The antiparasitic drug atovaquone inhibits arbovirus replication through the depletion of intracellular nucleotides

Arthropod-borne viruses represent a significant public health threat worldwide yet there are few antiviral therapies or prophylaxis targeting these pathogens. In particular, the development of novel antivirals for high-risk populations such as pregnant women is essential to prevent devastating disease such as that which was experienced with the recent outbreak of Zika virus (ZIKV) in the Americas. One potential avenue to identify new and pregnancy friendly antiviral compounds is to repurpose well-known and widely used FDA approved drugs. In this study, we addressed the antiviral role of atovaquone, a FDA Pregnancy Category C drug and pyrimidine biosynthesis inhibitor used for the prevention and treatment of parasitic infections. We found that atovaquone was able to inhibit ZIKV and chikungunya virus virion production in human cells and that this antiviral effect occurred early during infection at the initial steps of viral RNA replication. Moreover, we were able to complement viral replication and virion production with the addition of exogenous pyrimidine nucleosides indicating that atovaquone is functioning through the inhibition of the pyrimidine biosynthesis pathway to inhibit viral replication. Finally, using an ex vivo human placental tissue model, we found that atovaquone could limit ZIKV infection in a dose-dependent manner providing evidence that atovaquone may function as an antiviral in humans. Taken together, these studies suggest that atovaquone could be a broad-spectrum antiviral drug and a potential attractive candidate for the prophylaxis or treatment of arbovirus infection in vulnerable populations, such as pregnant women. Author Summary The ability to protect vulnerable populations such as pregnant women and children from Zika virus and other arbovirus infections is essential to preventing the devastating complications induced by these viruses. One class of antiviral therapies may lie in known pregnancy-friendly drugs that have the potential to mitigate arbovirus infections and disease yet this has not been explored in detail. In this study, we show that the common antiparasitic drug, atovaquone, inhibits arbovirus replication through intracellular nucleotide depletion and can impair ZIKV infection in an ex vivo human placental explant model. Our study provides a novel function for atovaquone and highlights that the rediscovery of pregnancy-acceptable drugs with potential antiviral effects can be the key to better addressing the immediate need for treating viral infections and preventing potential birth complications and future disease.


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Abstract 24 Arthropod-borne viruses represent a significant public health threat worldwide yet there are few 25 antiviral therapies or prophylaxis targeting these pathogens. In particular, the development of 26 novel antivirals for high-risk populations such as pregnant women is essential to prevent 27 devastating disease such as that which was experienced with the recent outbreak of Zika virus 28 (ZIKV) in the Americas. One potential avenue to identify new and pregnancy friendly antiviral 29 compounds is to repurpose well-known and widely used FDA approved drugs. In this study, we 30 addressed the antiviral role of atovaquone, a FDA Pregnancy Category C drug and pyrimidine 31 biosynthesis inhibitor used for the prevention and treatment of parasitic infections. We found that 32 atovaquone was able to inhibit ZIKV and chikungunya virus virion production in human cells and 33 that this antiviral effect occurred early during infection at the initial steps of viral RNA replication.

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Moreover, we were able to complement viral replication and virion production with the addition of 35 exogenous pyrimidine nucleosides indicating that atovaquone is functioning through the inhibition 36 of the pyrimidine biosynthesis pathway to inhibit viral replication. Finally, using an ex vivo human 37 placental tissue model, we found that atovaquone could limit ZIKV infection in a dose-dependent 38 manner providing evidence that atovaquone may function as an antiviral in humans. Taken   39 together, these studies suggest that atovaquone could be a broad-spectrum antiviral drug and a The ability to protect vulnerable populations such as pregnant women and children from Zika virus 49 and other arbovirus infections is essential to preventing the devastating complications induced by 50 these viruses. One class of antiviral therapies may lie in known pregnancy-friendly drugs that 51 have the potential to mitigate arbovirus infections and disease yet this has not been explored in 52 detail. In this study, we show that the common antiparasitic drug, atovaquone, inhibits arbovirus 53 replication through intracellular nucleotide depletion and can impair ZIKV infection in an ex vivo 54 human placental explant model. Our study provides a novel function for atovaquone and 55 highlights that the rediscovery of pregnancy-acceptable drugs with potential antiviral effects can 56 be the key to better addressing the immediate need for treating viral infections and preventing 57 potential birth complications and future disease. strain of ZIKV in the presence of atovaquone or a DMSO control and quantified infectious virion 144 production by plaque assay. We found that atovaquone significantly impaired virion production in 145 all cell types tested although the peak of inhibition varied between cell type (Figure 2A-C).

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One potential explanation for these results could be that atovaquone is toxic, and this 147 leads to reduced virus production. To address this, we first measured cell viability by a MTT cell 148 proliferation assay and found that atovaquone indeed had a dose-dependent reduction in cell 149 proliferation compared to the DMSO control ( Figure S1A) Figure S1B). Using this assay, we found that high concentrations of atovaquone, 155 particularly in 293T cells, did lead to more cell death than the DMSO control; however, lower 156 concentration, that did show effects by the MTT assay, had minimal effects on cell viability.

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Moreover, these results were confirmed in our data as although we observed a reduction in cell 158 growth by MTT assay we found that in Vero and 293T cells, virus production increased at higher 159 concentrations of atovaquone suggesting that the cells are still competent for virus production 160 under these conditions. Taken together, these results show that atovaquone is able to inhibit ZIKV 161 virion production in mammalian and human cell types.

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To expand on these findings, we addressed if atovaquone could inhibit chikungunya virus  Figure 3A). This inhibition in replication was further 170 confirmed in Vero and 293T cells where we observed reductions in infectious CHIKV virions after 171 treatment with atovaquone ( Figure 3B). These results suggest that atovaquone can inhibit 172 multiple arboviruses and has the potential to be used as a well-tolerated antiviral therapy.

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Using an MTT assay of cell proliferation measured through mitochondrial function we also saw that atovaquone was able to reduce mitochondrial function in all cell lines we analyzed, yet these

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Working virus stocks were generated by passaging virus over BHK-21 cells and viral titers were 379 quantified by plaque assay as described above.