LAWRENCE — A new study published in mBio details the vulnerability of coronaviruses to inhibitors of a small protein domain called Mac1, or the “macrodomain,” found in all coronaviruses such as SARS-CoV-2 and MERS-CoV.
The findings point toward potential antiviral therapies to combat future coronavirus pandemics and confirm the importance of Mac1 to the viability of the virus.
“The macrodomain is critical for the virus’s ability to cause disease,” said Anthony Fehr, associate professor of molecular biosciences at the University of Kansas, who led the research. “We’ve known for a long time, based on our work and that of others, this gene is really important for the virus. Several groups, including ours, have started efforts to develop antivirals against it. But until recently, there hadn’t been any proven compounds that could target this gene and affect the virus, at least in cell culture.”
In the new paper, Fehr’s KU lab reports developing molecules that bind to the Mac1 protein and inhibit coronavirus replication in cell cultures derived from mice and human lung tissue. This work builds on recent experiments in the Fehr lab that produced the first molecules effective against Mac1.
“This paper is similar to our earlier work, though with a completely different set of inhibitors,” Fehr said. “But this study had some cool twists and turns. One key development was the discovery of our top compound — we called it ‘4B.’ It looked very promising in how it fit into the Mac1 binding pocket, and its IC50 — the amount of drug needed to reduce viral activity by half — was significantly lower than all of our other compounds, meaning it should be a better inhibitor.”
Yet when the group first tested 4B in antiviral assays, it didn’t actually work.
“It showed no effect,” Fehr said. “Based on its biochemical properties, we believe it wasn’t able to cross the cellular membrane. Cell membranes are greasy, hydrophobic barriers that keep things inside cells but also prevent certain molecules from entering — especially charged molecules, like those with acid or base groups attached. Compound 4B had a notable acid group, which likely prevented it from getting into cells.”
To address this, the group modified the compound to improve its ability to enter coronavirus-infected cells.
“We converted the acid into an ester, using a couple of different types of modifications,” Fehr said. “Once we did that, we started to see robust antiviral activity. This was the ‘aha!’ moment. We were finally able to make the compound cell-permeable and functional in cell culture. That was a big step — taking a molecule that had strong activity in vitro, modifying it and showing it could now work in cells against the actual virus.”
Fehr credited many of his collaborators for the breakthrough on Mac1 targets.
“I have to give credit to co-authors Dana Ferraris, chemistry professor at McDaniel College in Westminster, Maryland; Lari Lehtiö, a biochemistry professor at Oulu University in Finland; and KU core facility directors Anuradha Roy and David Johnson. Each of these individuals and their labs put a lot of work into this paper,” Fehr said. “And of course the majority of the credit goes to the first author, Jessica J. Pfannenstiel, a graduate student in the KU Department of Molecular Biosciences, as she produced almost all the antiviral data and identified the drug-resistant mutations.”
While coronaviruses can develop resistance to the experimental KU inhibitors, it does so at a “fitness cost” — meaning future generations with more resistance are otherwise weakened and don’t fare well in mice. This mechanism suggests these inhibitors, if further developed into an antiviral, could render coronavirus harmless.
For this to happen, Fehr said more work is needed to refine the promising molecules.
“Mouse models are important, and before we can test our inhibitors in mice we need to improve their potency and stability so that they will survive the harsh environment of a living organism,” he said. His group is continuing to work towards these goals.
The KU researcher gave credit to funding from the National Institutes of Health and KU’s Chemical Biology of Infectious Disease program, led by KU faculty member Scott Hefty, for the breakthroughs.
“This program has been crucial in supporting multiple groups working on inhibitors for viruses, parasites and bacteria,” Fehr said. “It provides core facilities and resources, like the high-throughput screening lab led by Anuradha Roy, that enable robust assay development and compound testing. These tools position us well to rapidly respond to potential future coronavirus outbreaks by identifying promising inhibitors. While drug development is a long process, KU is equipped to quickly and effectively begin the process of identifying novel antimicrobial compounds.”
Fehr said that beyond drug discovery, involvement in COBRE helps faculty gain expertise that improves their ability to evaluate national antimicrobial research, strengthening the broader scientific community.
“As a university, our primary role is to provide knowledge and develop leaders — faculty, students and postdocs — with expertise to advance infectious disease research nationwide,” Fehr said.
Other KU contributors include Daniel Cluff, undergraduate researcher; Nathaniel Schemmel, undergraduate researcher; Joseph O’Connor, graduate research assistant; Pradtahna Saenjamsai, graduate research assistant; Srivatsan Parthasarathy, postdoctoral researcher; Mei Feng, formulation scientist at the Biopharmaceutical Innovation and Optimization Center; and Michael Hageman, Valentino J. Stella Distinguished Professor of Pharmaceutical Chemistry.
Other collaborators include undergraduate researchers Lavinia Sherrill, Iain Colquhoun, Gabrielle Cadoux and Devyn Thorne of McDaniel College in Westminster, Maryland; and research scientists Men Thi Hoai Duong, and Johan Pääkkönen of Oulu University in Finland.
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