Synergistic combination of orally available safe-in-man pleconaril, AG7404, and mindeudesivir inhibits enterovirus infections in human cell and organoid cultures

Abstract

Enteroviruses can infect various human organs, causing diseases such as meningitis, the common cold, hand-foot-and-mouth disease, myocarditis, pancreatitis, hepatitis, poliomyelitis, sepsis, and type 1 diabetes. Currently, there are no approved treatments for enterovirus infections. In this study, we identified a synergistic combination of orally available, safe-in-man pleconaril, AG7404, and mindeudesivir, that at non-toxic concentrations effectively inhibited enterovirus replication in human cell and organoid cultures. Importantly, the cocktail did not alter glucose and insulin levels in the culture medium of pancreatic β-cells and preserved the contraction rhythm of infected heart organoids. These findings highlight a promising drug cocktail for further preclinical studies and clinical trials targeting a broad range of enterovirus-mediated diseases.

Keywords: Antiviral drug combination; Broad-spectrum antivirals; Drug synergy; Enterovirus.

Fig. 1

Orally available triple drug combination for potential treatment of picornavirus infections. (a) Examples of picornaviruses, target organs and associated diseases. (b) Phylogenetic tree of enteroviruses used in our study. (c) The life cycle of picornavirus and inhibitors. (d) Example of published synergistic anti-enterovirus combination and orally available compound substitutes

Fig. 2

Comparison of antiviral activities of selected VP1 binders, 3 C protease and RNA synthesis inhibitors in cell cultures. Human A549, HE, MiaPaca-2 and RPE cells were treated for with increasing concentrations of compounds and infected with the CVB5, EV1, EV6, EV11 (moi 0.1) or mock. After 48 h, cell viability was determined using a CTG assay. Drug sensitivity scores (DSS) and their differences (DDSS) were calculated and shown as heatmaps (* - not determined because HE cells were not susceptible for CVB5 infection)

Fig. 3

Anti-enteroviral effect of combination of pleconaril, AG7404 and mindeudesivir in cell cultures. (a) Cells were treated with increasing concentrations of pleconaril, AG7404, mindeudesivir or their combinations, and infected with enterovirus (moi 0.1). After 48 h, cell viability was determined using a CTG assay. The 6 × 6 × 6 interaction landscapes were obtained for the combination. (b) Bliss synergy scores were calculated for most synergistic areas (MSA) and plotted

Fig. 4

Antiviral efficacy of combination of pleconaril, AG7404 and mindeudesivir in EndoC-βH5 cells. (a) Viability of mock- and virus- (moi 0.1) infected cells at 48 hpi was determined by CTG assay. (b) RT-qPCR analysis of viral RNA isolated from media of virus-infected cells. (c) Cells were treated with 0.1 µM pleconaril, 1 µM AG7404, 10 µM mindeudesivir or their combinations for and infected with the CVB5 (orange) or mock (blue). After 1 h of infection, the media was replaced with fresh media containing the drugs but not the virus. After 48 h, viability of the cells was determined using a CTG assay. (d) Cells were treated as in (c) and CVB5-infected. RT-qPCR analysis of viral RNA was performed. (e) Cells were treated as in (c). Glucose levels were measured in the cell media using glucometer. (f) Cells were treated as in (c). Human iso-insulin was measured using ELISA. (a-f) Mean ± SD, n = 3

Fig. 5

Effect of pleconaril, AG7404, and mindeudesivir combination on EV11- and mock-infected eGFP-expressing pancreatic organoids. (a) Organoids were differentiated for 4 days, treated with combination of 0.1 µM pleconaril, 5 µM AG7404, and 5 µM mindeudesivir, and infected with the EV11 or mock. After 72 h, fluorescent and bright-field microscopic images of eGFP-expressing organoids were taken. Scale bar 1 mm. (b) CTG reagent was added to the organoids and luminescence was measured. The responses were normalized to mock-infected vehicle-treated control. Mean ± SD, n = 3 (c) Median tissue culture infectious dose (TCID₅₀) was determined from organoids media obtained 72 h post infection before addition of CTG reagent (mean, technical replicate n = 2, maximum assay limit 3e⁵ TCID₅₀/mL). (d) Organoids were treated with increasing concentrations of pleconaril, AG7404, mindeudesivir or their combinations, and infected with the EV11 (moi 0.1). After 72 h, viability of virus-infected cells was determined using a CTG assay. The 4 × 4 × 4 interaction landscapes were obtained for the combination, and Bliss synergy scores were calculated

Fig. 6

Effect of pleconaril, AG7404, and mindeudesivir combination on CVB5- and mock-infected brain organoids. (a) Organoids were differentiated for 30 days, treated with combination of 0.1 µM pleconaril, 1 µM AG7404, and 10 µM mindeudesivir, and infected with the CVB5 (moi 0.1) or mock. After 1 h of infection, the media was replaced with fresh media containing the drugs but not the virus. After 72 h, CellToxGreen (CTxG, 1:1000) was added to visualize dead cells, and microscopic images of the organoids were taken. Scale bar, 1 mm. (b) Organoids were treated with 0.1 µM pleconaril, 1 µM AG7404, and 10 µM mindeudesivir or their combinations for 15 min, and infected with the CVB5 or mock. After 72 h, CTG reagent was added, luminescence was measured, and the responses were normalized to mock-infected vehicle-treated controls. Mean ± SD, n = 3. (c) Organoids were treated as in (a). After 72 h, RT-qPCR analysis of viral RNA isolated from media and organoids was performed. Mean ± SD, n = 3

Fig. 7

Effect of combination of pleconaril, AG7404, and mindeudesivir on CVB5- and mock-infected heart organoids. (a) Organoids were differentiated for 53 days, treated with combination of 0.1 µM pleconaril, 1 µM AG7404, and 10 µM mindeudesivir, and infected with the CVB5 or mock. 1 h after infection, the media was replaced with fresh media containing the drugs but not the virus. After 72 h, microscopic images of organoids were taken. Scale bar, 400 mm. (b) Differentiated organoids were treated with 0.1 µM pleconaril, 1 µM AG7404, and 10 µM mindeudesivir or their combinations for 15 min, and infected with the CVB5 or mock. After 72 h, movies of virus- and mock-infected organoids were recorded and transformed into cardiograms. (c) Organoids were treated and infected as in (a). After 72 h, RT-qPCR analysis of viral RNA isolated from media of CVB5-infeced organoids was performed. Mean ± SD, n = 3

Fig. 8

Effect of pleconaril, AG7404, and mindeudesivir combination on CVB5- and mock-infected eye organoids. (a) Organoids were differentiated for 20 days, treated with combination of 0.1 µM pleconaril, 1 µM AG7404, and 10 µM mindeudesivir, and infected with the CVB5 or mock. 1 h after infection, the media was replaced with fresh media containing the drugs but not the virus. After 72 h, microscopic images of organoids were taken. Scale bar, 400 mm. (b) Organoids were treated with 0.1 µM pleconaril, 1 µM AG7404, and 10 µM mindeudesivir or their combinations for 15 min, and infected with the CVB5 or mock. After 72 h, CTG reagent was added, luminescence was measured and the responses were normalized to mock-infected, non-treated controls. Mean ± SD, n = 3. (c) Organoids were treated and infected as in (a). After 72 h, RT-qPCR analysis of viral RNA isolated from media of CVB5-infeced organoids was performed. Mean ± SD, n = 3