Identification of novel thiazole derivatives as flaviviral protease inhibitors effective against Dengue (DENV2) and Japanese encephalitis viruses

Flaviviruses are the causative agents of viral hemorrhagic fever (VHF) globally and have demonstrated the capacity to result in fatal outcomes if not managed effectively. Among different types of flaviviruses, dengue (DENV) and Japanese encephalitis (JEV) viruses are the most common in tropical and subtropical countries. While vaccines have been developed and licensed for both DENV and JEV, effective treatment options remain sparse. Hence, there is a pressing need to develop small molecules that can target machineries crucial for virus replication and remain conserved across different flaviviruses, thereby could serve as a promising therapeutic option. This study outlines the synthesis of novel thiazole compounds as flavivirus NS2B-NS3 protease inhibitor and characterization of their antiviral activity against DENV and JEV. We synthesized a heterocyclic template derived from a substrate-based retrotripeptide dengue protease inhibitor, leading to 48 thiazole derivatives. Two compounds, 3aq and 3au demonstrated significant inhibition of dengue virus protease activity in vitro. Comprehensive characterization of these two compounds was conducted through biochemical assay, which revealed an uncompetitive mode of inhibition. Subsequent cell-based assays using Dengue and Japanese encephalitis viruses as representative flaviviruses revealed the potential of these compounds to block viral RNA synthesis, and viral replication exhibiting 50% inhibitory concentrations (IC50s) in the low-micromolar range. Time-course experiments unveiled that the two compounds impeded the accumulation of viral genomic RNA primarily at later stages of infection, aligning with their capacity to hinder NS2B-NS3 protease activity, polyprotein processing and viral genomic RNA replication. Finally, time of addition experiment showed the compounds remain effective even when added 9 hpi, thereby confirming their potential as promising antivirals. Together, our work presents the development and validation of flavivirus protease inhibitors with therapeutic potential against Dengue (DENV2) and Japanese encephalitis viruses.