The Impact of the S-Adenosylmethionine Analogue Sinefungin on Viral Life Cycles

Abstract

DNA and RNA methylation are essential epigenetic modifications that play a crucial role in regulating gene expression and cellular processes. Methylation is also significant in viral infections, influencing various stages of the viral life cycle and immune evasion. In this study, we investigated the antiviral potential of sinefungin, a potent methyltransferase inhibitor, against Herpes Simplex Virus 1 (HSV-1) and SARS-CoV-2. The cytotoxic effect of sinefungin was evaluated on VERO-76 cells by exposing them to concentrations ranging from 12.5 to 200 μg/mL for 24 h. The MTT assay results indicated that sinefungin reduced cell viability by approximately 21.7% at the highest concentration tested, with a CC₅₀ above 200 μg/mL. Our results demonstrated that sinefungin exhibited significant antiviral activity against both HSV-1 and SARS-CoV-2, with IC₅₀ values of 49.5 ± 0.31 μg/mL for HSV-1 and 100.1 ± 2.61 μg/mL for SARS-CoV-2. These results suggest that sinefungin may be a promising therapeutic candidate for treating viral infections, particularly those involving methylation-dependent processes.

1. Introduction

DNA and RNA methylation are fundamental epigenetic modifications that occur across all domains of life, playing pivotal roles in regulating a wide array of biological processes [1,2]. These modifications are catalyzed by methyltransferases, enzymes that transfer a methyl group from the cofactor S-adenosylmethionine (SAM) to specific nucleotide bases (adenine, cytosine, guanine, and uracil) [3]. In eukaryotes, DNA methylation regulates gene activity, chromatin structure, and genomic stability, guiding cellular differentiation and development by silencing repetitive elements. Meanwhile, RNA methylation stabilizes transcripts, modulates protein translation, and adapts gene expression to environmental and developmental stimuli [4]. DNA methylation is mainly part of restriction-modification systems in prokaryotes, where methyltransferases protect against foreign DNA. Orphan methyltransferases independently regulate gene expression, DNA replication, repair, and the cell cycle. On the other hand, RNA methylation in bacteria is essential for RNA stability, ribosome assembly, tRNA function, and mRNA regulation. It also helps bacteria adapt to stress and contributes to antibiotic resistance by modifying rRNA to block antibiotic binding [5]. Methylation is also a significant epigenetic modification in viruses, affecting various aspects of the viral life cycle [6]. In RNA and DNA viruses, methylation plays a crucial role in regulating the viral life cycle, enabling immune evasion and influencing interactions with the host [7]. In DNA viruses, DNA methylation regulates viral genome replication, gene expression, and the ability of the virus to evade the host’s immune system. This modification also plays a role in viral integration into the host genome. Additionally, methylation patterns within the viral genome help viruses persist in the host [8]. In this context, Zhang et al. performed bisulfite sequencing of the Epstein–Barr virus (EBV) genome, revealing that the viral genome undergoes extensive methylation during the latent phase. In contrast, infectious viral particles showed significant hypomethylation throughout the genome. In particular, their study highlighted that during the active phase of infection, the repression of EBNA latency genes was associated with hypermethylation in specific promoter regions [9]. Similarly, the E2 gene in the human papillomavirus (HPV) genome harbors CpG islands that serve as methylation loci. Methylation of these regions impairs the transcriptional regulatory function of the E2 protein, disrupting its ability to control the expression of the E6 and E7 oncogenes. This disruption results in the upregulation of E6 and E7, key drivers of HPV-associated oncogenesis [10,11]. In RNA viruses, methylation is essential to regulating the stability of the viral RNA, protecting it from degradation by host exonucleases. Furthermore, methylation plays a key role in modulating the host immune response by altering the recognition of the viral RNA and consequently evading detection by host immune receptors. This modification is also essential for efficient viral replication and translation. Furthermore, it influences the integration of the viral genome into the host DNA, a critical step in establishing infection and maintaining viral latency [12]. Courtney et al. demonstrated that IAV-PR8 mutants with reduced levels of m6A on HA segments showed significantly attenuated pathogenicity, as confirmed by in vivo tests in mice. Notably, only the wild-type virus caused lethality at the same doses tested (10 PFU) [13]. Similarly, Wang et al. and Liu et al. elucidated the impact of cytosine and adenine methylation on the SARS-CoV-2 genome, demonstrating their critical role in regulating viral replication, pathogenicity, and host antiviral responses [14,15]. Considering the significant impact of methylation modifications on the genome, directing drug development efforts toward targeting methylases holds promising potential. By modulating these enzymes, it may be possible to influence viral replication and pathogenicity and host immune responses, offering new therapeutic strategies to combat viral infections. Sinefungin, a potent inhibitor of SAM-dependent methyltransferases, has attracted considerable attention as a valuable tool in the study of methylation processes [16]. By interrupting the activity of methyltransferases, sinefungin offers a promising approach to modulating viral pathogenesis. Studies on the antiviral activity of sinefungin are limited. It has been shown to exhibit antiviral effects against Vaccinia virus and feline immunodeficiency virus-1 (FIV-1) [17,18]. Due to the distinct characteristics of these viruses, the observed antiviral activity suggests the potential for sinefungin to target a broader range of viral pathogens. In this context, we evaluated the antiviral activity of sinefungin against Herpes Simplex Virus 1 (HSV-1) and SARS-CoV-2, including its variants, due to the significant public health impact of these viruses. HSV-1, a DNA virus, is known for its ability to establish lifelong latency, while SARS-CoV-2 and its variants, rapidly mutating RNA viruses, have caused a global pandemic, with persistent therapeutic challenges [19,20]. The choice of these viruses underscores the urgent need for new therapeutic options, as both present unique challenges in terms of their viral persistence and mutation rates and the emergence of resistance. Studying the antiviral potential of sinefungin against these viruses could provide valuable insights into developing effective treatments for these evolving and widespread viral threats.

