Targeting dengue virus envelope protein: Insights from structural analysis and high-throughput screening

Dengue virus (DENV) poses a significant global health threat, causing millions of infections and thousands of deaths annually. The viral envelope (E) protein mediates fusion between the viral and host endosomal membranes, releasing the viral genome into the host cytoplasm and initiate RNA replication. Targeting E protein-mediated fusion is a promising antiviral strategy. In this study, we examined three-dimensional structures of the dengue virus serotype 2 (DENV-2) soluble envelope ectodomain (sE) in pre- and intermediate-fusion states, identifying three potential drug-binding regions: the known β-OG pocket, a fusion-loop-region within the sE dimer, and a stem-zipper-associated region within the sE trimer. Virtual screening of two large libraries of small molecules was performed targeting these regions. High-throughput screening using a high-content imaging (HCI)-based cell-cell fusion assay against DENV-2 identified several compounds with micromolar anti-fusion activity. Further testing narrowed this to three potential fusion inhibitors targeting the predicted drug-binding regions. By combining computational site analysis, high-throughput fusion screening, and infection-based assays, our study provides a rational framework for orthoflavivirus entry inhibitor discovery.