Pterostilbene Exhibits Broad‐Spectrum Antiviral Activity by Targeting the Enterovirus Capsid, Inactivating Viral Particles, Blocking Viral Binding, and Protecting Mice From Lethal EV‐A71 Challenge

Figures

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  FIGURE 1. Antiviral effects of stilbene analogues. Chemical structure of (a) resveratrol (Res), (b) pterostilbene (Pte), (c) oxyresveratrol (ORV), (d) piceatannol (Pic), and (e) pinostilbene (Pin). The antiviral activity of (f) Res, (g) Pte, (h) ORV, (i) Pic, and (J) Pin against EV‐D68 infection in RD cells. Cells were infected with 0.1 MOI EV‐D68 B3 for 1 h; after washing, cells were treated with increasing concentrations of stilbenes for 48 h. Cell viability was determined using the MTT assay. The antiviral activities of these stilbenes were calculated according to Equation (2) described in Section 2. N = 4. Data are expressed as the mean ± SEM.

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  FIGURE 2. Antiviral effects of pterostilbene and resveratrol. (a) Cytotoxicity of Pte and Res in RD cells. Cells were seeded in 96‐well plates and treated with increasing concentrations of Pte and Res for 48 h. Cell viability was determined using the MTT assay (N = 5). Antiviral activity of Pte and Res against (b) EV‐D68 MO/47 B1, (c) EV‐A71 C2, (d) EV‐A71 B4, (e) CV‐A16, and (f) CV‐B3 (MOI = 0.1) infection. RD cells were infected with enteroviruses and then treated with various concentrations of Pte or Res as indicated. The effects of Pte and Res on cell viability was determined using MTT assays. (g) Cell morphology of EV‐A71‐ and EV‐D68‐infected cells treated with Pte or Res (20 and 40 μM) under a phase‐contrast microscope (Olympus) (100× magnification). The mock group was treated with PBS, whereas the vehicle group was infected but not treated. N = 4. All experiments were performed at least in quadruplicate. Data are expressed as the mean ± SEM.

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  FIGURE 3. Pterostilbene inhibits viral progeny production, RNA expression, and VP1 protein production. The effect of Pte on infectious viral progeny production was investigated. RD cells were infected with (a) EV‐A71 C2 or (b) EV‐D68 B3 (0.1 MOI) for 1 h, and subsequently treated or not with Pte for 48 h. The culture supernatants were collected and the viral titers were determined using the TCID50 assay (N = 3). RD cells were infected with (c) EV‐A71 or (d) EV‐D68 at an MOI of 0.1 for 1 h, and then cells were treated with indicated concentrations of Pte for 24 h. The levels of viral RNA were quantified using RT‐PCR, with the expression of the VP1 gene being normalized to that of GAPDH (N = 3). The protein levels of VP1 were detected using western blot analysis. RD cells were infected with (e) EV‐A71 or (f) EV‐D68 at MOI of 0.1, 0.2, and 0.5 for 1 h, and then cells were treated with 20 μM Pte for 24 h. Densitometry of (g) EV‐A71 VP1 and (h) EV‐D68 VP1 western blot (N = 4). RD cells were mock‐infected or infected with (i) EV‐A71 or (j) EV‐D68 at a MOI of 0.1 for 1 h, and then cells were treated with Pte for 24 h. The amount of viral particles were assessed using immunofluorescence staining…

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  FIGURE 4. Time‐of‐addition and time‐of‐removal assays of pterostilbene in cells infected with EV‐A71 or EV‐D68. (a) Schematic layout of time‐of‐addition and time‐of‐removal assay with various stages of the virus life cycle. Cells were seeded in 12‐well plates and infected with (b) EV‐A71 or (c) EV‐D68 for 1 h. Following washing, for the time‐of‐addition assay, cells were treated with 20 μM of Pte at 0, 1, 3, and 5 hpi. For the time‐of‐removal assay, Pte was added at 0 hpi and subsequently removed at 0, 1, 3, and 5 hpi. At 8 hpi, the culture supernatants and cells were collected, and the viral titers were determined using the TCID50 assay. N = 4. Data are expressed as the mean ± SEM.

