A series of cyclic active‐site‐directed inhibitors of the NS2B‐NS3 proteases from Zika (ZIKV), West Nile (WNV), and dengue‐4 (DENV4) viruses has been designed. The most potent compounds contain a reversely incorporated d ‐lysine residue in the P1 position. Its side chain is connected to the P2 backbone, its α‐amino group is converted into a guanidine to interact with the conserved Asp129 side chain in the S1 pocket, and its C terminus is connected to the P3 residue via different linker segments. The most potent compounds inhibit the ZIKV protease with K i values <5 nM. Crystal structures of seven ZIKV protease inhibitor complexes were determined to support the inhibitor design. All the cyclic compounds possess high selectivity against trypsin‐like serine proteases and furin‐like proprotein convertases. Both WNV and DENV4 proteases are inhibited less efficiently. Nonetheless, similar structure‐activity relationships were observed for these enzymes, thus suggesting their potential application as pan‐flaviviral protease inhibitors.

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West Nile virus (WNV) and Dengue virus (DENV) replication depends on the viral NS2B-NS3 protease and the host enzyme furin, which emerged as potential drug targets. Modification of our previously described WNV protease inhibitors by basic phenylalanine analogs provided compounds with reduced potency against the WNV and DENV protease. In a second series, their decarboxylated P1-trans-(4-guanidino)cyclohexylamide was replaced by an arginyl-amide moiety. Compound 4-(guanidinomethyl)-phenylacetyl-Lys-Lys-Arg-NH inhibits the NS2B-NS3 protease of WNV with an inhibition constant of 0.11 µM. Due to the similarity in substrate specificity, we have also tested the potency of our previously described multibasic furin inhibitors. Their further modification provided chimeric inhibitors with additional potency against the WNV and DENV proteases. A strong inhibition of WNV and DENV replication in cell culture was observed for the specific furin inhibitors, which reduced virus titers up to 10,000-fold. These studies reveal that potent inhibitors of furin can block the replication of DENV and WNV.