Journal: eBioMedicine

Article Title: Tanomastat exerts multi-targeted inhibitory effects on viral capsid dissociation and RNA replication in human enteroviruses

doi: 10.1016/j.ebiom.2024.105277

Figure Lengend Snippet: Tanomastat targets EV-A71 RNA replication. ( a ) RD cells were infected with EV-A71 at M.O.I of 1 at 4 °C. After pre-adsorption, the infected cells were incubated for 2 h at 37 °C and post-treated with 0.1% DMSO vehicle control or Tanomastat at the relevant concentrations (from 1 μM to 50 μM). ( b ) In entry-bypass assay, RD cells were transfected with EV-A71 RNA and treated with 0.1% DMSO vehicle control or Tanomastat at the relevant concentrations (from 10 μM to 50 μM). In a and b , the infectious virus titres were determined by viral plaque assay. ( c–e ) RD cells were infected with EV-A71 at M.O.I of 1 and post-treated with 0.1% DMSO vehicle control or Tanomastat at the relevant concentrations (from 1 μM to 40 μM). Protein bands were separated by SDS-PAGE, followed by Western blot analysis using anti-EV-A71 VP2 monoclonal antibody, anti-EV-A71 VP1 polyclonal antibody, and anti- β -Actin monoclonal antibody. Band intensities below the detectable limit are denoted as not detectable (n.d.). The relative VP2 and VP1 band intensities were normalized against β -Actin. Results are representative of two independent experiments. ( f and g ) RD cells were transfected with EV-A71 3D polymerase replication competent or defective RNA replicons and treated with Tanomastat at the relevant concentrations (from 1 μM to 10 μM). 0.1% DMSO, CHX and GuHCl served as vehicle, general translation inhibitor and RNA replication-specific inhibitor controls, respectively. ( h ) RD cells were transfected with the EV-A71 bicistronic luciferase construct and treated with Tanomastat at the relevant concentrations (from 10 μM to 50 μM). 0.1% DMSO and apigenin served as vehicle and EV-A71 IRES translation inhibitor controls, respectively, Luminescence readings were used to derive the normalized F Luc/R Luc ratio, which is reflective of IRES activity. Each data point denotes the mean of triplicates, and the error bar denotes the standard deviation. One-way ANOVA followed by Dunnett’s test was used to determine the statistical significance of the treatments when compared against 0.1% DMSO vehicle control. P-values, mean difference, and 95% CI are reported in Supplementary Table S5 . Bar graphs represent mean ± standard deviation with ∗ P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Article Snippet: After blocking with 2% bovine serum albumin (BSA) (Sigma–Aldrich) dissolved in Tris-buffered saline-Tween 20, the membrane was incubated in the following primary antibodies: mouse anti-EV-A71 VP2 (Merck Millipore Cat#MAB979, RRID:AB_95300), rabbit anti-EV-A71 VP1 (Genetex Cat#GTX132339, RRID:AB_2886617), mouse anti-MMP-9 (Thermo Fisher Scientific Cat#MA5-15886, RRID:AB_11157246), and mouse anti- β -Actin (Sigma–Aldrich Cat#A2228, RRID:AB_476697).

Techniques: Infection, Adsorption, Incubation, Control, Transfection, Virus, Viral Plaque Assay, SDS Page, Western Blot, Luciferase, Construct, Activity Assay, Standard Deviation

Journal: eBioMedicine

Article Title: Tanomastat exerts multi-targeted inhibitory effects on viral capsid dissociation and RNA replication in human enteroviruses

doi: 10.1016/j.ebiom.2024.105277

Figure Lengend Snippet: Tanomastat displays in vivo prophylactic efficacy and viral load inhibition at tested drug doses in sucking BALB/c mice challenged with lethal dose of EV-A71. ( a ) 5-day-old BALB/c neonatal mice were infected with EV-A71 at a dose of 2 × 10 7 per mice via i.p. At 0 d.p.i, a single dose of 10 mg/kg or 30 mg/kg Tanomastat was administered via oral gavage 2 h pre-infection. A second dose was administered 24 h post-infection, and subsequent doses were administered daily up to 120 h.p.i. DMSO was used as treatment vehicle control. The ( b ) percent survival, ( c ) body weight, and ( d ) clinical scorings were recorded for up to 14 d.p.i. EV-A71-infected mice were sacrificed on 5 d.p.i for quantification of viral titres in ( e ) brain and ( f ) hind limb muscle tissues, as well as for histopathology evaluation in spinal cord and hind limb muscle tissues. H&E staining showed severe necrosis in ( g ) DMSO control-treated mice, whereas mild necrosis was observed in 10 mg/kg and 30 mg/kg Tanomastat-treated mice. IHC staining muscle tissue using anti-EV-A71 VP2 monoclonal antibody showed extensive antigen positive in ( h ) DMSO control-treated mice, whilst similarly low antigen distribution was present in 10 mg/kg and 30 mg/kg Tanomastat-treated mice. Mantel–Cox test or Kruskal–Wallis test was used to determine the statistical significance of the treatments when compared against DMSO vehicle control. P-values, HR, and 95% CI are reported in Supplementary Table S7 . Horizontal lines in scatter dot plots represent geometric mean ± geometric standard deviation with ∗ P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Data displayed are representative images of each group (n ≥ 7). Magnification for H&E and IHC staining are conducted at 10X.

Article Snippet: After blocking with 2% bovine serum albumin (BSA) (Sigma–Aldrich) dissolved in Tris-buffered saline-Tween 20, the membrane was incubated in the following primary antibodies: mouse anti-EV-A71 VP2 (Merck Millipore Cat#MAB979, RRID:AB_95300), rabbit anti-EV-A71 VP1 (Genetex Cat#GTX132339, RRID:AB_2886617), mouse anti-MMP-9 (Thermo Fisher Scientific Cat#MA5-15886, RRID:AB_11157246), and mouse anti- β -Actin (Sigma–Aldrich Cat#A2228, RRID:AB_476697).

Techniques: In Vivo, Inhibition, Infection, Control, Histopathology, Staining, Immunohistochemistry, Standard Deviation