Targeting SARS-CoV-2 replication with inhibitors of lipid metabolism

As the COVID-19 pandemic continues to inflict devastating effects worldwide, it is critically important to understand the biology of SARS-CoV-2 to identify new ways to target the virus. One approach to develop efficient clinical interventions is to repurpose medicines developed for other diseases to treat COVID-19. Such an approach could accelerate the timelines for COVID-19 treatment availability for patients across the globe.

Recently, my team at the Institute for Biomedical Sciences at Georgia State University, in collaboration with Synthego and Axion Biosystems Inc., investigated such an approach. We built on the current understanding of SARS-CoV-2 viral replication to test whether small molecules that block fatty acid metabolism could inhibit viral replication. Our findings were recently published in Cell Reports.¹

Coronavirus replication and lipid metabolism

Coronaviruses rely on host cell membranes to enter cells. Once inside the cell, the virus establishes specialised structures called replication organelles, which are then used as the location for assembling new virus particles. Viral entrance into the cell, formation of replication organelles and the eventual release of new viruses require hijacking of the host cell membranes to complete the viral replication cycle.

Some proteins involved in fatty acid metabolism, which is crucial for the formation and modification of cell membranes, have previously been shown to inhibit the replication of numerous viruses.^2-6 This study aimed to identify proteins in the fatty acid metabolism pathway that are critical for SARS‑CoV-2 replication.

One such protein that plays a role in several steps of membrane biology is VPS34, a class III phosphoinositol-3 kinase (PI3K) that has also been a target to inhibit viral replication.^7,8 We used small molecule inhibitors of VPS34 to evaluate the ability of SARS-CoV-2 to replicate in Vero E6 cells, a cell line often used for viral replication studies. When these cells were infected with SARS‑CoV-2 and treated with VPS34-IN1 and PIK-III, well‑characterised VPS34 inhibitors, the virus was unable to efficiently replicate. We further conducted additional assays to validate that VPS34 inhibitors halt replication at the early stages of the viral replication cycle.

We also investigated the roles of fatty acid synthase (FASN) and long chain Acyl-CoA synthetase, two other proteins involved in fatty acid metabolism, in viral replication. SARS-CoV-2-infected Vero E6 cells were treated with two inhibitors shown to have antiviral activity. Treatment of cells with Orlistat, a US Food and Drug Administration (FDA)-approved drug that inhibits gastric lipases and FASN and Triacsin C, an inhibitor of long chain Acyl-CoA synthetase, significantly impaired SARS-CoV-2 replication in Vero E6 cells.^3,5,9,10 Assays performed to understand when inhibition occurs indicated that treatment with these compounds hinders replication after the virus enters the cell.

These findings were confirmed in a human airway cell line, Calu-3, which shows increased susceptibility to infection and has previously been established as a standard for infection studies with SARS-CoV-1, Middle East respiratory syndrome coronavirus (MERSCoV) and SARS-CoV-2.^11-13 Treatment of infected Calu-3 cells with both VPS34 and fatty acid metabolism inhibitors provided further evidence that specific proteins involved in fatty acid metabolism are important for efficient SARS‑CoV-2 replication and may be potential therapeutic targets.

Validating the role of FASN in SARS-CoV-2 replication

To validate the role of these targets, we chose to test the effect of FASN gene function on SARS‑CoV-2 replication. Firstly, CRISPR-mediated FASN knockout cells were rapidly developed. Gene knockouts were then generated using multi‑guide technology, wherein up to three spatially designed guides are used to generate a large fragment deletion, which is known to increase likelihood of functional knockouts. The knockout cell pools showed high editing efficiencies and were amenable to directly assay without cloning.

Consistent with the previous results in the Vero E6 and Calu-3 cell lines, SARS-CoV-2 replication was substantially lower in the FASN knockout cells compared with wild type cells. Viral protein production also diminished in the FASN knockout cell line. Furthermore, supplementing cells with fatty acids rescued the impaired viral replication effect. These data suggest that fatty acid metabolism is necessary for efficient, sustained replication during SARS-CoV-2 infection.

Potential therapeutic targets

Our data show proteins involved in lipid metabolism could serve as therapeutic targets for SARS-CoV-2 infection. In addition, these data provide key insights into SARS-CoV-2-host interaction. Although this study provided a greater understanding of how SARS-CoV-2 uses the host’s cell membranes to propagate, there is much more to learn about how this virus interacts with our cells and spreads throughout the body. Further studies could help uncover how uninhibited VPS34 promotes SARS‑CoV-2 infection and determine the mechanism of action for inhibitors of VPS34 and fatty acid metabolism in impairing viral replication.

References

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13. Tseng, C.T., Tseng, J., Perrone, L., et al. (2005). Apical entry and release of severe acute respiratory syndrome-associated coronavirus in polarized Calu-3 lung epithelial cells. Virol. 79, 9470–9479.