ABSTRACT
Recently, we have shown that C6-ceramides efficiently suppress viral replication by trapping the virus in lysosomes. Here, we use antiviral assays to evaluate a synthetic ceramide derivative α-NH2-ω-N3-C6-ceramide (AKS461) and to confirm the biological activity of C6-ceramides inhibiting SARS-CoV-2. Click-labeling with a fluorophore demonstrated that AKS461 accumulates in lysosomes. Previously, it has been shown that suppression of SARS-CoV-2 replication can be cell-type specific. Thus, AKS461 inhibited SARS-CoV-2 replication in Huh-7, Vero, and Calu-3 cells up to 2.5 orders of magnitude. The results were confirmed by CoronaFISH, indicating that AKS461 acts comparable to the unmodified C6-ceramide. Thus, AKS461 serves as a tool to study ceramide-associated cellular and viral pathways, such as SARS-CoV-2 infections, and it helped to identify lysosomes as the central organelle of C6-ceramides to inhibit viral replication.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/metabolism , Ceramides/pharmacology , Ceramides/metabolism , Virus Replication , Antiviral Agents/pharmacologyABSTRACT
Recently, we have described novel pyridyl indole esters and peptidomimetics as potent inhibitors of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) main protease. Here, we analysed the impact of these compounds on viral replication. It has been shown that some antivirals against SARS-CoV-2 act in a cell line-specific way. Thus, the compounds were tested in Vero, Huh-7, and Calu-3 cells. We showed that the protease inhibitors at 30 µM suppress viral replication by up to 5 orders of magnitude in Huh-7 cells, while in Calu-3 cells, suppression by 2 orders of magnitude was achieved. Three pyridin-3-yl indole-carboxylates inhibited viral replication in all cell lines, indicating that they might repress viral replication in human tissue as well. Thus, we investigated three compounds in human precision-cut lung slices and observed donor-dependent antiviral activity in this patient-near system. Our results provide evidence that even direct-acting antivirals may act in a cell line-specific manner.
Subject(s)
COVID-19 , Hepatitis C, Chronic , Humans , Antiviral Agents/pharmacology , SARS-CoV-2 , Protease Inhibitors/pharmacology , Indoles/pharmacologyABSTRACT
BACKGROUND: Herpesviruses are common animal and human pathogens that cause severe health problems in children, immunocompromised patients, and infected animals with a host range from fish to mammals. Anthocyanin-containing plant extracts have been described as potent antivirals, which might cause fewer harmful side effects than direct-acting antivirals. Here, we report that an extract of Aristotelia chilensis (Molina) Stuntz (Elaeocarpaceae) (MBE) with a high content of the anthocyanin delphinidin suppresses lytic replication of equine, murine and human herpesviruses of replication in vitro. METHODS: We treated cultured cells with MBE and purified individual anthocyanins present in the extract to determine the most active compound at different concentrations. We subsequently infected the cultures with human herpesviruses 1 (HSV-1) or 8 (HHV-8), murine cytomegalovirus (CMV), or equine herpesviruses 1 (EHV-1) and determined the number of infected cells and viral infectivity. RESULTS: MBE inhibited the HSV-1, murine CMV, and EHV-1 by up to 2 orders of magnitude. In the presence of the stabilizing randomly methylated-beta-cyclodextrin, the inhibitory concentration could be lowered significantly. We identified delphinidin as an active antiviral compound and showed that the non-glycosylated delphinidin solved and stabilized with sulfobutylether-beta-cyclodextrin allowed usage of approximately 50 times lower concentrations. CONCLUSION: Glycosylated delphinidin derivatives were identified as active antiviral compounds of MBE. This suggests that plant extracts rich in delphinidin-anthocyanins have potent antiviral properties that could be used in treatment and prevention.
Subject(s)
Cytomegalovirus Infections , Elaeocarpaceae , Hepatitis C, Chronic , Herpesvirus 1, Human , Child , Humans , Animals , Horses , Mice , Anthocyanins/pharmacology , Anthocyanins/analysis , Antiviral Agents/pharmacology , Plant Extracts/pharmacology , MammalsABSTRACT
Recently, Tummino et al. reported that 34 compounds, including Chloroquine and Fluoxetine, inhibit SARS-CoV-2 replication by inducing phospholipidosis, although Chloroquine failed to suppress viral replication in Calu-3 cells and patients. In contrast, Fluoxetine represses viral replication in human precision-cut lung slices (PCLS) and Calu-3 cells. Thus, it is unlikely that these compounds have similar mechanisms of action. Here, we analysed a subset of these compounds in the viral replication and phospholipidosis assays using the Calu-3 cells and PCLS as the patient-near system. Trimipramine and Chloroquine induced phospholipidosis but failed to inhibit SARS-CoV-2 replication in Calu-3 cells, which contradicts the reported findings and the proposed mechanism. Fluoxetine, only slightly induced phospholipidosis in Calu-3 cells but reduced viral replication by 2.7 orders of magnitude. Tilorone suppressed viral replication by 1.9 orders of magnitude in Calu-3 cells without causing phospholipidosis. Thus, induction of phospholipidosis is not correlated with the inhibition of SARS-CoV-2, and the compounds act via other mechanisms. However, we show that compounds, such as Amiodarone, Tamoxifen and Tilorone, with antiviral activity on Calu-3 cells, also inhibited viral replication in human PCLS. Our results indicate that antiviral assays against SARS-CoV-2 are cell-line specific. Data from Vero E6 can lead to non-transferable results, underlining the importance of an appropriate cell system for analysing antiviral compounds against SARS-CoV-2. We observed a correlation between the active compounds in Calu-3 cells and PCLS.
Subject(s)
COVID-19 , Tilorone , Humans , Fluoxetine , SARS-CoV-2 , Antiviral Agents/pharmacology , Cell Line , ChloroquineABSTRACT
Aspirin, with its active compound acetylsalicylic acid (ASA), shows antiviral activity against rhino- and influenza viruses at high concentrations. We sought to investigate whether ASA and its metabolite salicylic acid (SA) inhibit SARS-CoV-2 since it might use similar pathways to influenza viruses. The compound-treated cells were infected with SARS-CoV-2. Viral replication was analysed by RTqPCR. The compounds suppressed SARS-CoV-2 replication in cell culture cells and a patient-near replication system using human precision-cut lung slices by two orders of magnitude. While the compounds did not interfere with viral entry, it led to lower viral RNA expression after 24 h, indicating that post-entry pathways were inhibited by the compounds.
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.