Is niclosamide a potential SARS-CoV-2 antiviral inhibitor?
Since the beginning of the coronavirus disease 2019 (COVID-19) pandemic, there has been a scramble to identify antiviral drugs that could assist in combating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, many of the most potent candidates against the virus are limited by poor bioavailability or toxicity. Niclosamide is an antihelminthic drug, historically used to treat tapeworm infestations, though has been investigated and repurposed for use against SARS-CoV-2. In a new study recently uploaded to the preprint server bioRxiv* by Garrett et al. (July 12, 2021), the drug demonstrates the ability to reorganize the lipid profile of SARS-CoV-2 infected Vero E6 cells, thereby limiting virus replication. Niclosamide has previously been shown to suppress MERS-CoV replication by inhibiting autophagosome-lysosome fusion through disrupting autophagy regulator proteins. This made it of interest as an early drug candidate against SARS-CoV-2. Specifically, the Beclin1 - S-phase kinase-associated protein 2 (Bec1-SKP2) pathway, though as with other 4-aminoquinolines (hydroxychloroquine and chloroquine), the drug appears to have other functional effects that result in broad-spectrum anti-infective properties. To investigate the mechanism of action of the drug, the group exposed Vero E6 cells to the drug, characterizing the autophagic or lipophagic pathways that are targeted in the presence or absence of SARS-CoV-2 infection, and performed global lipid extraction and analysis to identify their role in antiviral effects or cell death. The group firstly established that SARS-CoV-2 alters the host cell lipidome, as has been demonstrated by previous studies. Pre-existing host lipids and their signaling mechanisms are co-opted by SARS-CoV-2 for cell entry and subsequent replication. The group assessed the lipid profile of infected cells at several time points. Lipids bearing longer fatty acid chains with lower saturation levels were elevated at 16 hours, while saturated fatty acids, monounsaturated fatty acids, and long-chain fatty acids were notably downregulated. At 48 hours, these latter fatty acids instead become elevated. Triglyceride lipids and cholesterol fatty acids are significantly downregulated at 48 hours, thought to be due to their use in the construction of viral particles. There was also an increase in plasminogens, as observed in several other viruses such as Zika. Administering niclosamide to the cells in the absence of SARS-CoV-2 infection resulted in lowered triglyceride at both time points, and similarly downregulates the ether lipid profile during infection. This suggests that the drug can disrupt lipid droplet formation, potentially depriving SARS-CoV-2 of its major source of triglycerides. Niclosamide also causes a decrease in plasminogens in uninfected cells, and induces autophagy in infected cells. Similarly to chloroquine, the drug disrupts lysosomal function, resulting in an increase in autophagic flux. The effect, however, was seen to be proportional to the viral burden, stage of replication, and cellular state. Phosphatidylethanolamine (PE) levels in infected cells were noted to be unaffected by treatment with niclosamide at 16 hours, though significantly elevated at 48 hours. PE is a lipid that is involved in assembling factors that go on to initiate autophagy, and therefore the significant increase at this time-point suggests that the drug enhances autophagy machinery upon viral replication. There was also a notable reduction in ganglioside numbers upon administration of the drug, which is involved in the initiation of autophagic cell death. Niclosamide may, therefore, increase cell survival upon infection, and this factor could have contributed to the low toxicity observed. The influence of the drug on plasminogen levels may also contribute to lessening the cytokine storm induced by SARS-CoV-2 by reducing the inflammatory response of the cell. In conclusion, niclosamide was able to lower SARS-CoV-2 viral burden in Vero E6 cells by stimulating autophagy, and raising PE levels while lowering the number of ether lipids and triglycerides in the cell. These lipids are essential to virus formation, and thus the ability of the virus to enter the cell, replicate, and undergo egress is limited. Unfortunately, the drug is unlikely to perform well when given in vitro, as it is somewhat limited by poor bioavailability. This is not an issue when used to treat parasite infection, as the drug can be given orally and pass through the intestines without needing to be absorbed systemically. However, these insights into the mechanism behind to antiviral activity of the drug may allow future improvements that enable the drug or a similar molecule to be employed against COVID-19. *Important notice  bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.