Niclosamide as a chemical probe for analyzing SARS-CoV-2 modulation of host cell lipid metabolism

Garret TJ, Coatsworth H, Mahmud I, Hamerly T, Stephenson CJ, Ayers JB, Yazd HS, Miller MR, Lednicky JA, and Dinglasan RR. (2023). Niclosamide as a chemical probe for analyzing SARS-CoV-2 modulation of host cell lipid metabolism. Frontiers in Microbiology

Autophagy is a cellular process that includes the lysosomal degradation pathway and is used by the body to eliminate damaged or dysfunctional organelles and cells. This is an essential processes during viral infection, therefore autophagy has been a target when attempting to identify drugs that can be repurposed to be used as an antiviral medication. A timely example of this is the response to pandemic viruses such as SARS-CoV-2. Repurposing an already available drug requires less time than developing a brand new compound, making it an obvious option to try when a quick response to a new virus is needed. Garret et al were interested in lipophagy, a selective form of autophagy that targets lipid droplets, during SARS-CoV-2 infection since there are known changes to the lipidome caused by this virus. Because of this, they wanted to use niclosamide, an anti-parasitic drug that is known to suppress MERS-CoV propagation through inhibition of autophagosome-lysosome fusion, as a chemical probe and determine its effect on the virus-induced lipidome changes.

For this in vitro study, Vero E6 cells were used since, unlike normal mammalian cells, they are interferon deficient. Interferons are a group of signaling proteins released in response to the presence of many viruses causing neighbouring cells to heighten their viral defense. In addition, these cells have some contact inhibition after forming a monolayer, which makes this strain ideal for propagating viruses that replicate slowly. First, the authors looked at the lipidomic and transcriptomic profile of Vero E6 cells in the absence of viral infection and with and without niclosamide to develop a baseline response at 16 hours and 48 hours, two timepoints chosen within the virus intracellular life cycle. A decrease in plasmalogens and triglyceride expression was seen at 48 hours in the cells treated with niclosamide compared to those without the treatment. As well, no difference in gene expression was identified across the 48 hours, suggesting that this change to the lipidome is not due to age itself.

When specifically looking at the effect of niclosamide on lipidome, 520 differently expressed lipids were identified with distinct profiles seen between the control-treated and niclosamide-treated cells. A large decrease was seen in plasmalogens and triglycerides at 16 hours and 48 hours, with the latter being the most evident, when compared to the cells in the control group. Some difference was seen in the lipidome in the control-treated group between 16 hours and 48 hours, but the authors state that this was expected since some cell growth is anticipated during this time period. They suggest that the differences seen between the control and niclosamide-treated groups could indicate that niclosamide disrupts the lipid pathway, especially for plasmalogens and triglycerides.

The role of lipids in the infection cycle of SARS-CoV-2 in Vero E6 cells was also studied. A dose that mimics natural exposure that would be experienced by aerosols in an enclosed area was chosen to deliver an MOI of 0.001 infectious virus particles/Vero E6 cell based on previous experiments they performed in this cell line. A correlation matrix showed clustering of cells infected with SARS-CoV-2 and those infected with SARS-CoV-2 and received the niclosamide treatment. As well, different lipid profiles were detected between 16 hours and 48 hours in the infected cells and between the infected cells also treated with niclosamide at the two timepoints. Specific classes of lipids were also looked at and SARS-CoV-2 altered 720 lipids form 34 classes of lipids and the four that were most abundant were phosphatidylcholine, phosphatidylethanolamine, plasmalogens, and triglycerides. Interestingly, alterations to different lipid classes were associated with being earlier or later in the infection life cycle. Triglycerides and cholesterol esters were elevated in early stages of infection while longer chain fatty acids, saturated fatty acids, and monounsaturated fatty acids were significantly downregulated at 16 hours of infection. Plasmalogens and diacylglycerides were found to be elevated at 48 hours of infection, however a similar increase in plasmalogen levels was also seen in uninfected cells at 48 hours, so this increase may not be related to infection and simply from cell growth. It was hypothesized that niclosamide might reverse some of the lipid metabolism dysregulation and it was tested by treating at the same time as SARS-CoV-2 infection or at 24 hours post-infection for 24 hours. The authors demonstrated that when treating at 24 hours post-infection, they significantly impacted the lipidome compared to when treated at the same time of infection which suggests that the antiviral effect of niclosamide depends on the time of treatment. When the cells were infected and treated with niclosamide there was a reduction in plasmalogens and diacylglycerides, which normally increased with SARS-CoV-2 infection, at the 48 hour timepoint.

Garret et al were interested in how SARS-CoV-2 infection affects the lipidome and whether the anti-parasitic drug, niclosamide, could be repurposed and reverse any of the effects of viral infection. They first established a baseline to determine just the effects of niclosamide on Vero E6 cells alone and found that there was a decrease in plasmalogens and triglycerides after 48 hours of treatment and determined this was because of the treatment and not simply a cause of age since there were no transcriptional changes. The authors also determined the optimal time for niclosamide treatment was 24 hours post-treatment as opposed to at the same time of viral infection. In addition, it was demonstrated that SARS-CoV-2 infection causes an increase in plasmalogen and diacylglyceride levels, but this is reversed when niclosamide is added to the cell culture 24 hours post-infection. Limitations of this work are that it was all performed in transformed cells that are interferon deficient which does not exactly model to how typical cells in the body would act, although did allow for the virus to propagate and enable these studies. As well, it was performed in cells and further work in animal models would be required to confirm these affects in vivo. Further research into repurposing drugs that can have antiviral roles could reduce the time required to find treatments for novel viruses.