To the untrained eye, the curls, folds and creases of the single strand of RNA that make up the coronavirus genome look like a jumble of spaghetti or tangled threads. But for researchers like Amanda Hargrove, professor of chemistry at Duke University, the complex forms RNA takes when it folds into itself could have untapped therapeutic potential in the fight against COVID-19.
In a study published Nov. 26 in the journal Science Advances, Hargrove and his colleagues identified chemical compounds that can cling to these 3D structures and block the virus’s ability to replicate.
“These are the first molecules with antiviral activity that specifically target virus RNA, so this is a completely new mechanism in this direction,” Hargrove said.
Even more than 18 months after the start of the pandemic, this is good news. We have vaccines to prevent COVID-19, but effective and easy-to-administer drugs to help people survive and recover once they have been infected remain limited.
The virus is declining in some parts of the world, but cases continue to rise in others where vaccines are scarce. And even in regions with easy access to vaccines, reluctance to face the COVID-19 vaccine means that many of the world’s eight billion people remain vulnerable to infection.
To infect your cells, the coronavirus must break in, deliver its genetic instructions in the form of RNA, and hijack the body’s molecular machinery to build new copies of itself. The infected cell becomes a virus factory, reading the 30,000 nucleotide “letters” of the virus’s genetic code and producing the proteins the virus needs to replicate and spread.
Most antivirals – 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 his colleagues take a different approach. They identified the first molecules that target the viral genome itself – not just the linear sequence of A, C, G, and U, but the complex three-dimensional structures into which the RNA strand folds.
When the first terrifying clues of the pandemic started to grab headlines, the team including Hargrove, Blanton Tolbert of Case Western Reserve University and Gary Brewer and Mei-Ling Li of Rutgers were already investigating potential drug candidates to fight a disease. another RNA virus – Enterovirus 71, a common cause of hand, foot and mouth disease in children.
They had identified a class of small molecules called amilorides that can bind to hairpin folds in the genetic material of the virus and throw a key in the replication of the virus.
To see if the same compounds could work against coronaviruses as well, they first tested 23 amiloride-based molecules against another much less deadly coronavirus, which is responsible for many colds. They identified three compounds that, 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 got similar results when they tested the molecules on cells infected with SARS-CoV-2, the virus that causes COVID-19.
Other work has shown that the molecules prevent the virus from building up by binding to a site within the first 800 letters of the viral genome. Most of this stretch of RNA does not code for the proteins themselves, but drives their production.
The region folds back on itself to form multiple bulges and hairpin structures. Using computer modeling and a technique called nuclear magnetic resonance spectroscopy, the researchers were able to analyze these RNA structures in 3D and determine where the chemical compounds bonded.
Researchers are still trying to figure out exactly how these compounds stop the virus from multiplying, once they are linked to its genome.
When it comes to using RNA as a drug target, Hargrove says the field is still in its early stages. Part of the reason is that RNA structures are unstable. They bounce much more than their protein counterparts, making it difficult to design molecules that can interact with them in specific ways.
“The binding sleeve you are looking for may not even be present most of the time,” Hargrove said.
Moreover, 85% of the RNA of an infected cell does not belong to the virus, but to ribosomes – cellular particles made up of RNA and proteins – of its human host. “There is a sea of competition,” said Hargrove.
But Hargrove is hopeful. The first small molecule drug that works by binding directly to non-ribosomal RNA, rather than proteins, was just approved by the FDA last August to treat people with a devastating disease called spinal atrophy . “So while there are a lot of challenges, it’s not impossible,” Hargrove said.
The researchers have a patent pending on their method. They want to modify the compounds to make them more potent, then test them in mice “to see if that might be a viable drug candidate,” Hargrove said.
This isn’t the first time coronaviruses have caused an epidemic, and it likely won’t be the last, researchers say. Over the past two decades, the same family of viruses was responsible for SARS, which emerged in China and spread to more than two dozen countries in 2002, and MERS, first reported in Saudi Arabia in 2012.
The researchers determined that the RNA loops and bulges they identified remained largely unaffected by the evolution of related coronaviruses in bats, rats, and humans, including those that caused the outbreaks of SARS and MERS. This means their method might be able to fight more than SARS-CoV-2, the virus that causes COVID-19.
Obviously, more antivirals would be valuable weapons, so when the next pandemic hits we will be better prepared. Having more drugs on hand would have another advantage: fighting resistance. Viruses mutate over time. Being able to combine drugs with different mechanisms of action would make it less likely that the virus could develop resistance to all of them simultaneously and become untreatable, Hargrove said.
“It’s a new way of thinking about antivirals for RNA viruses,” Hargrove said.
Reference: Zafferani M, Haddad C, Luo L, et al. Amilorides inhibit the replication of SARS-CoV-2 in vitro by targeting RNA structures. Sci Adv. 7 (48): eabl6096. doi: 10.1126 / sciadv.abl6096
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