N-Desmethylclozapine, Fluoxetine, and Salmeterol Inhibit Postentry Stages of the Dengue Virus Life Cycle

Around 10,000 people die each year due to severe dengue disease, and two-thirds of the world population lives in a region where dengue disease is endemic. There has been remarkable progress in dengue virus vaccine development; however, there are no licensed antivirals for dengue disease, and none appear to be in clinical trials. We took the approach of repositioning approved drugs for anti-dengue virus activity by screening a library of pharmacologically active compounds. We identified N-desmethylclozapine, fluoxetine hydrochloride, and salmeterol xinafoate as dengue virus inhibitors based on reductions in the numbers of infected cells and viral titers. Dengue virus RNA levels were diminished in inhibitor-treated cells, and this effect was specific to dengue virus, as other flaviviruses, such as Japanese encephalitis virus and West Nile virus, or other RNA viruses, such as respiratory syncytial virus and rotavirus, were not affected by these inhibitors. All three inhibitors specifically inhibited dengue virus replication with 50% inhibitory concentrations (IC(50)s) in the high-nanomolar range. Estimation of negative-strand RNA intermediates and time-of-addition experiments indicated that inhibition was occurring at a postentry stage, most probably at the initiation of viral RNA replication. Finally, we show that inhibition is most likely due to the modulation of the endolysosomal pathway and induction of autophagy.