Small molecules targeting coxsackievirus A16 capsid inactivate viral particles and prevent viral binding

Figures

• 

  Fig. 1. Drug screening analysis and antiviral dose-response activity of CHLA and PUG against CVA16. a RD cells were challenged with CVA16 (50 PFU/well) in the presence of the test compounds and then analyzed by a plaque assay 72 h post infection; the results are expressed as percent (%) CVA16 infectivity. b, c RD cells were infected with CVA16 (50 PFU/well) in the presence of b CHLA (0.01, 0.1, 0.5, 1, 2, 5, 10, 20, and 25 µM) or c PUG (0.01, 0.1, 0.5, 1, 2, 5, 10, 25, and 50 µM) and analyzed for virus-induced plaque formation after 72 h of incubation; the data are expressed as % CVA16 inhibition. Chemical structures of CHLA and PUG are shown in the respective panels. For all experiments, a DMSO (0.25%) treatment served as negative control, and the data shown are the means ± standard deviation (SD) from three independent repeats. *p < 0.05 compared to the ‘virus only’ group

• 

  Fig. 2. Time-of-drug-addition effect of CHLA and PUG against CVA16 infectivity. RD cells were treated with CHLA (20 µM) or PUG (25 µM) at different times of CVA16 inoculation (50 PFU/well). a For the pretreatment, cells were incubated with the test compounds for 1 or 4 h and then were washed before CVA16 infection. b For coaddition assays, cells were administered with drugs and virus simultaneously for 1 h and were subsequently washed. c In the postinfection experiment, cells were infected with CVA16 for 1 h, washed, and treated with the test compounds. For all of the above experiments, a DMSO (0.25%) treatment was included as negative control for each condition, and viral plaques were stained and counted after 72 h of incubation. The data shown are the means ± SD from three independent experiments. *p < 0.05 compared to the respective ‘virus only’ group

• 

  Fig. 3. CHLA and PUG abolish CVA16 binding to the host cell. a Schematic of the experiment. b RD cells (2 × 105 cells/well) were infected with CVA16 (MOI = 100) in the presence or absence of CHLA (20 µM), PUG (25 µM), soluble heparin (500 µg/ml), or DMSO (0.25%, negative control) for 3 h at 4 °C. The inocula were removed and the cells were then collected into tubes, washed twice, fixed, and stained with anti-VP1 antibody followed by an Alexa 488-conjugated secondary antibody for flow cytometry detection of surface-bound viruses. The quantified data from the detected fluorescence signals were plotted as the means ± SD from three independent experiments in bar graph as ‘Viral binding (%)’. *p < 0.05 compared to the ‘DMSO’ control treatment. The representative flow cytometry histograms of CHLA c, PUG d, and heparin e treatments are shown

• 

  Fig. 4. CHLA and PUG inactivate cell-free CVA16 virus particles. a Schematics of the experiment. b CVA16 (106 PFU/ml) was treated with CHLA (20 µM) or PUG (25 µM) and incubated for 1 h at 37 °C, or were mixed immediately (0 h), and then diluted 50-fold to non-effective concentrations of the test compounds before inoculating the RD cells (final virus concentration = 50 PFU/well) and being analyzed for viral infectivity by a plaque assay. DMSO (0.25%) served as a negative control. The data shown are the means ± SD from three independent experiments. *p < 0.05 compared to the respective ‘virus only’ group

• 

  Fig. 5. CHLA and PUG target the CVA16 capsid near the pocket entrance. a, b Molecular docking analysis of CHLA (a; green) and PUG (b; blue) on the pentamer of CVA16 (PDB: 5C4W); zoomed panels from the yellow demarcation are shown. VP1 = orange, VP2 = gray, and VP3 = white; polar contacts are shown as black dashes. Residues that make-up the pocket entrance are colored red (Ile94, Asp95, Gln207, Met212, Met213, Lys257, Thr258). c, d Alternate close-up side view into the canyon where CHLA and PUG bind near the pocket entrance. Unique residues from the polar contacts of the tannins to amino acids in VP1 (yellow font), VP2 (white font), and in VP3 (black font) are indicated. The white dashed line indicates the opening of the pocket region