Saini, M.; Aschenbrenner, J. C.; Ruiz, F. X.; Chopra, A.; Chandran, A. V.; Marples, P. G.; Balcomb, B. H.; Fearon, D.; von Delft, F.; Arnold, E.

Dengue viruses (DENV) infect approximately 400 million people each year, and there are currently no effective therapeutics available. To explore potential starting points for antiviral drug development, we conducted a large-scale crystallographic fragment screen targeting the RNA-dependent RNA polymerase (RdRp) domain of the non-structural protein 5 (NS5) from DENV serotype 2. Our screening, which involved 1,108 fragments, identified 60 hit compounds across various known binding sites, including the active site, N pocket, and RNA tunnel. Additionally, we discovered a novel binding site and a fragment-binding hotspot in thumb site II. These structural findings open amenable avenues for developing non-nucleoside inhibitors and offer valuable insights for future structure-based drug design aimed at DENV and other flaviviral RdRps.