biostudies-literatureStarvaggi JPRIN_2022PNRR24https://www.ebi.ac.uk/biostudies/studies/S-EPMC12845239biostudies-literatureUnknown19(1)Background: 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. Results: Enzymatic studies revealed that S-proline derivatives bearing electron-withdrawing substituents on the aromatic ring, particularly that with a trifluoromethyl group in meta position (i.e., compound 3, IC50 = 5.0 µM), were the most potent against DENV NS2B/NS3, while nitro-substituted inhibitors were mostly effective only against the ZIKV protease. R-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 24 and 27 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. Conclusions: 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.Pharmaceuticals (Basel, Switzerland)Development of Novel Proline- and Pipecolic Acid-Based Allosteric Inhibitors of Dengue and Zika Virus NS2B/NS3 Protease.PMC12845239P2022KCW3LP2022KCW3L_001 and P2022KCW3LP2022KCW3L_002Lang JKlein CStarvaggi JBelgiovine VNicolotti ODi Chio CEttari RPreviti STrisciuzzi DDi Maro SZappala MfalseDevelopment of Novel Proline- and Pipecolic Acid-Based Allosteric Inhibitors of Dengue and Zika Virus NS2B/NS3 Protease.Background: 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. Results: Enzymatic studies revealed that S-proline derivatives bearing electron-withdrawing substituents on the aromatic ring, particularly that with a trifluoromethyl group in meta position (i.e., compound 3, IC50 = 5.0 µM), were the most potent against DENV NS2B/NS3, while nitro-substituted inhibitors were mostly effective only against the ZIKV protease. R-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 24 and 27 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. Conclusions: 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.2025-01-01T00:00:00Z2025 Dec2026-06-13T03:19:47.754Z2026-06-13T03:12:01.199ZS-EPMC128452394159962610.3390/ph19010024