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  FIGURE 5. Effects and mechanisms of action of pterostilbene. (a) Schematic diagram of the attachment assay. Cells were incubated with drug‐virus mixtures at 4°C for 1 h, followed by cell harvesting for detecting viral RNA expression using RT‐qPCR. The interference effects of Pte on the attachment of (b) EV‐A71 and (c) EV‐D68 at an MOI of 5 are presented as the relative expression level compared with that of the virus control group. (d) Schematic diagram of the inactivation assay. The compound‐virus mixture was homogenized at 4°C for 1 h, followed by centrifugal filtration using a 50 kDa‐MWCO unit. The inactivation effects of Pte against (e) EV‐A71 and (f) EV‐D68 are expressed as a ratio relative to that of the virus control group at a final titer of 1 × 105 PFU/mL, as determined using TCID50. (g) Experimental procedure of the receptor pulldown assays. Viruses (TCID50 = 108) were preincubated with Pte or DMSO, and then subjected to MWCO filtration before binding to receptor protein‐conjugated beads; 1 μg of virus served as an input control. The expression of VP1 with precipitated (h) hSCARB2, (i) hPSGL‐1, and (j) hICAM‐5 was determined using western blotting. Densitometry of EV‐A71 or…

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  FIGURE 6. Docking pose analysis of pterostilbene in the VP1 hydrophobic pockets of EV‐A71 and EV‐D68. The docking sites of Pte was simulated using the VP1 hydrophobic pocket of (a) EV‐A71 (3ZFF) and (d) EV‐D68 (7TAF). Pte is shown as blue stick structures. The corresponding binding free energies (ΔG) and ‐CDOCKER energies are described in units of kcal/mol. VP1 is depicted as a grey cartoon with a hydrophobic surface (blue‐white‐brown gradient). Hydrophobic pockets are presented as brown surfaces. (b) and (e) In the key interactive residues, the labelled interacting residues on VP1 are shown as green stick structures. (c) and (f) In the 2D‐interaction diagram, the hydrophobic π‐alkyl, alkyl, π‐π stacked, and π‐sulfur interactions are drawn as pink, purple, hotpink, and yellow dashed lines, respectively. Hydrogen bonds are presented in cyan. while van der Waal forces are shown in green. VP1 is shown in cartoon representation in blue with side chains of residues forming the hydrophobic pocket shown as sticks.

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  FIGURE 7. Multiple sequence alignment of VP1 capsid proteins from EV‐A71 (PDB: 3ZFF), EV‐D68 (PDB: 7TAG), CV‐A16 (PDB: 5ABJ), and CV‐B3 (PDB: 1COV). The numbering of amino acids was according to EV‐A71 primary amino acid sequence. Dark blue amino acids indicate complete conservation, medium blue residues represent strong similarity, and light blue residues show moderate similarity in chemical properties. Pterostilbene‐binding sites within the VP1 hydrophobic pocket were marked with red boxes. This figure was generated using BIOVIA Discovery Studio 2024 (Dassault Systems, San Diego, CA, USA).

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  FIGURE 8. Oral administration of pterostilbene protects against EV‐A71 infection. (a) Schematic diagram of in vivo experiment. hSCARB2 mice (21‐d‐old) were intragrastrically (i.g.) infected with EV‐A71, and then inoculated (i.g.) once‐daily with 20 and 50 mg/kg Pte for 1 h after Day 0–6, consecutively for a total of 7 day. (b) Representative photographs of healthy mice in the EV‐A71 + Pte (50 mg/kg) group. In contrast, limb paralysis was observed among mice in the EV‐A71 alone group. (c) Body weight, (d) survival rate, and (e) clinical scores were recorded daily after infection for 14 day. Clinical scores were graded as: 0, healthy; 1, hair loss, wasting, or ruffled hair; 2, limb weakness; 3, paralysis in only one limb; 4, paralysis in 2–4 limbs; 5, death. N = 6–7. Data are expressed as the mean ± SEM. **: p < 0.01, ***: p < 0.001 versus the virus control group.