An international team of researchers has found that three commonly used antiviral and antimalarial drugs are effective in vitro to prevent the replication of SARS-CoV-2, the virus that causes COVID-19. The work also underscores the need to test compounds against multiple cell lines to rule out false negative results.
The team, which included researchers from North Carolina State University and Collaborations Pharmaceuticals, looked at three antiviral drugs that have been shown to be effective against Ebola and Marburg virus: tilorone, quinacrine, and pyronaridine.
“We were looking for compounds that could block the virus from entering the cell,” says Ana Puhl, lead scientist at Collaborations Pharmaceuticals and corresponding co-author of the research. “We chose these compounds because we know that other antivirals that work successfully against Ebola are also effective SARS-CoV-2 inhibitors.”
The compounds were tested in vitro against SARS-CoV-2, as well as against the common cold virus (HCoV 229E) and murine hepatitis virus (MHV). The researchers used a variety of cell lines that represented potential targets for SARS-CoV-2 infection in the human body. They infected the cell lines with the different viruses and then looked at how well the compounds prevented viral replication in the cells.
The results were mixed, with the efficacy of the compounds depending on whether they were used in human-derived cell lines or monkey-derived cell lines, known as Vero cell lines.
“In human-derived cell lines, we found that all three compounds worked in a similar way to remdesivir, which is currently used to treat COVID-19,” says Frank Scholle, associate professor of biology at NC State and a co-author of the research. . “However, they were not entirely effective in Vero cells.”
“The researchers saw similar results when these compounds were initially tested against Ebola,” says Sean Ekins, CEO of Collaborations Pharmaceuticals and a corresponding co-author of the research. “They were effective in cell lines of human origin, but not in Vero cells. This is important because Vero cells are one of the standard models used in this type of testing. In other words, different cell lines can have different responses to a compound. It points out the need to test compounds in many different cell lines to rule out false negatives. “
The next steps of the research include testing the effectiveness of the compounds in a mouse model and further work to understand how they inhibit viral replication.
“One of the most interesting findings here is that these compounds not only prevent the virus from potentially binding to cells, but they can also inhibit viral activity because these compounds act on lysosomes,” says Puhl. “Lysosomes, which are important for normal cell function, are hijacked by the virus to enter and exit the cell. So if that mechanism is disrupted, it cannot infect other cells. “
“It is also interesting that these compounds are effective not only against SARS-CoV-2, but also against related coronaviruses,” says Scholle. “It could give us a head start on therapies as new coronaviruses emerge.”
The work appears in ACS Omega and was supported in part by the NC State Institute for Comparative Medicine and the National Institutes of Health. NC State college students James Levi and Nicole Johnson, as well as Ralph Baric of the University of North Carolina at Chapel Hill, contributed to the work. Other collaborating institutions were: the Oswaldo Cruz Institute and the University of Campinas, both in Brazil; Utah State University; the University of Maryland; and SRI International.
Note to editors: A summary follows.
“Reuse of Ebola and Marburg virus inhibitors, tilorone, quinacrine and pyronaridine: in vitro activity against SARS-CoV-2 and possible mechanisms”
DOI: 10.1021 / acsomega.0c05996
Authors: Ana Puhl, Sean Ekins, Collaborations Pharmaceuticals; Frank Scholle, James Levi, Nicole Johnson, North Carolina State University; et al
Published: March 12, 2021 at ACS Omega
The severe acute respiratory coronavirus 2 (SARS-CoV-2) is a recently identified virus that has caused more than 2.5 million deaths worldwide and more than 116 million cases worldwide in March 2021. The Small molecule inhibitors that reverse the severity of the disease have been difficult to discover. One of the key approaches that has been widely applied in an effort to accelerate drug translation is drug reuse. Some drugs have shown in vitro activity against Ebola viruses and have shown activity against SARS-CoV-2 in vivo. Most notably, remdesivir-targeted RNA polymerase demonstrated in vitro activity and efficacy in the early stage of disease in humans. Testing other small molecule drugs that are active against Ebola viruses (EBOV) would seem like a reasonable strategy to assess their potential for SARS-CoV-2. Previously, we have reused pyronaridine, tilorone, and quinacrine (for anti-malaria, influenza, and antiprotozoal uses, respectively) as Ebola and Marburg virus inhibitors in vitro on HeLa and EBOV cells adapted to mice in vivo. We have now tested these three drugs on various cell lines (VeroE6, Vero76, Caco-2, Calu-3, A549-ACE2, HUH-7, and monocytes) infected with SARS-CoV-2, as well as other viruses (including MHV and HCoV 229E). The compilation of these results indicated considerable variability in the antiviral activity observed across the cell lines. We found that tilorone and pyronaridine inhibited virus replication in A549-ACE2 cells with IC50 values of 180 nM and IC50 of 198 nM, respectively. We used microscale thermophoresis to test the binding of these molecules to the peak protein, and tilorone and pyronaridine bind to the peak receptor binding domain protein with Kd values of 339 and 647 nM, respectively. The human Cmax for pyronaridine and quinacrine is greater than the IC50 observed in A549-ACE2 cells. We also provide new insights into the mechanism of these compounds, which is probably lysosomotropic.