Piperazine-derived small molecules as potential Flaviviridae NS3 protease inhibitors. In vitro antiviral activity evaluation against Zika and Dengue viruses García-Lozano, María del Rosario Rodríguez-Martínez, Alejandro Rodríguez-Martínez Flavivirus NS3 protease Small molecules inhibitors Privileged structures Acyl and urea piperazine derivatives Live virus phenotypic assay Molecular docking Molecular modeling This work has been supported by Ministerio de Ciencia, Innovación y Universidades, Plan Estatal 2017-2020 Retos - Proyectos I + D + i (PID2019-104767RB-I00). MRGL also thanks Ministerio de Economía y Competitividad, Instituto de Salud Carlos III (grants PI19/00589, PI19/01404, PI16/01842, PI17/00535 and GLD19/00100) for financial support. This work was supported by funds from MIUR Ministero dell’Istruzione, dell’Università della Ricerca Italiano, project PRIN 2017, ORIGINALE CHEMIAE in Antiviral Strategy—Origin and Modernization of Multi-Component Chemistry as a Source of Innovative Broad-Spectrum Antiviral Strategy (cod. 2017BMK8JR) and from Tuscany region, project Tuscany Antiviral Research Network -TUSCAVIR.NET (Bando Ricerca Salute 2018). We would like to thank Giulietta Venturi for making the ZIKV H/PF/2013 strain available for this study. This work has also been funded by the Fundación Séneca de la Región de Murcia under Project 20988/PI/18. This research was partially supported by the computer resources and the technical support provided by the supercomputing infrastructure of the NLHPC (ECM-02), Powered@NLHPC and the Extremadura Research Centre for Advanced Technologies (CETA − CIEMAT), funded by the European Regional Development Fund (ERDF). CETA − CIEMAT is part of CIEMAT and the Government of Spain. Authors thank CITIUS (Centro de Investigación, Tecnología e Innovación de la Universidad de Sevilla) for its important contribution in recording NMR and Mass spectra and in elemental analysis determination. Supplementary data to this article can be found online at https://doi.org/10.1016/j.bioorg.2023.106408 Since 2011 Direct Acting antivirals (DAAs) drugs targeting different non-structural (NS) viral proteins (NS3, NS5A or NS5B inhibitors) have been approved for clinical use in HCV therapies. However, currently there are not licensed therapeutics to treat Flavivirus infections and the only licensed DENV vaccine, Dengvaxia, is restricted to patients with preexisting DENV immunity. Similarly to NS5 polymerase, the NS3 catalytic region is evolutionarily conserved among the Flaviviridae family sharing strong structural similarity with other proteases belonging to this family and therefore is an attractive target for the development of pan-flavivirus therapeutics. In this work we present a library of 34 piperazine-derived small molecules as potential Flaviviridae NS3 protease inhibitors. The library was developed through a privileged structures-based design and then biologically screened using a live virus phenotypic assay to determine the half-maximal inhibitor concentration (IC50) of each compound against ZIKV and DENV. Two lead compounds, 42 and 44, with promising broad-spectrum activity against ZIKV (IC50 6.6 µM and 1.9 µM respectively) and DENV (IC50 6.7 µM and 1.4 µM respectively) and a good security profile were identified. Besides, molecular docking calculations were performed to provide insights about key interactions with residues in NS3 proteases’ active sites. 2023-05-05T10:25:18Z 2023-05-05T10:25:18Z 2023-02-04 journal article M. del Rosario García-Lozano et al. Piperazine-derived small molecules as potential Flaviviridae NS3 protease inhibitors. In vitro antiviral activity evaluation against Zika and Dengue viruses. Bioorganic Chemistry 133 (2023) 106408 [https://doi.org/10.1016/j.bioorg.2023.106408] https://hdl.handle.net/10481/81350 10.1016/j.bioorg.2023.106408 eng http://creativecommons.org/licenses/by-nc-nd/4.0/ open access Attribution-NonCommercial-NoDerivatives 4.0 Internacional Elsevier