2. Materials and Methods

2.1. Cells, Viruses, and Compounds

The African green monkey kidney cell line (Cercopithecus aethiops, VERO-76) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Waltham, MA, USA), 100 U/mL penicillin, 100 μg/mL streptomycin, 4.5 g/L glucose, and 2 mM L-glutamine (Gibco; Thermo Fisher Scientific, Waltham, MA, USA) in a humidified atmosphere containing 5% CO₂ at 37 °C. The HSV-1 strain VR-1493 was purchased from the ATCC, while SARS-CoV-2 was kindly provided by the Lazzaro Spallanzani National Institute for Infectious Diseases [21]. Both viruses were propagated in VERO-76 monolayers in FBS-free DMEM at 37 °C in 5% CO₂ until a cytopathic effect was observed and then stored at −80 °C. Sinefungin was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in nuclease-free water to a final concentration of 1 mg/mL (

Table 1. Chemical structure and molecular properties of sinefungin.

	Sinefungin
Chemical structure
Molecular formula	C15H23N7O5
Molecular weight	381.39 g/mol
Appearance	Colorless
Density	1.2 g/cm3
Purity	95% (HPLC)
Solubility in water	20 mg/mL

).

2.2. The Cytotoxicity Assay

The cytotoxicity of sinefungin was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, St. Louis, MO, USA) assay. The VERO-76 cells were seeded at a density of 2 × 10⁴ cells/well into 96-well plates and incubated at 37 °C in 5% CO₂. After 24 h, the cells were treated with increasing concentrations (12.5–200 μg/mL) of sinefungin. Following 24 h of exposure, the cells were washed once with phosphate-buffered saline 1× (PBS 1×, Gibco; Thermo Fisher Scientific, Waltham, MA, USA), and 100 μL of fresh DMEM containing 0.3 mg/mL of MTT reagent was added to each well. The plates were incubated for 3 h at 37 °C in 5% CO₂; then, the MTT solution was removed, and formazan crystals were solubilized with 100 μL of 100% DMSO. The optical density was measured at 570 nm with a reference wavelength of 630 nm using a TECAN Infinite 200 PRO (TECAN, Männedorf, Switzerland). Cell viability was expressed as a percentage compared to the untreated controls (CTRL−), while the positive control consisted of cells treated with 100% DMSO (CTRL+).

