# The Impact of the S-Adenosylmethionine Analogue Sinefungin on Viral Life Cycles
## 3 Results
### Antiviral Activity  
Sinefungin's antiviral efficacy was assessed in co-treatment assays against HSV-1 (enveloped DNA virus) and SARS-CoV-2 (enveloped RNA virus). Viral particles were co-incubated with sinefungin (25-200 \(\upmu\)g/mL) directly on the cell monolayer for 1 h. Subsequently, the mixture was removed, and the compound was added to the CMC 3% supplemented with sinefungin for 24 (SARS-CoV-2) and 48 (HSV-1) hours to evaluate the plaque formation (Figure 2). A significant reduction in the cytopathic effect was observed for both viruses. Against HSV-1, sinefungin achieved 81% viral inhibition at 200 \(\upmu\)g/mL and maintained >50% inhibition up to 50 \(\upmu\)g/mL. For SARS-CoV-2, the inhibition reached 70% and 54% at 200 and 100 \(\upmu\)g/mL, with residual activity (28% inhibition) at 50 \(\upmu\)g/mL. The non-linear regression analysis yielded IC\({}_{50}\) and IC\({}_{90}\) values of 49.5 \(\pm\) 0.31 and 229.03 \(\pm\) 4.11 \(\upmu\)g/mL for HSV-1 and 100.1 \(\pm\) 2.61 and 522.7 \(\pm\) 6.31 \(\upmu\)g/mL for SARS-CoV-2, indicating dose-dependent antiviral activity that was more pronounced against HSV-1. To clarify the mode of action, the virus yield reduction was assessed in the infected VERO-76 cells. For HSV-1, the sinefungin treatment (25-200 \(\upmu\)g/mL) was evaluated at 10,  
Figure 1: Sinefungin’s cytotoxicity to VERO-76 cells. Following 24 h of exposure to increasing concentrations of sinefungin (12.5–200 \(\upmu\)g/mL), a dose-dependent reduction in cell viability was observed, with a maximum effect of 21.7% at 200 \(\upmu\)g/mL. No concentration tested induced complete cytotoxicity (CC\({}_{50}\)\(>\) 200 \(\upmu\)g/mL). The data represent the mean \(\pm\) standard deviation (SD) of three independent experiments: \(\mathbf{\mathrm{\text{\text{\text{\text{\text{\text{\text{\text{\text{\text{ \text{\text{\texttext{\text  
20, and 30 h post-infection (Figure 3). After 10 h, the untreated control titers reached 1.25 \(\times\) 10\({}^{3}\) PFU/mL. The sinefungin treatment at 200 \(\mathrm{\SIUnitSymbolMicro g}\)/mL reduced the titers to 73 \(\mathrm{PFU/mL}\) (\(\mathrm{log_{10}}\) fold reduction \(\approx\) 1.23); 100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL yielded 410 \(\mathrm{PFU/mL}\) (\(\mathrm{log_{10}}\) fold reduction \(\approx\) 0.48), while 50 and 25 \(\mathrm{\SIUnitSymbolMicro g}\)/mL resulted in minor reductions to 830 and 1100 \(\mathrm{PFU/mL}\), respectively (\(\mathrm{log_{10}}\) fold reduction \(<\) 0.18). At 20 h, the control titers reached 4.9 \(\times\) 10\({}^{4}\) PFU/mL; sinefungin at 200 \(\mathrm{\SIUnitSymbolMicro g}\)/mL reduced the titers to 750 \(\mathrm{PFU/mL}\) (\(\mathrm{log_{10}}\) fold reduction = 1.82), while 100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL yielded 1030 \(\mathrm{PFU/mL}\) (\(\mathrm{log_{10}}\) fold reduction = 1.77). At 50 and 25 \(\mathrm{\SIUnitSymbolMicro g}\)/mL, the titers were 5.5 \(\times\) 10\({}^{4}\) and 4.8 \(\times\) 10\({}^{4}\) PFU/mL, respectively, with no significant reduction. At 30 h, the control titer was 6.0 \(\times\) 10\({}^{6}\) PFU/mL. Sinefungin at 200 \(\mathrm{\SIUnitSymbolMicro g}\)/mL reduced the titers to 5.4 \(\times\) 10\({}^{3}\) PFU/mL (\(\mathrm{log_{10}}\) fold reduction = 3.05), while 100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL resulted in 7.4 \(\times\) 10\({}^{5}\) PFU/mL (\(\mathrm{log_{10}}\) fold reduction \(\approx\) 0.91). Lower concentrations (50 and 25 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) showed minimal or no reduction (titers: 4.8 \(\times\) 10\({}^{6}\) and 5.5 \(\times\) 10\({}^{6}\) PFU/mL; \(\mathrm{log_{10}}\) fold reduction \(<\) 0.1). For SARS-CoV-2, the viral titers were assessed at 6, 12, and 24 h post-infection (Figure 4). At 6 h, the untreated controls reached 1.5 \(\times\) 10\({}^{5}\) PFU/mL. Treatment at 200 \(\mathrm{\SIUnitSymbolMicro g}\)/mL resulted in 1.01 \(\times\) 10\({}^{3}\) PFU/mL (\(\mathrm{log_{10}}\) fold reduction = 2.17); 100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL yielded 2.0 \(\times\) 10\({}^{4}\) PFU/mL (\(\mathrm{log_{10}}\) fold reduction = 0.88).