A nitrogen-containing diphyllin derivative C156-P1 exhibited broad-spectrum antiviral activity against <i>Flaviviridae</i> viruses by preventing endosomal acidification

Dengue virus (DENV) represents a significant public health threat, with its four serotypes estimated to account for approximately 96 million symptomatic infections annually. Currently, there are no antiviral agents available for the prevention or treatment of DENV infection. Here, we initially screened 12 diphyllin derivatives and identified C156-P1, a nitrogen-containing compound, as a potent agent against DENV infection. Further, C156-P1 exhibited inhibitory effects against the viruses of the <i>Flaviviridae</i> family, including four serotypes of DENV (DENV-1 to DENV-4) in multiple human and monkey cell lines, as well as Zika virus, Japanese encephalitis virus, yellow fever virus, and hepatitis C virus. In addition, C156-P1 also showed inhibition of the infections of herpes simplex virus type 1 and vesicular stomatitis virus, but not adenovirus and Sendai virus. Mechanistic studies demonstrated that C156-P1 inhibited DENV-2 after cell entry but before the endosomal membrane fusion step. C156-P1 inhibited vacuolar-type ATPase activity by perturbing the expression of ATP6V0A2 subunit, thereby suppressing endosomal acidification. Consequently, DENV was restricted in the late endosome, inhibiting virus fusion with endosomal membranes and resulting in infection inhibition. C156-P1 treatment also suppressed both IFN-I responses and endosomal TLR3 activation induced by DENV-2 infection. Furthermore, administration of C156-P1 in AG129 mice significantly reduced DENV-2 infection and effectively increased the survival rate of the mice. Taken together, our study demonstrates that the novel nitrogen-containing diphyllin derivative C156-P1 functions as a broad-spectrum antiviral agent by inhibiting endosomal acidification, thus representing a promising host-targeting antiviral candidate for future development.