2.3. The Plaque Reduction Assay in Infected Monolayers

To assess the antiviral activity of sinefungin, the VERO-76 cells were seeded in DMEM supplemented with 10% FBS at a density of 2.2 × 10⁵ cells/well into 12-well plates and incubated at 37 °C in 5% CO₂. After 24 h, a co-treatment plaque reduction assay was performed. The cells were simultaneously exposed to sinefungin (25–200 μg/mL) and the virus (HSV-1 or SARS-CoV-2) at a multiplicity of infection (MOI) of 0.01 for 1 h at 37 °C in 5% CO₂. The virus–compound mixture was removed, and the cells were washed twice with PBS 1× to eliminate the extracellular virus. The cells were overlaid with carboxymethylcellulose 3% (CMC, Sigma-Aldrich, St. Louis, MO, USA) diluted in DMEM containing sinefungin (25–200 μg/mL) and incubated for 24 h (SARS-CoV-2) and 48 h (HSV-1). Then, the cell monolayers were fixed with 4% formaldehyde and stained with 0.5% crystal violet (both from Sigma-Aldrich). Each plaque lysis was counted to define the plaque-forming units (PFU)/mL, and viral inhibition was calculated by comparing the treated wells to the untreated controls. In the co-treatment assay, the positive control was F. rubiginosa extract, previously reported to inhibit both viruses at 10 μg/mL completely. In the post-infection assays, remdesivir (5 μM) was used against SARS-CoV-2 and acyclovir (5 μM) against HSV-1 [21,22].

2.4. The Virus Yield Reduction Assay

The VERO-76 cells were seeded at a 2.2 × 10⁵ cells/well density into 12-well plates and cultured in DMEM with 10% FBS at 37 °C and 5% CO₂. The medium was then removed, and the cells were infected with HSV-1 or SARS-CoV-2 at an MOI of 0.01 in FBS-free DMEM containing sinefungin (25–200 μg/mL). After 1 h of incubation at 37 °C, the cells were washed twice with PBS 1× to remove unbound viral particles and overlaid with medium containing 10% FBS with or without sinefungin (25–200 μg/mL) and incubated at 37 °C and 5% CO₂. The incubation periods were 6, 12, and 24 h for SARS-CoV-2 and 10, 20, and 30 h for HSV-1. The supernatants were collected, and the viral titers were determined via a plaque assay for each virus strain at all tested concentrations and time points. The percentage of plaques was calculated relative to that in the untreated control.

2.5. Molecular Validation

The inhibition observed was confirmed through a molecular analysis (qPCR). The test was set up as the viral yield reduction assay (as described in Section 2.4), with an incubation time of 10 h for HSV-1 and 6 h for SARS-CoV-2. For HSV-1, the expression of the late gene UL27 (F: 5′-GCCTTCTTCGCCTTTCGC-3′; R: 3′-CGCTCGTGCCCTTCTTCTT-5′) was analyzed; for SARS-CoV-2, the expression of the S gene (F: 5′-AGGTTGATCACAGGCAGACT-3′; R: 3′-GCTGACTGAGGGAAGGAC-5′) encoding the spike protein was evaluated. Threshold cycle (Ct) values were normalized to the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH, F: 5′-CCTTTCATTGAGCTCCAT-3′, R: 3′-CGTACATGGGAGCGTC-5′) and calculated using the 2^−ΔΔCt method.

2.6. The Statistical Analysis

All experiments were performed in biological duplicates and technical triplicates. The results are expressed as the mean ± standard deviation (SD), calculated using GraphPad Prism software (Version 10.2.3). A one-way ANOVA followed by Dunnett’s multiple comparisons test was used for the statistical analysis, with a p-value ≤ 0.05 considered significant. Cytotoxic (CC₅₀) and inhibitory (IC₅₀, IC₉₀) concentrations were determined using a non-linear regression analysis with GraphPad Prism (Version 10.2.3).

3. Results

3.1. Cell Viability

To evaluate the cytotoxicity of sinefungin, VERO-76 cells were selected and also used for the antiviral tests (Figure 1). The cells were exposed to sinefungin at concentrations ranging from 12.5 to 200 μg/mL for 24 h. The cytotoxic effect was evaluated relative to CTRL−. No concentration tested induced complete cytotoxicity. Sinefungin reduced the cell viability by approximately 21.7% at 200 μg/mL, 16.6% at 100 μg/mL and 8.8% at 50 μg/mL. Lower concentrations (25 and 12.5 μg/mL) showed negligible effects, with reductions in viability below 5%. CC₅₀, determined using the non-linear regression analysis, was greater than the highest concentration tested (CC₅₀ > 200 μg/mL).

