Journal: Nature Communications

Article Title: A rationally designed 2C inhibitor prevents enterovirus D68-infected mice from developing paralysis

doi: 10.1038/s41467-025-61083-8

Figure Lengend Snippet: a Representative frames from 500-ns-MD simulations of the complexes between Jun571 and the EV-D68 2C (protein = blue ribbon and gray sticks; ligand’s carbons are shown in orange; nitrogen, oxygen, and sulfur are shown in blue and red, and yellow respectively; hydrogen bonding interactions are shown with yellow dashes). b Interactions between Jun571 and the residues at the allosteric site of EV-D68 2C. c Stabilizing interactions inside the binding area of 2C with Jun571 ; hydrogen bonding interactions bar is depicted in green, ionic hydrogen bonding interactions in red, water bridges in blue, and lipophilic interactions in gray. d RMSD plots of Cα carbons of 2C (blue diagram) and the heavy atoms of the ligand Jun571 (red diagram).

Article Snippet: Enterovirus D68 (EV-D68) strains US/MO/14-18947, US/KY/14-18953, US/MO/14-18949, US/IL/14-18952, and US/IL/14-18956 (ATCC); Coxsackievirus B3 (CVB3) Nancy strain (VR-30); Enterovirus A71 (EV-A71) Tainan/4643/1998 (BEI Resources, NR-471); Coxsackievirus A6 (CVA6, VR-1801); and human rhinovirus 16 (RV16, VR-283) were sourced from ATCC or BEI Resources.

Techniques: Binding Assay

Journal: Nature Communications

Article Title: A rationally designed 2C inhibitor prevents enterovirus D68-infected mice from developing paralysis

doi: 10.1038/s41467-025-61083-8

Figure Lengend Snippet: a Antiviral EC 50 values were determined in the CPE assay with EV-D68 US/MO/14-18947 in RD cells. Cytotoxicity CC 50 values were determined using the neutral red uptake method in RD cells. The results are the mean ± standard deviation (SD) of two independent experiments with three repeats in each experiment. *The antiviral activity EC 50 values of Jun5532 and Jun5253 were reported earlier . SI selectivity index. b Antiviral activity of selected 2C inhibitors against EV-A71 Tainan strain in plaque assay. c Antiviral activity of selected 2C inhibitors against CVB3 Nancy strain in plaque assay. The EC 50 values in the plaque assay are the mean ± SD of two biological replicates. Source data are provided as a Source Data file.

Article Snippet: Enterovirus D68 (EV-D68) strains US/MO/14-18947, US/KY/14-18953, US/MO/14-18949, US/IL/14-18952, and US/IL/14-18956 (ATCC); Coxsackievirus B3 (CVB3) Nancy strain (VR-30); Enterovirus A71 (EV-A71) Tainan/4643/1998 (BEI Resources, NR-471); Coxsackievirus A6 (CVA6, VR-1801); and human rhinovirus 16 (RV16, VR-283) were sourced from ATCC or BEI Resources.

Techniques: Standard Deviation, Activity Assay, Plaque Assay

Journal: Nature Communications

Article Title: A rationally designed 2C inhibitor prevents enterovirus D68-infected mice from developing paralysis

doi: 10.1038/s41467-025-61083-8

Figure Lengend Snippet: a Antiviral activity of Jun6504 against five contemporary EV-D68 strains in RD cells. EC 50 values against EV-D68 strains were determined using the cytopathic effect (CPE) assay in RD cells after 60 h of compound incubation. The EC 50 values are the mean ± SD of three biological replicates. b Antiviral activity of Jun6504 against poliovirus, CVA16, CVA6, and CVB3 in CPE assay. The EC 50 values are the mean ± SD of two biological replicates. c Antiviral activity of Jun6504 against EV-A71 and EV-D68 in plaque assay. The EC 50 values are the mean ± SD of two biological replicates. d Cytotoxicity of Jun6504 in HeLa, RD, and Vero cells. The results are from two biological replicates. e DSF assay of Jun6504 with EV-A71, EV-D68, and CVB3 2 C proteins. The results are from two biological replicates. f Jun6504 time-of-addition experiment in a single cycle of EV-D68 replication. g Viral titers in the time-of-addition experiment. EC 50 and CC 50 curve fittings using log 10 (concentration of Jun6504 ) vs. percentage of positive control with variable slopes were performed in Prism 8. Error bars denote mean ± SD of two biological replicates. Source data are provided as a Source Data file.

Article Snippet: Enterovirus D68 (EV-D68) strains US/MO/14-18947, US/KY/14-18953, US/MO/14-18949, US/IL/14-18952, and US/IL/14-18956 (ATCC); Coxsackievirus B3 (CVB3) Nancy strain (VR-30); Enterovirus A71 (EV-A71) Tainan/4643/1998 (BEI Resources, NR-471); Coxsackievirus A6 (CVA6, VR-1801); and human rhinovirus 16 (RV16, VR-283) were sourced from ATCC or BEI Resources.

