Relatively few specific anti-coronavirus disease 2019 (COVID-19) treatments exist. Until recently, hospitals were forced to either treat the symptoms using mechanical ventilation and oxygen supplements or use relatively untested emergency-approved drugs, many of which were later shown to have no effect. In a study published in Viruses, researchers from the New York University Grossman School of Medicine have screened several compounds that could show the ability to inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
Study: Atovaquone and Berberine Chloride Reduce SARS-CoV-2 Replication In Vitro. Image Credit: Corona Borealis Studio/Shutterstock
The researchers tested 12 compounds known to have antiviral/antimicrobial activity. These were tested across concentrations that previous studies had shown to be effective in Vero E6 cells. Cells were pre-treated for two hours with the compounds during and after viral infection to ensure the effect across all stages of viral replication was accounted for.
The first approved compound the scientists tested, hydroxychloroquine, was initially thought to have strong anti-SARS-CoV-2 properties, but more recent studies have questioned the efficacy when applied to infected humans. In this screen, the compound showed a significant ability to decrease SARS-CoV-2 titers while also showing low toxicity levels.
Azithromycin, another drug that received emergency approval, showed no significant effect on viral titers, and additional studies have shown no efficacy in COVID-19 patients. Many of the other candidates tested showed no impact on SARS-CoV-2 infection, including sofosbuvir, ribavirin, lisinopril, and verapamil. Others showed significant cytotoxicity that made them inappropriate in humans, such as imipramine, atorvastatin, lovastatin, and zinc chloride.
The researchers had more luck with atovaquone, which reduced SARS-CoV-2 titers compared to the control and maintained cell viability. Brequinar, another pyrimidine synthesis inhibitor, revealed the same results. Atovaquone, in particular, is particularly effective against Zika and chikungunya, although it reduces in effectivity at the highest concentrations. This was also seen in the most recent tests against SARS-CoV-2. When applied at these highest concentrations, crystals were observed forming in the media – potentially resulting in a lower concentration in the solution. It is also significantly cytotoxic at these concentrations.
When atovaquone is used as a malaria prophylaxis drug, it is often prescribed alongside proguanil-chloride, and the researchers examined the effect of this combination on infected Vero E6 cells. They also used both drugs individually. Atovaquone showed the same effects as previous experiments, proguanil-chloride was ineffective, but together they performed slightly better than atovaquone did individually. The combination was also not cytotoxic.
Due to the mechanisms by which atovaquone works (inhibiting the mitochondrial electron transport chain and an enzyme in the pyrimidine biosynthesis pathway), the researchers were worried that the antiviral effect could be tied to cell arrest. By treating the cells at a range of concentrations and rescuing them with uridine, they found that uridine rescued cell death at all but the lowest cell concentrations, but not enough to attribute the entire viral inhibition to cell arrest.
To ensure the compound could successfully inhibit viral replication in a more realistic setting, the scientists examine the ability of atovaquone to reduce SARS-CoV-2 infection in Calu-3, a human lung epithelial cell line – the cells that are typically infected when humans contract COVID-19. Once again, atovaquone showed significant ability to inhibit SARS-CoV-2 replication, but the synergistic effects of atovaquone and proguanil did not appear.
For even more accurate testing, the experiments were repeated on 3D human airway epithelial cultures to provide the most realistic model, as well as human 2D colonic organoids, as SARS-CoV-2 has shown the ability to infect gut organoids. Unfortunately, atovaquone showed no effects in the colonic colonies. The effects seen in the airway colonies were not statistically significant – although the researchers profess that the data could suggest that atovaquone interferes with SARS-CoV-2 replication in these colonies.
Finally, the scientists tested some holistic medicine. Berberine chloride has been shown to have some antiviral activity and is an orally bioavailable molecule derived from plant matter. In Vero E6 cells, the cells did appear to inhibit viral replication, but in Calu3 cells, this effect only showed at the highest concentration. Strangely, the cell viability rose with the concentration of berberine chloride in Calu3 cells. Repetition with CHIKV, a virus berberine chloride, has been shown to inhibit, the results were similar.
The authors argue that these results show that berberine chloride is a ‘broad-spectrum antiviral compound’ with multiple modes of action – which may be slightly premature, even if the results are promising. However, the atovaquone results are very suggestive of a powerful antiviral effect. While in-human trials are still necessary, this compound could prove very valuable against the spread of COVID-19.