified chemical compounds that can attach to these 3D structures and block the virus’ ability to replicate.
These are the first molecules with antiviral activity that specifically target the virus’s RNA, so it’s a completely new mechanism in that sense. “
Amanda Hargrove, professor of chemistry, Duke University
Even more than 18 months after the pandemic started, that’s good news. We have vaccines to prevent COVID-19, but effective, easy-to-administer drugs that help people survive and recover from infection remain limited.
The virus is declining in some parts of the world, but cases are still increasing in others where vaccines are scarce. And even in regions with easy access to vaccines, reluctance to adopt COVID-19 vaccines means many of the eight billion people worldwide remain susceptible to infection.
To infect your cells, the coronavirus has to break in, deliver its genetic instructions in the form of RNA, and hijack the body’s molecular machinery to make new copies of itself. The infected cell becomes a virus factory, reads the 30,000 nucleotide “letters” of the virus’s genetic code, and produces the proteins the virus needs to replicate and spread.
Most antiviral drugs – including remdesivir, molnupiravir, and paxlovid, the only antiviral drugs for COVID-19 that have been approved by the FDA or are pending approval – work by binding to these proteins. But Hargrove and colleagues are going a different way. They identified the first molecules that target the viral genome itself – and not just the linear sequence of A’s, C’s, G’s, and U’s, but also the complex three-dimensional structures into which the RNA strand folds.
When the first terrifying indications of the pandemic hit the headlines, the team led by Hargrove, Blanton Tolbert from Case Western Reserve University and Gary Brewer and Mei-Ling Li from Rutgers were already investigating potential drug candidates to fight another RNA virus – Enterovirus 71, one common cause of hand, foot and mouth disease in children.
They had identified a class of small molecules called amilorides that attach to hairpin-like folds in the virus’s genetic material and can interfere with the virus’s replication.
To see if the same compounds could work against coronaviruses, they first tested 23 amiloride-based molecules against another, far less deadly coronavirus, which is responsible for many colds. They identified three compounds that, when added to infected monkey cells, reduced the amount of virus within 24 hours of infection without causing collateral damage to their host cells. They also showed greater effects at higher doses. The researchers obtained similar results when they tested the molecules on cells infected with SARS-CoV-2, the virus that causes COVID-19.
Further work showed that the molecules prevented the virus from building up by binding to a location in the first 800 letters of the viral genome. Most of this RNA segment does not itself code for proteins, but drives their production.
The region folds in on itself to form multiple bulges and hairpin-like structures. Using computer models and a technique called nuclear magnetic resonance spectroscopy, the researchers were able to analyze these 3D RNA structures and pinpoint where the chemical compounds bind.
Researchers are still trying to figure out how these compounds stop the virus from multiplying once they’re tied to its genome.
When it comes to using RNA as a drug target, the field is still at an early stage, according to Hargrove. One reason for this is that RNA structures are unstable. They bounce around a lot more than their protein counterparts, making it difficult to develop molecules that can interact with them in specific ways.
“The tie-bag you’re looking for may not even be there most of the time,” said Hargrove.
In addition, 85% of the RNA of an infected cell does not belong to the virus, but to the ribosomes – cellular particles made of RNA and proteins – of its human host. “There’s a sea of competition,” said Hargrove.
But Hargrove is hopeful. The first small molecule drug that works by binding directly to non-ribosomal RNA, not proteins, was approved by the FDA just last August for the treatment of people with a devastating disease called spinal muscular atrophy. “While there are many challenges, it is not impossible,” said Hargrove.
The researchers have applied for a patent for their method. They want to modify the compounds to make them more potent and then test them on mice “to see if this could be a viable drug candidate,” Hargrove said.
This isn’t the first time coronavirus has caused an outbreak, and it probably won’t be the last, researchers say. For the past two decades, the same family of viruses was responsible for SARS, which appeared in China and spread to more than two dozen countries in 2002, and MERS, which was first reported in Saudi Arabia in 2012.
The researchers found that the loops and bulges of RNA they identified remained essentially unchanged through evolution in related coronaviruses in bats, rats, and humans, including those that caused the SARS and MERS outbreaks. That means their method can potentially do more than just fight SARS-CoV-2, the virus that causes COVID-19.
Obviously, more antivirals would be valuable weapons so that we are better prepared for the next pandemic. Having more medication on hand would have another advantage: combating resistance. Viruses mutate over time. Being able to combine drugs with different mechanisms of action would make the virus less likely to develop resistance to all and impossible to treat at the same time, Hargrove said.
“This is a new way of thinking about antivirals against RNA viruses,” said Hargrove.
Researchers worked at seven institutions for this study, including Rutgers University, Case Western Reserve University, Washington University School of Medicine in St. Louis, University of Nebraska-Lincoln, University of Glasgow, and University of Michigan.
Zafferani, M., et al. (2021) Amilorides inhibit SARS-CoV-2 replication in vitro by targeting RNA structures. Scientific advances. doi.org/10.1126/sciadv.abl6096.