De Novo Discovery of Bicyclic Competitive Inhibitors of Zika Virus Protease from Peptide-Bismuth Phage Display Screening.

No specific vaccines or therapeutics are currently available for the prevention or treatment of Zika virus infections. The viral protease NS2B-NS3 is essential for the replication of Zika and other orthoflaviviruses, making it a target for antiviral drug development. Traditional discovery of competitive inhibitors has relied on substrate recognition sequences, typically yielding multibasic peptides. Herein, a de novo strategy is presented for identifying competitive inhibitors using peptide phage display in combination with Bi(III)-mediated in situ formation of bicyclic peptides. In an initial screening, phages displaying a library of randomized peptide-bismuth bicycles are eluted by interrupting the phage-target interactions at low pH. This approach yields a small number of peptides biased toward the active site, characterized by dibasic motifs, but only one low-ranking sequence shows modest inhibitory activity. To enhance specificity, a second screening campaign employs competitive phage elution using the dibasic boronate inhibitor CN-714 that covalently binds to the catalytically active serine residue S135 of NS2B-NS3. This strategy enriches a larger pool of competitive inhibitors sharing the characteristic dibasic substrate recognition motif. The most potent peptide-bismuth bicycle identified and synthesized features a completely novel sequence, exhibits an inhibition constant of 3.9 µM and displays remarkable proteolytic stability over 24 h.