Targeting a genomic RNA G-quadruplex of dengue virus with small molecules as an alternative to protein-targeted therapeutics

BACKGROUND: Given the increasing global incidence of dengue virus (DENV) infections and the lack of approved antiviral therapies, developing a new antiviral strategy against DENV is urgently required. However, despite considerable efforts to develop protein-targeted antivirals, clinical translation has largely failed, highlighting the need for alternative, non-protein targets. METHODS: Here, we investigated a novel therapeutic approach that targets RNA G-quadruplexes (G4s), highly conserved secondary structures across viral strains, including all serotypes. Bioinformatic predictions and biophysical analyses identified conserved G4-forming sequences within the DENV genome. RESULTS: Among the tested representative G4-binding ligands, BRACO-19 showed the highest stabilizing effect on G4 structures, particularly at the G4-3 region corresponding to the NS3 gene with high binding affinity. Functionally, BRACO-19 treatment significantly reduced viral translation resulting in strong antiviral effects in a mouse model of DENV infection. Consistently, recombinant DENV carrying a G4-3-disrupting mutation showed enhanced viral gene expression and attenuated sensitivity to BRACO-19. These findings establish DENV RNA G4 as a druggable structural element and position BRACO-19 as a promising lead compound for dengue therapeutics. Furthermore, we also demonstrated that BRACO-19 exhibits broad-spectrum activity against all DENV serotypes. This RNA structure-based approach offers an alternative to protein-targeted therapeutics, helping to mitigate mutational escape and serotype variability in dengue virus. CONCLUSION: Collectively, our study highlights a novel therapeutic strategy that directly targets conserved RNA secondary structures, paving the way for the development of broad-spectrum antivirals against flaviviruses by targeting a non-canonical nucleic acid structure.