Nitazoxanide (NTZ), a compound approved by the Food and Drug Administration for the treatment of diarrhea in patients infected by the Cryptosporidium and Giardia intestinalis species, is used extensively around the world with minimal toxicity. First described in 1975 by Jean Francois Rossignol, it was initially developed as a veterinary antihelminthic with activity against intestinal nematodes, cestodes, and liver trematodes. Remarkably, recent studies report that NTZ also exhibits antiviral activity against both RNA and DNA viruses. This is extraordinary because viruses that store their genetic information in RNA replicate differently from viruses whose genetic information is encoded in DNA, suggesting that NTZ can confer an unusually broad antiviral activity. Such broad antiviral activity has been attributed to effects on innate immune signaling; however, other reports have shown that NTZ may exert its effects through different virus-specific mechanisms. As such, the mechanism of action of NTZ against viruses remains unclear.
Although NTZ has been previously shown to inhibit some DNA viruses, including human cytomegalovirus, its effects on poxviruses, one of the largest known animal DNA viruses are unknown. Of the poxviruses that thrive in human hosts, variola virus was notorious as the cause of smallpox. Although smallpox has been eradicated, the potential for its re-emergence remains. Vaccinia virus (VACV) played a crucial role in the eradication of smallpox as the active constituent of the smallpox vaccine. VACV is widely used as the model poxvirus in the laboratory, functioning as a vector to deliver other antigens, as well as infecting and killing tumor cells.
So, how did NTZ end up in the hands of Dr. Adam Geballe? He explained: “This project started when a colleague of mine in VIDD, David Fredricks, alerted me to a paper indicating that the drug nitazoxanide, which is FDA-approved for treatment of some protozoal infections, inhibits some RNA viruses by activating protein kinase R (PKR), a host defense protein my lab has been working on for several years. We are always on the lookout for tools for manipulating the PKR pathway, so we did some pilot studies, which led us to conclude that the drug does not act though PKR, as had been suggested. However, we did confirm that it has extraordinarily broad antiviral activity, including against large DNA viruses. We therefore set out to try to elucidate the mechanism by which the drug inhibits vaccinia virus.” With help from Drs. Julian Simon and David Hockenbery and their laboratories, Sarah Hickson in the Geballe laboratory (Human Biology Division) systematically evaluated a large number of possible explanations for the drug’s effects. In their paper recently published in the journal Virology, they found that NTZ blocks a metabolic response that is required by many viruses to replicate efficiently.
Firstly, to determine if NTZ inhibits VACV, the authors infected human fibrobasts with a recombinant virus expressing lacZ and added medium containing varying concentrations (5uM to 20uM) of NTZ. Using beta-galactosidase activity assays, they showed that NTZ inhibited new virus production in a dose-dependent manner. Tizoxanide, the active metabolite of NTZ, demonstrated a similar level of inhibition. This trend was also observed in other cell lines from other species suggesting the inhibition is not species-specific. The authors observed that NTZ inhibited VACV production not only after a high multiplicity of infection, but also when added as late as 6 hrs post-infection, suggesting that NTZ acts after the virus gains entry into the cells. They then queried at which stage of the viral lifecycle NTZ exerts its effects by performing qPCR on genes indicative of the VACV replicative cycle. They also showed that NTZ prevents the shut off of host protein synthesis. These data suggest that NTZ inhibits the VACV replicative cycle at an early phase after infection.
Next, to further investigate the mechanism of action of NTZ on VACV inhibition, the Geballe lab turned to PKR, an appealing target that is part of the host defense system. Previous studies showed that NTZ can activate PKR, leading to inhibition of translation. However, the authors did not see activation of PKR by NTZ in this new study. They then studied the interaction of NTZ and the PKR pathway using a series of knockout experiments. From those results, they concluded that NTZ truly has no effect on the PKR pathway regardless of VACV infection, and that PKR is not necessary for the VACV-inhibitory activity of NTZ. Sarah Hickson explored more potential mechanisms of action based on what is known about NTZ and VACV, but found that neither a variety of mitochondrial functions, interferon signaling pathways or glutamine utilization were the answer.
NTZ inhibits the metabolic pathway that produces acetyl-CoA from pyruvate in bacteria. A similar process takes place in vertebrates, as acetyl-CoA is necessary in several metabolic pathways including fatty acid and cholesterol synthesis. Since palmitate is a precursor for more complex fatty acids, the authors tested whether palmitate could suppress the NTZ antiviral effect. Intriguingly, palmitate significantly decreased inhibition of VACV production by NTZ, suggesting that NTZ may interfere with fatty acid metabolism.
This study is the first to demonstrate the ability of NTZ to inhibit a poxvirus, and establishes the potential use of NTZ to control poxviral infections. Although the exact mechanism of action of the broad antiviral effect of NTZ remains unclear, the authors have uncovered a metabolic impact that will be further explored. Dr. Geballe affirmed his lab’s future efforts: “We are continuing to try to understand the drug’s antiviral mechanism since it might be a useful target for broadly acting antiviral drugs.”
Hickson SE, Margineantu D, Hockenbery DM, Simon JA, Geballe AP. 2018. Inhibition of vaccinia virus replication by nitazoxanide. Virology 518:398—405.
Funding was provided by the National Institutes of Health.
Fred Hutch/UW Cancer Consortium faculty members Adam Geballe, David Hockenbery and Julian Simon contributed to this research.