Discovery of 2-aryl-1H-pyrazole-S-DACO analogs as novel inhibitors targeting the DENV NS5 protein

Dengue virus (DENV), a member of the flavivirus genus, is a mosquito-borne pathogen that has raised significant public health concerns in recent years. Currently, no effective anti-DENV therapeutic is available for clinical use. In this study, we synthesized 40 novel 2-aryl-1H-pyrazole-S-DACO derivatives and identified eight compounds with anti-DENV activity. Among them, the most promising compounds, 4b and 1, demonstrated substantial in vitro antiviral efficacy without cell type specificity, significantly inhibiting DENV-2 replication at both protein and RNA levels. The mechanism of action revealed that both 4b and 1 exhibited antiviral effects following viral entry into host cells, while CETSA experiments confirmed their ability to bind to the DENV NS5 protein. However, these two compounds do not inhibit the activity of DENV NS5-RdRp nor interact with NS5-MTase. Integrating findings from molecular docking studies, we hypothesize that these compounds target 'cavity B' of the DENV NS5 polymerase and exert their antiviral effects by disrupting the interaction between DENV NS5 and NS3. This class of compounds represents a promising basis for further optimization and development.