3.2. 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 μg/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 μg/mL and maintained >50% inhibition up to 50 μg/mL. For SARS-CoV-2, the inhibition reached 70% and 54% at 200 and 100 μg/mL, with residual activity (28% inhibition) at 50 μg/mL. The non-linear regression analysis yielded IC₅₀ and IC₉₀ values of 49.5 ± 0.31 and 229.03 ± 4.11 μg/mL for HSV-1 and 100.1 ± 2.61 and 522.7 ± 6.31 μg/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 μg/mL) was evaluated at 10, 20, and 30 h post-infection (Figure 3). After 10 h, the untreated control titers reached 1.25 × 10³ PFU/mL. The sinefungin treatment at 200 μg/mL reduced the titers to 73 PFU/mL (log₁₀ fold reduction ≈ 1.23); 100 μg/mL yielded 410 PFU/mL (log₁₀ fold reduction ≈ 0.48), while 50 and 25 μg/mL resulted in minor reductions to 830 and 1100 PFU/mL, respectively (log₁₀ fold reduction < 0.18). At 20 h, the control titers reached 4.9 × 10⁴ PFU/mL; sinefungin at 200 μg/mL reduced the titers to 750 PFU/mL (log₁₀ fold reduction = 1.82), while 100 μg/mL yielded 1030 PFU/mL (log₁₀ fold reduction = 1.77). At 50 and 25 μg/mL, the titers were 5.5 × 10⁴ and 4.8 × 10⁴ PFU/mL, respectively, with no significant reduction. At 30 h, the control titer was 6.0 × 10⁶ PFU/mL. Sinefungin at 200 μg/mL reduced the titers to 5.4 × 10³ PFU/mL (log₁₀ fold reduction = 3.05), while 100 μg/mL resulted in 7.4 × 10⁵ PFU/mL (log₁₀ fold reduction ≈ 0.91). Lower concentrations (50 and 25 μg/mL) showed minimal or no reduction (titers: 4.8 × 10⁶ and 5.5 × 10⁶ PFU/mL; log₁₀ 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 × 10⁵ PFU/mL. Treatment at 200 μg/mL resulted in 1.01 × 10³ PFU/mL (log₁₀ fold reduction = 2.17); 100 μg/mL yielded 2.0 × 10⁴ PFU/mL (log₁₀ fold reduction = 0.88). At 50 and 25 μg/mL, the titers were 7.2 × 10⁴ and 1.0 × 10⁵ PFU/mL, respectively (log₁₀ fold reduction < 0.35). At 12 h, the control titers increased to 1.53 × 10⁹ PFU/mL. Sinefungin at 200 and 100 μg/mL reduced the titers to 8.0 × 10⁴ and 7.2 × 10⁶ PFU/mL, respectively (log₁₀ fold reduction = 4.28 and 2.33). At 50 and 25 μg/mL, the titers were 9.04 × 10⁶ and 1.53 × 10⁹ PFU/mL, respectively (log₁₀ fold reduction = 2.23 and 0). At 24 h, the control titers further increased to 1.71 × 10¹² PFU/mL. Treatment with 200 and 100 μg/mL resulted in titers of 1.12 × 10⁶ and 6.51 × 10⁶ PFU/mL (log₁₀ fold reduction = 6.18 and 5.42). At 50 and 25 μg/mL, the viral titers were 5.9 × 10¹⁰ and 1.5 × 10¹² PFU/mL, with modest or no reduction (log₁₀ fold reduction = 1.46 and 0.06). Overall, the data demonstrate the potent antiviral activity of sinefungin against HSV-1, particularly at 200 μg/mL, with increasing effectiveness over time. Against SARS-CoV-2, sinefungin also exerted significant antiviral activity in a time- and dose-dependent manner, though this was slightly less pronounced than that for HSV-1. To confirm the results obtained from the plaque reduction assay, qPCR was performed (Figure 5). For HSV-1, the expression of the UL27 gene, a late gene encoding the structural glycoprotein B (gB), was evaluated. For SARS-CoV-2, the expression levels of the S gene, which encodes the spike protein involved in viral entry into host cells, were analyzed. A virus-sinefungin co-treatment assay was conducted for the molecular analysis. In the SARS-CoV-2-infected cells, treatment with sinefungin at 200 µM resulted in an approximately 50-fold reduction in the S gene expression compared to that in the virus control. At 100 µM and 50 µM, the reductions were 2.36-fold and 0.98-fold, respectively, indicating a loss of antiviral efficacy at lower concentrations. For HSV-1, the reduction in the UL27 gene expression was even more pronounced, further confirming the plaque reduction assay findings observed in the infected monolayers. Treatment with 200 µM of sinefungin led to a 96.1-fold decrease in gene expression, while reductions of 8.47-fold and 1.26-fold were observed at 100 µM and 50 µM, respectively.