Techniques: Activity Assay, Incubation, Plaque Assay, Concentration Assay, Positive Control

Journal: Nature Communications

Article Title: A rationally designed 2C inhibitor prevents enterovirus D68-infected mice from developing paralysis

doi: 10.1038/s41467-025-61083-8

Figure Lengend Snippet: a Schematic illustration of the serial passage of Jun6504 in RD cells with EV-D68 US/MO/14-18947. The graph was created with Biorender.com. Li, K (2025) https://BioRender.com/0kxixma . b Electropherogram traces of 2C protein coding region at residues 112, 183, and 323 from viruses at passages 0, 3, 6, and 9. c Antiviral EC 50 curves of Jun6504 against recombinant EV-D68 viruses in plaque assay. The EC 50 values are mean ± SD from two biological replicates. d Thermal shift binding assay of Jun6504 with EV-D68 2C WT, D183V, D323G, and D183V/D323G. The red line indicates a 0.5 °C shift (Δ T m = 0.5 °C). A ΔTm greater than 0.5 °C generally indicates ligand binding to the protein . The assay was performed in two biological replicates. e Interactions between Jun6504 and the residues at the allosteric site of EV-D68 2C. f Stabilizing interactions inside the binding area of 2C with Jun6504 ; hydrogen bonding interactions bar is depicted in green, ion hydrogen bonding interactions in red, water bridges in blue, and lipophilic interactions in gray. g RMSD plots of Cα carbons of 2C (blue diagram) and of the heavy atoms of the ligand Jun6504 (red diagram). h Interactions between Jun6504 and the residues at the allosteric site of EV-D68 2C-D183V. i Stabilizing interactions inside the binding area of 2C-D183V with Jun6504 ; hydrogen bonding interactions bar is depicted in green, ion hydrogen bonding interactions in red, water bridges in blue and lipophilic interactions in gray. j RMSD plots of Cα carbons of 2C-D183V (blue diagram) and of the heavy atoms of the ligand Jun6504 (red diagram). Source data are provided as a Source Data file.

Article Snippet: Enterovirus D68 (EV-D68) strains US/MO/14-18947, US/KY/14-18953, US/MO/14-18949, US/IL/14-18952, and US/IL/14-18956 (ATCC); Coxsackievirus B3 (CVB3) Nancy strain (VR-30); Enterovirus A71 (EV-A71) Tainan/4643/1998 (BEI Resources, NR-471); Coxsackievirus A6 (CVA6, VR-1801); and human rhinovirus 16 (RV16, VR-283) were sourced from ATCC or BEI Resources.

Techniques: Recombinant, Plaque Assay, Binding Assay, Ligand Binding Assay

Journal: Nature Communications

Article Title: A rationally designed 2C inhibitor prevents enterovirus D68-infected mice from developing paralysis

doi: 10.1038/s41467-025-61083-8

Figure Lengend Snippet: a Schematic illustration of the serial passage of Jun6504 in RD cells with EV-D68 US/MO/14-18947. b Antiviral EC 50 curves of Jun6504 against Jun6504 -treated and DMSO-treated viruses at P7 and P10. The assay was performed in two biological replicates. c Sequencing results of the 2C protein from Jun6504 -treated viruses at P7 and P10. d DSF binding assay of Jun6504 against EV-D68 2C WT, F190L. D183V/F190L, and D183V/F190L/D323G. The assay was performed in two biological replicates. e Docking model of Jun6504 in EV-D68 2C. f Interactions between Jun6504 and the residues at the allosteric site of EV-D68 2C-F190L mutant. g Stabilizing interactions inside the binding area of EV-D68 2C-F190L with Jun6504 ; hydrogen bonding interactions bar is depicted in green, ion hydrogen bonding interactions in red, water bridges in blue and lipophilic interactions in gray. h RMSD plots of Cα carbons of EV-D68 2C-F190L (blue diagram) and of the heavy atoms of the ligand Jun6504 (red diagram). Source data are provided as a Source Data file.

Article Snippet: Enterovirus D68 (EV-D68) strains US/MO/14-18947, US/KY/14-18953, US/MO/14-18949, US/IL/14-18952, and US/IL/14-18956 (ATCC); Coxsackievirus B3 (CVB3) Nancy strain (VR-30); Enterovirus A71 (EV-A71) Tainan/4643/1998 (BEI Resources, NR-471); Coxsackievirus A6 (CVA6, VR-1801); and human rhinovirus 16 (RV16, VR-283) were sourced from ATCC or BEI Resources.

Techniques: Sequencing, Binding Assay, Mutagenesis

Journal: Nature Communications

Article Title: A rationally designed 2C inhibitor prevents enterovirus D68-infected mice from developing paralysis

