Laura Demuru
| Identification, Design, and Optimization of New Allosteric Inhibitors Against ZIKV and WNV NS2B-NS3pro |
| Demuru Laura1, Lupia Antonio1, Nieddu Salvatore1, Esposito Francesca1, Bryant Sharon D.2, Onali Alessia1, Sanna Erica1, Atzeni Giulia1, Cottiglia Filippo1, Corona Angela1, Tramontano Enzo1, Maccioni Elias1, Meleddu Rita1, Distinto Simona1 1 Department of Life and Environmental Sciences, University of Cagliari, Cittadella Universitaria, Monserrato, Italy; 2 InteLigand GmbH, Mariahilfer Straße 74B/11, 1070 Vienna, Austria; |
| Zika virus (ZIKV) and West Nile virus (WNV) are mosquito-borne members of the Flaviviridae family, recognized as emerging human pathogens. Aedes mosquitoes primarily transmit ZIKV, whereas Culex species predominantly transmit WNV. The global distribution and public health impact of these viruses underscore the urgent need for effective preventive and therapeutic strategies. The viral NS2B-NS3 protease (NS2B-NS3pro) is essential for viral replication, as it cleaves the viral polyprotein into structural and non-structural components [1]. NS3pro is a chymotrypsin-like enzyme featuring a catalytic triad composed of S135, H51, and D75 in its N-terminal region. The structural similarity between the NS3pro active site and various host serine proteases, combined with the limited effectiveness of covalent peptide-based inhibitors, has made it difficult to develop selective competitive inhibitors, making allosteric inhibitors the preferred strategy [2]. Currently, no approved antivirals exist for ZIKV and WNV. In this study, an integrated approach combining several in silico methods with biochemical assays was employed to identify four potential inhibitors from commercial and FDA-approved compound databases targeting NS3pro. Considering the high conservation among the flaviviral proteases, the compounds were also tested against ZIKVpro and WNVpro. Three compounds exhibited IC50 values below 30 μM against ZIKVpro, while four showed IC50 values ranging from 11 to 33 μM. Based on these results, the scaffold of the active compound 47P was selected for further optimization, leading to the synthesis of a library of 42 analogues. Among the fourteen compounds tested so far, three demonstrated improved inhibitory activity against ZIKV NS3pro, and four showed enhanced activity against WNV NS3pro. Additionally, the known allosteric inhibitor NSC135618 was synthesized, and enzyme kinetic studies are ongoing to elucidate the mechanism of inhibition of the newly identified compounds. |
| References [1] Shiryaev, S.A., et al., Dual function of Zika virus NS2B-NS3 protease. PLOS Pathogens, 2023. 19(11): p. e1011795. [2] Santos, N.P., et al., Characterization of an Allosteric Pocket in Zika Virus NS2B-NS3 Protease. J. Chem. Inf. Model., 2022. 62(4): p. 945-957. Acknowledgments: This work was funded by PNRR – START-UP D.M. 737/2021 IHOPE: Identification and Biological Evaluation of Broad-Spectrum InHibitors Of NS2B/NS3 ProteasE to fight flavivirus outbreaks. CUP: F25F21002720001 |