4. Discussion

Epigenetic-mechanism-targeting drugs represent promising new therapeutic strategies for the treatment of a broad spectrum of diseases, including malignancies, developmental disorders, and both acute and chronic viral infections [23,24]. Recent evidence has highlighted the critical role of methylation as a key epigenetic modification in viral genomes, with significant implications for the regulation of viral pathogenesis [25,26]. In both RNA and DNA viruses, methylation events, whether they occur on viral genomes or are mediated by the host’s epigenetic machinery, modulate critical aspects of the viral life cycle. These include control of viral gene expression, latency, replication efficiency, and the ability to evade the host’s immune surveillance [27,28]. Furthermore, methylation can influence virus–host interactions by altering the host’s transcriptional landscape or mimicking host epigenetic marks, thereby facilitating persistent or chronic infections. The important role of viral genome methylation in regulating the replication cycle and contributing to pathogenic mechanisms highlights the need to direct the development of new therapeutic agents towards the enzymes and proteins that drive these epigenetic processes. Targeting these components could provide an innovative strategy for altering viral replication and modulating virus–host interactions, thus opening new avenues for antiviral therapy [29,30]. Sinefungin has emerged as a potential antiviral agent. This adenosine derivative, particularly the delta-(5′-adenosyl) derivative of ornithine, exhibits potent inhibition of SAM-dependent methyltransferases. By interfering with the activity of these enzymes, sinefungin offers a promising approach to modulating viral pathogenesis [31]. Although the antiviral effects of sinefungin are still limited, our study aimed to evaluate its activity against HSV-1 and SARS-CoV-2, representing enveloped and non-enveloped viral models with DNA and RNA genomes, respectively. Initially, the cytotoxic impact of sinefungin was evaluated on VERO-76 cells by exposing them to concentrations ranging from 12.5 to 200 μg/mL for 24 h. The MTT results indicated that sinefungin reduced the cell viability by approximately 21.7% at the highest concentration tested, with CC₅₀ greater than 200 μg/mL. In support of our findings, Kuroda et al. reported that sinefungin did not significantly affect the cell viability of CRFK cells at a concentration of 100 μg/mL [17]. In contrast, higher cytotoxicity was observed in the 3T3, HepG2, and A549 cell lines. Bobrovs et al. reported CC₅₀ values of 38.7 μg/mL, higher than 38 μg/mL and 28 μg/mL, respectively [32]. After evaluating the cytotoxicity used to establish a non-toxic concentration range, the antiviral activity of sinefungin was subsequently evaluated against HSV-1 and SARS-CoV-2. Our results demonstrated that sinefungin showed strong antiviral activity against HSV-1, reaching 81% inhibition at 200 μg/mL and maintaining >50% inhibition up to 50 μg/mL. In the case of SARS-CoV-2, sinefungin induced 70% and 54% inhibition at 200 and 100 μg/mL, respectively, with a residual antiviral activity of 28% at 50 μg/mL. The IC₅₀ values were 49.5 ± 0.31 and 100.1 ± 2.61 μg/mL for HSV-1 and SARS-CoV-2, respectively. Considering the known inhibitory activity of sinefungin against SAM-dependent methyltransferases and the critical role of these enzymes in viral replication, we evaluated the effect of this compound on the viral yield in HSV-1- and SARS-CoV-2-infected VERO-76 cells at different times (6–30 h) and doses (25–200 μg/mL). Sinefungin treatment at 10, 20, and 30 h post-infection resulted in dose- and time-dependent reductions in the viral yield. At 10 hpi, treatment with 200 μg/mL induced a reduction in the viral titers of approximately 1.23 log₁₀, while 100 μg/mL reduced the titers by ~0.48 log₁₀. Lower