Tomatidine derivatives suppress Dengue virus infection through entry and post-entry inhibition

ABSTRACT Dengue is a rapidly spreading disease transmitted by mosquitoes and is caused by the Dengue virus (DENV), affecting approximately 390 million individuals globally. Approved vaccines for use have limited efficacy, and there are no approved antivirals. Tomatidine, a steroidal alkaloid obtained from Solanum species, has demonstrated broad-spectrum antiviral properties, leading to the investigation of its derivatives for their anti-DENV activity. In the current study, the anti-DENV potential of tomatidine and 11 such derivatives was assessed using a combination of in vitro and in silico approaches. Vero cells were used to evaluate the cytotoxicity and antiviral effects of the derivatives in pre-, co-, and post-treatment settings. The results indicated that the derivatives 5d, 5f, and 5c possessed the most potent antiviral effects; 5d achieved a 2.81 log decrease in viral RNA, while both 5f and 5c suppressed viral activity under both pre- and post-treatment conditions (5f has a selectivity index of 43.36), including the entry phase, as indicated by the entry assay. Furthermore, effective inhibition was noted between 0 and 9 h post-infection (hpi) and 15–21 hpi via a time-of-addition assay, indicating dual-phase activity. Additionally, docking and molecular dynamics studies revealed stable interactions of compounds 5c and 5d with the non-structural proteins NS1 and NS5–RdRp of DENV, reinforcing the experimental observations. In conclusion, tomatidine derivatives, especially 5d, 5f, and 5c, showed significant potential as anti-DENV therapeutics, necessitating additional mechanistic and in vivo studies to determine their clinical efficacy. IMPORTANCE Dengue is a major global health threat, infecting hundreds of millions of people each year, yet there are no antiviral drugs available for this disease. Identifying safe and effective therapeutics is therefore a global public health priority. In this study, we discovered three promising tomatidine-derived compounds (5d, 5f, and 5c) that markedly reduced Dengue virus replication in laboratory experiments. These compounds acted at multiple stages of the viral life cycle, suggesting that they could both limit the establishment of infection and suppress viral spread after the infection has begun. Computational analyses further indicated that these molecules target key viral proteins while also influencing host pathways that support viral infection, providing insights into how they may exert their antiviral effects. Together, our findings identify tomatidine derivatives as promising lead candidates for dengue antiviral drug development and provide a strong foundation for future preclinical and clinical investigations.