Synthesis, Structure–Activity Relationships, and Antiviral Activity of Allosteric Inhibitors of Flavivirus NS2B–NS3 Protease

Flaviviruses, including Zika, dengue, and West Nile viruses, are important human pathogens. The highly conserved NS2B-NS3 protease of Flavivirus is essential for viral replication and therefore a promising drug target. Through compound screening, followed by medicinal chemistry studies, a novel series of 2,5,6-trisubstituted pyrazine compounds are found to be potent, allosteric inhibitors of Zika virus protease (ZVpro) with IC₅₀ values as low as 130 nM. Their structure-activity relationships are discussed. The ZVpro inhibitors also inhibit homologous proteases of dengue and West Nile viruses, and their inhibitory activities are correlated. The most potent compounds 47 and 103 potently inhibited Zika virus replication in cells with EC₆₈ values of 300-600 nM and in a mouse model of Zika infection. These compounds represent novel pharmacological leads for drug development against Flavivirus infections.