concentrations (50 and 25 μg/mL) resulted in minimal inhibition (reduction < 0.18 log₁₀). At 20 hpi, the inhibition increased, with 200 and 100 μg/mL producing reductions of 1.82 and 1.77 log₁₀, respectively, while no significant reduction was observed at 50 or 25 μg/mL. At 30 hpi, the maximal antiviral activity was observed, with 200 μg/mL reducing the viral titers by 3.05 log₁₀ and 100 μg/mL by ~0.91 log₁₀. Again, lower concentrations showed minimal effects (<0.1 log₁₀). For SARS-CoV-2, sinefungin treatment at 6, 12, and 24 hpi also showed a time- and dose-dependent antiviral effect. At 6 hpi, 200 μg/mL reduced the viral titers by 2.17 log₁₀, while 100 μg/mL achieved a reduction of 0.88 log₁₀. Concentrations of 50 and 25 μg/mL induced reductions of less than 0.35 log₁₀. At 12 hpi, the antiviral efficacy significantly increased, with 200 and 100 μg/mL producing reductions of 4.28 and 2.33 log₁₀, respectively. A comparable reduction of 2.23 log₁₀ was also observed at 50 μg/mL, while 25 μg/mL was ineffective. At 24 h post-infection, sinefungin showed its strongest antiviral effect: 200 μg/mL reduced the viral yield by 6.18 log₁₀ and 100 μg/mL reduced it by approximately 5.42 log₁₀. A reduction of 1.46 log₁₀ was observed at 50 μg/mL, while 25 μg/mL produced only minimal inhibition (0.06 log₁₀). There is limited evidence regarding the antiviral activity of sinefungin. Kuroda et al. demonstrated an effect of sinefungin on the viral yield following the infection of CRFK cells with Feline Calicivirus (FCV), Feline infectious peritonitis virus (FIPV), FHV-1, Pseudorabies virus (PRV) and Equine herpes virus type 1 (EHV-1). In response to treatment with 100 μg/mL of sinefungin, the plaque counts for FCV and FIPV were reduced by 67.7% and 7.8%, respectively, compared to those in control infection. In contrast, sinefungin showed greater efficacy against FHV-1, with a plaque reduction of 0.04%. In contrast, PRV and EHV-1, both belonging to the Varicellovirus genus, demonstrated reduced susceptibility to sinefungin, with plaque yields of 52.4% and 21.9%, respectively. The effect of sinefungin on FHV-1 has been extensively studied. The results have shown that sinefungin did not impact the adhesion of FHV-1 to the host cells but could interfere with post-adsorption biochemical processes, with an IC₅₀ value of 9.5 μg/mL [17]. Pugh et al. demonstrated antiviral activity of sinefungin against the smallpox virus, showing significant inhibition of the intracellular phase of infection. At a 200 μg/mL concentration, sinefungin induced a 77% reduction in viral replication. This effect was attributed to the compound’s inhibition of guanine-7-methyltransferase, an enzyme crucial for viral RNA methylation, thereby disrupting key processes in the viral life cycle [18]. Moreover, Li et al. demonstrated that sinefungin acted as an inhibitor of VSV mRNA cap methylation in vitro. Based on this observation, the researchers evaluated its ability to inhibit viral replication. The results revealed a dose-dependent reduction in the viral yield, with an approximate IC₅₀ of 220 μM [33]. Due to the broad-spectrum antiviral activity of sinefungin and its effect on a critical target involved in different stages of the viral life cycle, this compound has significant potential as a therapeutic agent. Sinefungin inhibits key enzymatic processes, particularly those related to DNA and RNA methylation, a modification essential for genomic stability, protein translation, and the regulation of viral RNA. By targeting these crucial processes, sinefungin impacts a broad spectrum of RNA and DNA viruses, positioning it as a promising candidate for the treatment of various viral infections [34]. Furthermore, its action on the viral methylation mechanism positions sinefungin as a novel therapeutic approach that may overcome the current limitations associated with virus-specific treatments.