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The Acid Ceramidase Is a SARS-CoV-2 Host Factor
Nina Geiger1, Louise Kersting2, Jan Schlegel3
1Institute of Virology und Immunobiology, Julius-Maximilians-Universität Würzburg, Versbacher Str. 7, 97078 Würzburg, Germany.
Abstract:
SARS-CoV-2 variants such as the delta or omicron variants, with higher transmission rates, accelerated the global COVID-19 pandemic. Thus, novel therapeutic strategies need to be deployed. The inhibition of acid sphingomyelinase (ASM), interfering with viral entry by fluoxetine was reported. Here, we described the acid ceramidase as an additional target of fluoxetine. To discover these effects, we synthesized an ASM-independent fluoxetine derivative, AKS466. High-resolution SARS-CoV-2-RNA FISH and RTqPCR analyses demonstrate that AKS466 down-regulates viral gene expression. It is shown that SARS-CoV-2 deacidifies the lysosomal pH using the ORF3 protein. However, treatment with AKS488 or fluoxetine lowers the lysosomal pH. Our biochemical results show that AKS466 localizes to the endo-lysosomal replication compartments of infected cells, and demonstrate the enrichment of the viral genomic, minus-stranded RNA and mRNAs there. Both fluoxetine and AKS466 inhibit the acid ceramidase activity, cause endo-lysosomal ceramide elevation, and interfere with viral replication. Furthermore, Ceranib-2, a specific acid ceramidase inhibitor, reduces SARS-CoV-2 replication and, most importantly, the exogenous supplementation of C6-ceramide interferes with viral replication. These results support the hypotheses that the acid ceramidase is a SARS-CoV-2 host factor.
Insights
Fluoxetine and its derivative AKS466 target acid ceramidase, inhibiting SARS-CoV-2 replication by altering lysosomal pH and ceramide levels. This reveals acid ceramidase as a crucial host factor for viral proliferation.
Area of Science:
- Virology
- Biochemistry
- Drug Discovery
Background:
- SARS-CoV-2 variants necessitate new therapeutic strategies.
- Fluoxetine was previously shown to inhibit viral entry by targeting acid sphingomyelinase (ASM).
- The role of acid ceramidase in SARS-CoV-2 infection was unexplored.
Purpose of the Study:
- To identify acid ceramidase as a novel target of fluoxetine.
- To investigate the mechanism by which fluoxetine and its derivative AKS466 inhibit SARS-CoV-2 replication.
- To explore the potential of targeting acid ceramidase for COVID-19 treatment.
Main Methods:
- Synthesis of an ASM-independent fluoxetine derivative (AKS466).
- High-resolution SARS-CoV-2-RNA Fluorescence In Situ Hybridization (FISH) and RTqPCR.
- Biochemical assays to assess enzyme activity, lysosomal pH, and cellular localization.
- Treatment with specific inhibitors and exogenous supplementation.
Main Results:
- AKS466 down-regulates SARS-CoV-2 gene expression.
- SARS-CoV-2 infection deacidifies lysosomal pH via ORF3 protein; AKS466 and fluoxetine reverse this.
- AKS466 and fluoxetine inhibit acid ceramidase, elevate endo-lysosomal ceramide, and impede viral replication.
- Specific acid ceramidase inhibitor Ceranib-2 and C6-ceramide supplementation reduce viral replication.
Conclusions:
- Acid ceramidase is a SARS-CoV-2 host factor essential for viral replication.
- Fluoxetine and AKS466 exert antiviral effects by inhibiting acid ceramidase and modulating lysosomal function.
- Targeting acid ceramidase presents a promising therapeutic avenue against SARS-CoV-2.
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