Rational design and in vivo validation of capsid inhibitors for enterovirus D68

Fig. 1. Discovering and optimizing quinoline-based inhibitors as potential EV-D68 VP1 capsid inhibitors. a Chemical structure of the screening hit Z281155752 identified from virtual screening (EC50 and CC50, mean ± SD of three biological replicates). b Superimposed binding poses of pleconaril and Z281155752 at the VP1 hydrophobic pocket. The chemical structure of pleconaril is shown underneath. c Summary of lead optimization. d Chemical structures of Jun11787 and Jun11695, two of the most promising lead compounds prioritized for the in vivo mouse model study (EC50 and CC50, mean ± SD of three biological replicates). Source data are provided as a Source Data file.

Fig. 2. Jun11695 and Jun11787 inhibit multiple enterovirus strains and have synergistic antiviral effects with telaprevir. a Antiviral activity and cytotoxicity of Jun11695, Jun11787, and pleconaril, against five strains of EV-D68 in CPE assay (EC50 and CC50, mean ± SD of three biological replicates). b Antiviral activity of pleconaril, Jun11695, and Jun11787 against EV-D68 US/MO/14-18947 in plaque assay (EC50, mean ± SD of four biological replicates). c Antiviral activity of pleconaril, Jun11695, and Jun11787 against EV-A71 Tainan/4643/1998 in plaque assay (EC50, mean ± SD of four biological replicates). d Antiviral activity of pleconaril, Jun11695, and Jun11787 against CVB3 Nancy in plaque assay (EC50, mean ± SD of four biological replicates). e Inhibition of EV-D68 US/MO/14-18947 by Jun11695 and Jun11787 at different MOIs in the viral titer reduction assay (mean ± SD, n = 3, independent experiments). f Combination therapy of Jun11695 and Jun11787 with telaprevir against EV-D68 US/MO/14-18947 (n = 3, independent experiments). Source data are provided as a Source Data file.

Fig. 3. Quinoline-based inhibitors inhibit EV-D68 replication in the early stage as capsid VP1 binders. a The structures of EV-D68 in complex with Jun11537, Jun11541, Jun11695, and Jun11787 are shown as a ribbon diagram with the inhibitor displayed in its atomic structure in each panel (gold). The four viral proteins in each protomer are shown in blue (VP1), green (VP2), red (VP3), and orange (VP4). Icosahedral fivefold, threefold, and twofold axes are indicated with a star, triangle, and ellipse, respectively. The corresponding cryoEM map are shown around the inhibitors, and chemical structures for Jun11537, Jun11541, Jun11695, and Jun11787 are shown in the right panel. VP1 and VP2 are shown as cartoons in blue and green, with the inhibitor in yellow. Residues that are interfacing with the inhibitor are shown as sticks. b Schematic illustration of the time-of-addition experiment. c Quantification of immunofluorescence of the intracellular viral VP1 capsid protein during the time-of-addition experiment with Jun11695, Jun11787, and pleconaril (mean ± SD, n = 3, independent experiments). A two-sided ordinary one-way ANOVA comparison was employed for statistical test using GraphPad Prism…

Fig. 4. Jun11695 and Jun11787 show favorable in vivo PK in BALB/c mice. a In vivo clearance of Jun11695 in female BALB/c mice (n = 7) after I.P. injection at 50 mg/kg. b Pharmacokinetic parameters of Jun11695. c In vivo clearance of Jun11787 in female BALB/c mice (n = 6) after I.P. injection at 50 mg/kg. d Pharmacokinetic parameters of Jun11787. Adult female BALB/c mice were given a single i.p. injection of Jun11695 or Jun11787 at 50 mg/kg. Blood was obtained at up to 12 h after administration of the compound. The plasma concentrations of Jun11695 and Jun11787 and the internal standard were analyzed by LC-MS/MS. Values are mean ± SEM, n = 7 for Jun11695 and n = 7 for Jun11787. Source data are provided as a Source Data file.

Fig. 5. Jun11695 and Jun11787 display in vivo antiviral efficacy in the EV-D68 infection-induced neurological mouse model. a Immediate treatment model showing paralysis scores by group (control N = 6, Jun11787 N = 7, Jun11695 N = 7). Jun11695 plot is hidden behind Jun11787 at zero on y-axis. ****p < 0.0001, repeated measures ANOVA. b Immediate treatment model showing average animal weight by group (same mice as panel a). ***p = 0.001, repeated measures ANOVA c, 24 h delayed treatment model showing paralysis scores by group (control N = 17, Jun11787 N = 17, Jun11695 N = 17). ****p < 0.0001, repeated measures ANOVA d, 24 h delayed treatment model showing average pup weight by group (same mice as panel c). *p = 0.0093 repeated measures ANOVA. e Viral titer in the spinal cord from mice treated with Jun11787 in the 24 h delayed treatment model. Tissue was collected at 4, 6, and 8 days post-infection from the same animals used for muscle viral titers (control dpi4 N = 8, Jun11787 dpi4 N = 6; control dpi6 N = 8, Jun11787 dpi6 N = 6; control dpi8 N = 9, Jun11787 dpi8 N = 7). f Viral titer in the muscle from mice treated with Jun11787 in the 24 h delayed treatment model. The limit of detection in…