Combination of vemurafenib, pleconaril, and AG7404 attenuates enterovirus replication in vitro and in vivo

Figures

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  Figure 1.. Vemurafenib inhibits replication of some enteroviruses in cell cultures. (A) RD, RPE, or MP2 cells were treated with increasing concentrations of vemurafenib or DMSO and infected with enteroviruses (100 TCID50) or mock. The viability of the cells was determined at 48 hpi with the Cell Titer Glo assay (mean ± SD, n = 3). (B) Data presented in (A) was used to calculate EC50 values for vemurafenib. (C) Cells were treated with 3 μM vemurafenib or DMSO, infected with viruses (moi 0.1) or mock. After 48 h, cell culture supernatants were collected, and RNA was extracted. Viral RNA was analyzed by RT-qPCR (mean ± SD, n = 3). Welch’s t-test statistics (*P < 0.05, **P < 0.01).

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  Figure 2.. Drug-resistance test. (A) Schematic representation of the experiment. (B) EV1 (starting 100 TCID50) was passaged six times in RD cells in the presence of 5 μM vemurafenib or DMSO (n = 3) Viability of cells was determined using a CTG assay. (C) Cell culture supernatants were collected, and RNA was extracted and sequenced. Mutations found in EV1 genomes after 6 passages of the virus in RD cells in the presence or absence of vemurafenib were determined. (D) Some mutations were mapped onto structure of 3A protein (orange) in complex with host ACDB3 domain (blue) of Golgi resident protein GCP60 (PDB ID: 5LZ6).

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  Figure 3.. Anti-enteroviral effect of pleconaril (P), AG7404 (A), vemurafenib (V) and their combinations in RD, RPE, and MP2 cell cultures. Cells were treated with 0.2 μM P, 1 μM A, 5 μM V or their combinations, and infected with enteroviruses (100 TCID50). After 72 h, cell viability was determined using a CTG assay (mean, n = 3).

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  Figure 4.. Antiviral efficacy of combination of pleconaril (P), AG7404 (A), and vemurafenib (V) in mice and human organoids. (A) 10 mg/kg V, 5 mg/kg A, and 20 mg/kg P were administered IP 5 consecutive days to 3-week-old Swiss albino mice. CVB4 (6 × 10 6 TCID50 in 100 µl) was inoculated on Day 2 by IP route. Pancreas was recovered on Day 6. The organs were weighed and ground in 500 µl of PBS. After centrifugation, the supernatant of the crushed organs was collected for viral titration. Titers were normalized to tissue weight. Results are expressed as log10 TCID50 per gram. (B) Pancreatic organoids (hPOs) were treated with 0.1 µM P, 1 µM A, 5 µM V or their combinations, and infected with the EV1 (100 TCID50). After 72 h, CTG reagent was added, luminescence was measured. The 4 × 4 × 3 interaction landscapes were obtained for the combination, and Bliss synergy scores were calculated. (C) Retinal (hROs) and brain (hBOs) organoids were treated with 0.2 μM P, 1 μM A, 5 μM V or their combinations, and infected with EV11 (100 TCID50). After 72 h, culture supernatants were collected, and RNA was extracted. Viral RNA was analyzed by RT-qPCR (mean ± SD, n = 3); *P < 0.1, **P < 0.05, ***P < 0.01, ****P…

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  Figure 5.. Targeting multiple stages of the enterovirus life cycle is a promising strategy for broadly active therapeutic combinations. (A) Representative anti-enteroviral inhibitors. (B) These agents primarily block viral entry, translation and polyprotein processing, or RNA replication within replication organelles. (C) Several combinations show synergistic or additive effects across model systems.