Fig. 1: Summary of the HTS campaign.
From: An allosteric inhibitor of the Zika virus NS2B-NS3 protease with oral efficacy in mouse models
a Schematic representation of the wt ZIKV genome and the engineered replicon used to generate stable cell lines for the phenotypic screen. NanoLuciferase (NanoLuc), Ubiquitin (UBI) and neomycin phosphotransferase resistance (NEO). b High-throughput screening (HTS) funnel (Supplementary Figs. 1 and 2); phenotypic screening using ZIKV replicon and cell viability (CellTiter-Glo) assays, generation of compound-resistant replicon cell lines, characterization of resistant clones and identification of IRBM-Z-1 as an NS3 protease inhibitor. Replicon assays were carried out using Vero cells. IRBM-Z-1 chemical structure; the N-acylsydnone imine core highlighted in blue. c Dose–response curves and IC50 and EC₅₀ values of IRBM-Z-1 in the ZIKV replicon assay (red curve), NS2B-NS3 protease enzymatic assay (blue curve), and Vero cell proliferation assay (black curve). d Dose–response curves and IC₅₀ values of IRBM-Z-1 against ZIKV and DENV2 NS2B-NS3 protease activity, including wt and mutants with substitutions at residue 156. In both panels, percent inhibition of enzymatic activity is plotted against compound concentration (nM). EC₅₀ values were determined using Prism software. All data represent the mean ± SD from at least three independent experiments. e Representative BLI (biolayer interferometry) sensorgrams (blue curves) and 1:1 fitting (red curves) showing the IRBM-Z-1 and ZIKV NS2B-NS3 protease interaction. Binding parameters represent the mean ± SD of six independent experiments. f Mechanism of action of IRBM-Z-1 on the NS2B-NS3 protease. The solid red line represents the IC50 value at substrate concentration [S] = KM, dashed red lines indicate the confidence interval (IC50 [S] = KM ± 3 fold SD). IC50 values are shown in Supplementary Fig. 12. All data represent the mean ± SD from at three independent experiments.
