Development of Novel Proline- and Pipecolic Acid-Based Allosteric Inhibitors of Dengue and Zika Virus NS2B/NS3 Protease

<b>Background:</b> In this study, we report a novel series of proline- and pipecolic acid-based small molecules designed as allosteric inhibitors of the NS2B/NS3 serine proteases from dengue and Zika viruses, key targets in antiviral drug discovery. <b>Results:</b> Enzymatic studies revealed that <i>S</i>-proline derivatives bearing electron-withdrawing substituents on the aromatic ring, particularly that with a trifluoromethyl group in <i>meta</i> position (i.e., compound <b>3</b>, IC₅₀ = 5.0 µM), were the most potent against DENV NS2B/NS3, while nitro-substituted inhibitors were mostly effective only against the ZIKV protease. <i>R</i>-configured pipecolic acid-based derivatives were the only ones active against DENV NS2B/NS3, even if the mid-micromolar range; however, they demonstrated improved cellular efficacy since inhibitors <b>24</b> and <b>27</b> exhibiting strong activity in a DENV2 protease reporter gene assay (EC₅₀ = 5.2 and 5.1 µM, respectively). All compounds showed no cytotoxicity (CC₅₀ > 100 µM) and were selective for the viral protease over off-target serine proteases. Structure-based approaches were exploited to map the druggable allosteric site close to Asn152. <b>Conclusions:</b> Our findings led us to identify proline and pipecolic acid-based inhibitors as promising leads for the development of selective flaviviral NS2B/NS3 allosteric inhibitors.