Antiviral Effect of Piperine on Chikungunya Virus: In Vitro Evidence and In Silico Analysis of E1-E2 Binding

Chikungunya virus (CHIKV) is an emerging arbovirus that causes acute and chronic disease with significant public health concerns. Although recent vaccines have been licensed, no specific antiviral treatments are currently available. Targeting viral entry remains a promising strategy, particularly by inhibiting the E1-E2 glycoprotein complex, which mediates host cell attachment and membrane fusion. Natural products such as piperine have demonstrated broad-spectrum bioactivity, including antiviral properties, making them attractive candidates for drug discovery. This study employed a multidisciplinary approach, combining in vitro assays and computational modeling to evaluate the antiviral potential of piperine against CHIKV. Cytotoxicity assays were conducted in VERO cells, followed by plaque reduction assays to assess piperine's effects at different stages of CHIKV infection. In parallel, molecular docking, MD simulations, and MM/GBSA calculations revealed a stable and energetically favorable binding of piperine to the E1-E2 fusion pocket. The results demonstrated that piperine exerts a time-dependent antiviral effect, with the most pronounced inhibition observed when administered during or after infection, suggesting activity beyond the initial entry step. Docking studies revealed that piperine binds within the E1-E2 fusion pocket, forming stable interactions with key residues such as MET88, LEU16, and TYR15, which are functionally important for CHIKV fusion. MD simulations confirmed that piperine maintains stable interactions at this interface and subtly alters the conformational dynamics of the glycoprotein complex. Cytotoxicity analysis showed that piperine is safe at low concentrations (0.001-10 μg/mL), while higher doses (≥100 μg/mL) induced oxidative stress and DNA damage in a dose-dependent manner. Collectively, these findings highlight piperine as a promising antiviral candidate targeting the CHIKV E1-E2 glycoprotein complex. Future studies should focus on structure-based optimization, pharmacokinetics, and in vivo efficacy to support the development of piperine-based antiviral therapies.