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 dose-dependent inhibition against a broad range of enteroviruses in vitro . ( a ) Chemical structure of Tanomastat. ( b ) RD cells were treated with Tanomastat at the relevant concentrations (from 10 μM to 200 μM) to determine the cytotoxicity profile. 0.1% DMSO was used as vehicle control. Cell viability is expressed as a percentage relative to 0.1% DMSO vehicle control. The dashed line represents the 80% relative cell viability threshold which is indicative of non-cytotoxicity. In post-infection treatment assays, RD cells were infected with ( c ) EV-A71 ( d ) EV-A71 (Strain H), ( e ) EV-A71 (Genotype B5), ( f ) EV-A71 (Genotype C4), ( g ) CV-A6, ( h ) CV-A16, ( i ) CV-B5, ( j ) ECHO-7, ( k) CV-A24, and ( l ) EV-D68 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 50 μM). The total infectious virus titres were determined by viral plaque assay. Virus titres below the detectable limit are denoted as not detectable (n.d.). 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 S2 . Bar graphs represent mean ± standard deviation with ∗ P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Article Snippet: Tanomastat (Cayman Chemical) was reconstituted in 100% DMSO to an initial 25 mg/mL stock concentration and stored at −20 °C for in vivo experiments in mice.

Techniques: Inhibition, In Vitro, Control, Infection, Virus, Viral Plaque 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 targets viral internalization, an early stage during EV-A71 replication. In time-of-addition and removal studies, RD cells were infected with EV-A71 at M.O.I of 1 and treated with ( a ) 0.1% DMSO vehicle control or ( b ) 25 μM Tanomastat at 2-h intervals from 0 to 10 h time points. ( c ) In pre-infection treatment assay, RD cells were pre-treated with 0.1% DMSO vehicle control or Tanomastat at the relevant concentrations (from 0.1 μM to 50 μM) and infected with EV-A71 at M.O.I of 1. ( d ) In co-infection treatment assay, EV-A71 virus particles were pre-incubated with 0.1% DMSO vehicle control or Tanomastat at the relevant concentrations (from 0.1 μM up to 50 μM), and then used to infect RD cells at M.O.I of 1. In a–d , the infectious virus titres were determined by viral plaque assay. ( e and f ) In virus attachment assay, EV-A71 virus particles were pre-incubated with 0.1% DMSO vehicle control or Tanomastat at the relevant concentrations (5 μM and 50 μM), and then used to infect RD cells at M.O.I of 1 at 4 °C. ( g and h ) In virus internalization assay, RD cells were infected with EV-A71 at M.O.I of 1 at 4 °C and treated with 0.1% DMSO vehicle control or Tanomastat at the relevant concentrations (5 μM and 50 μM) at 37 °C. In e–h , the relative fold change in VP1 gene expression and infectious virus titres were determined by RT-qPCR and viral plaque assay, respectively. The relative fold change in VP1 gene expression was normalized against β -Actin. 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 S3 . Line and bar graphs represent mean ± standard deviation with ∗ P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Article Snippet: Tanomastat (Cayman Chemical) was reconstituted in 100% DMSO to an initial 25 mg/mL stock concentration and stored at −20 °C for in vivo experiments in mice.

Techniques: Infection, Control, Virus, Incubation, Viral Plaque Assay, Gene Expression, Quantitative RT-PCR, 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 limits capsid-viral RNA dissociation upon internalization of EV-A71 into RD cells. ( a ) Time-course live cell assay of capsid-viral RNA dissociation was observed for a prolonged period of 60 min post-infection at M.O.I of 20. Tracking of single viral capsid showed dissociation of the capsid (green) and release of viral RNA (red). Overlapping viral RNA and capsid (yellow) indicates the start of dissociation. The purple/white arrows over the time course tracks a single virus particle up to capsid-viral RNA dissociation. ( b ) Snapshot of time-course live cell assay of capsid and viral RNA intracellular at 20-, 30-, and 60-min post-infection at M.O.I of 20.10 μM Pleconaril served as capsid-viral RNA dissociation inhibitor positive control. Images were processed using Imaris 10·1 software indicated by capsid (green), viral RNA (red), and nucleus (blue). ( c ) Capsid-viral RNA dissociation is indicated by colocalization of capsid to viral RNA with a distance of less than or equal to 1 μm (white arrow). ( d ) Mean of viral RNA, capsid and viral particle dissociation counts for 0·1% DMSO, positive control, pleconaril, and 50 μM Tanomastat treatment upon infection at M.O.I of 20. ( e ) 0·1% DMSO, positive control, pleconaril, and 50 μM Tanomastat treatment upon infection at M.O.I of 20. Green arrow indicates lower or higher mean viral RNA to mean viral capsid counts. Two-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 S4 . Line graphs represent mean ± standard deviation with ∗ P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Article Snippet: Tanomastat (Cayman Chemical) was reconstituted in 100% DMSO to an initial 25 mg/mL stock concentration and stored at −20 °C for in vivo experiments in mice.

Techniques: Infection, Virus, Positive Control, Software, Control, 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 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: Tanomastat (Cayman Chemical) was reconstituted in 100% DMSO to an initial 25 mg/mL stock concentration and stored at −20 °C for in vivo experiments in mice.

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 does not display in vivo toxicity at tested drug doses. 5-day-old BALB/c neonatal mice were challenged with 10 mg/kg and 30 mg/kg Tanomastat via oral gavage daily for six days. DMSO was used as treatment vehicle control. The ( a ) percent survival, ( b ) body weight, and ( c ) clinical scores were recorded daily for up to 15 days. One-way ANOVA followed by Dunnett’s test was used to determine the statistical significance of the treatments when compared against DMSO vehicle control, with ∗ P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Article Snippet: Tanomastat (Cayman Chemical) was reconstituted in 100% DMSO to an initial 25 mg/mL stock concentration and stored at −20 °C for in vivo experiments in mice.

Techniques: In Vivo, Control

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: Tanomastat (Cayman Chemical) was reconstituted in 100% DMSO to an initial 25 mg/mL stock concentration and stored at −20 °C for in vivo experiments in mice.

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