doi: 10.1038/s41467-025-61083-8

Figure Lengend Snippet: All control animals received vehicle alone (90% corn oil + 10% DMSO). All Jun6504 groups received 50 mg/kg of Jun6504 in the vehicle. All error bars represent SEM. a Paralysis scores are significantly lower in the Jun6504 group in the immediate treatment model (* p = 0.0004 Wilcoxon rank-sum; Control N = 10 [DPI14 = 5.35 ± 0.89], Jun6504 N = 10 [DPI14 = 0.40 ± 0.42]). b Weights from EV-D68-infected mice shown in Fig. a. Weight gain is also significantly increased by Jun6504 (* p = 0.003 t -test, Control DPI14 = 7.92 ± 0.46 g vs Jun6504 DPI14 = 9.29 ± 0.14 g). c Paralysis scores are significantly lower in Jun6504 group in 24-h delayed treatment model (* p = 0.0043 Wilcoxon rank-sum; Control N = 18 [DPI14 = 6.42 ± 0.63], Jun6504 N = 17 [DPI14 = 1.59 ± 0.61]). d Weights from the EV-D68-infected mice shown in Fig. c. Weight gain is also significantly increased by Jun6504 (* p = 0.0052 t -test; Control DPI14 = 8.65 ± 0.23 g vs Jun6504 DPI14 = 9.59 ± 0.27 g). e Pictorial representation of tissue harvest time points before treatment at DPI1 or after 4x daily injections on DPI5. The graph was created with Biorender.com. Li, K (2025) https://BioRender.com/kmwsykp . f Viral titer in muscle tissue from left quadriceps (infected muscle) prior to treatment (blue bar) and after 4 days of treatment (red and green bars). There is significantly more virus in the quadriceps muscle from untreated pups at DPI5 compared to Jun6504 -treated pups (pooled t -test * p > 0.0001; Control N = 9 mean 119,544 ± 33,194TCID 50 vs Jun6504 N = 9 mean 8762 ± 3080TCID 50 ). The limit of detection in this assay is 1580TCID 50 . g Viral titer in spinal cords from the same animals shown in panel ( f ). There is significantly more virus in the spinal cord of untreated pups at DPI5 compared to Jun6504 - treated pups (pooled t -test * p > 0.0484; Control mean 19,834 ± 11,519TCID 50 vs Jun6504 mean 4520 ± 1796TCID 50 ). All TCID 50 values in untreated spinal cords from DPI1 are at the limit of detection. h Concentration of Jun6504 in spinal cords collected from uninfected pups. Jun6504 does enter the spinal cord (* p = 0.0151 Wilcoxon rank-sum, vehicle control N = 4 [50.0 ± 0.0] vs Jun6504 N = 5 [574.6 ± 100.5]). All vehicle control data is at the limit of detection. All statistics shown in Fig. 7 are two-sided ( a – d , f – h ). Source data are provided as a Source Data file.

Article Snippet: Enterovirus D68 (EV-D68) strains US/MO/14-18947, US/KY/14-18953, US/MO/14-18949, US/IL/14-18952, and US/IL/14-18956 (ATCC); Coxsackievirus B3 (CVB3) Nancy strain (VR-30); Enterovirus A71 (EV-A71) Tainan/4643/1998 (BEI Resources, NR-471); Coxsackievirus A6 (CVA6, VR-1801); and human rhinovirus 16 (RV16, VR-283) were sourced from ATCC or BEI Resources.

Techniques: Control, Infection, Virus, Concentration Assay

Journal: Nature Communications

Article Title: A rationally designed 2C inhibitor prevents enterovirus D68-infected mice from developing paralysis

doi: 10.1038/s41467-025-61083-8

Figure Lengend Snippet: a IHC stain of a transverse section through the lumbar spinal cord of Jun6504 -treated animal with no paralysis in hindlimbs. Four panels to the right show increased zoom of the white dashed box where neuronal nuclei, choline acetyltransferase, and DAPI are green, red, and blue signals, respectively. b IHC stain of transverse section through lumbar spinal cord of corn oil control-treated animal with complete paralysis in both hindlimbs. Four panels to the right show increased zoom of the white dashed box where neuronal nuclei, choline acetyltransferase, and DAPI are green, red, and blue signals, respectively. c IHC stain of transverse section through cervical spinal cord of corn oil control-treated animal with complete paralysis in both hindlimbs, but no paralysis in forelimbs. Four panels to the right show an increased zoom of the white dashed box. Anti-neuronal nuclei is green, anti-Enterovirus D68 viral protein 2 is red, and DAPI is blue. d IHC stain of transverse section through lumbar spinal cord of Jun6504 - treated animal with complete paralysis in left hindlimb, but no paralysis in the right hindlimb. Four panels to the right show an increased zoom of the white dashed box. Anti-neuronal nuclei is green, anti-Enterovirus D68 viral protein 2 is red, and DAPI is blue. Scale bars in all images are 100 microns in length. Five serial sections (separated by 200 µm intervals) were collected for each spinal cord. A single representative image is shown in Fig. 8, but N = 5 spinal cords were stained for ChAT and N = 3 were stained for VP2.

Article Snippet: Enterovirus D68 (EV-D68) strains US/MO/14-18947, US/KY/14-18953, US/MO/14-18949, US/IL/14-18952, and US/IL/14-18956 (ATCC); Coxsackievirus B3 (CVB3) Nancy strain (VR-30); Enterovirus A71 (EV-A71) Tainan/4643/1998 (BEI Resources, NR-471); Coxsackievirus A6 (CVA6, VR-1801); and human rhinovirus 16 (RV16, VR-283) were sourced from ATCC or BEI Resources.

Techniques: Staining, Control