Novel Anti-Enterovirus A71 Compounds Discovered by Repositioning Antivirals from the Open-Source MMV Pandemic Response Box

Abstract

The open-source drug library, namely, MMV Pandemic Response Box, contains 153 antiviral agents, a chemically and pharmacologically diverse mixture of early-stage, emerging anti-infective scaffolds, and mature compounds currently undergoing clinical development. Hence, the Pandemic Response Box might contain compounds that bind and interfere with target molecules or cellular pathways that are conserved or shared among the closely related viruses with enterovirus A71 (EV-A71). This study aimed to screen antiviral agents included in the Pandemic Response Box for repurposing to anti-EV-A71 activity and investigate the inhibitory effects of the compounds on viral replication. The compounds' cytotoxicity and ability to rescue infected cells were determined by % cell survival using an SRB assay. The hit compounds were verified for anti-EV-A71 activity by virus reduction assays for viral RNA copy numbers, viral protein synthesis, and mature particle production using qRT-PCR, Western blot analysis, and CCID₅₀ assay, respectively. It was found that some of the hit compounds could reduce EV-A71 genome replication and protein synthesis. D-D7 (2-pyridone-containing human rhinovirus 3C protease inhibitor) exhibited the highest anti-EV-A71 activity. Even though D-D7 has been originally indicated as a polyprotein processing inhibitor of human rhinovirus 3C protease, it could be repurposed as an anti-EV-A71 agent.

Keywords: antivirus drugs; enterovirus; enterovirus A71; hand-foot-mouth disease; repurposed drugs.

Figure 1

The results of the cytotoxicity test of the selected 18 compounds. RD cell monolayers were incubated with 1 μM of the tested compounds for 24 h. The names of the compounds were according to the location in the plate layout of the Pandemic Response Box (plate-well number). DMEM and DMSO were cell culture medium alone and supplemented with DMSO, served as maximum % cell survival and vehicle control, respectively. Cytotoxicity of the compounds was determined by SRB assay and calculated for % cell survival relative to the maximum % cell survival control. The % cell survival values are derived from three independent experiments and presented in Box-and-whisker and dot plots. Each dark grey circle represents the individual data points, the middle bar is the median, and the boxes represent quartile data distribution. The dashed line represents the cut-off point derived from a mean − 1SD of % cell survival of the DMSO controls.

Figure 2

Evaluation of potential anti-EV-A71 activity of the selected 18 antiviral compounds from the MMV Pandemic Response Box. RD cell monolayers were infected with EV-A71 at 1 M.O.I. by adsorbing with the virus inoculum for 1 h and then incubated with fresh medium containing 1 μM of the tested compounds for 24 h. The D-A7 (rupintrivir) and D-H7 (T-1106) compounds served as positive anti-EV-A71 activity control. The infected RD cell monolayers incubated with cell culture medium supplemented with DMSO (infDMSO) served as negative anti-EV-A71 activity control. Uninfected RD cells incubated with cell culture medium alone (DMEM) and DMSO served as maximum % cell survival and vehicle control, respectively. The SRB assay determined % cells rescued from EV-A71-induced cytopathic effect (CPE) and was calculated for % cell survival relative to the maximum % cell survival control. The % cell survival values are derived from three independent experiments and presented in Box-and-whisker and dot plots as described in Figure 1. The dashed line represents the cut-off point relative to the positive anti-EV-A71 activity control (D-H7).

Figure 3

The hit compounds were verified for anti-EV-A71 activity by virus reduction assays for viral RNA copy numbers (A,B), viral protein synthesis (C), and mature particle production (D) using qRT-PCR, Western blot analysis, and CCID₅₀ assay, respectively. The infected cells incubated with a cell culture medium supplemented with DMSO (infDMSO) were used as negative inhibition control and for comparison. The uninfected cells served as negative EV-A71 infection control (Uninfected). The log₁₀ EV-A71 RNA copy numbers μL⁻¹ and log₁₀ CCID₅₀/0.1 mL values of (A,B,D) are the means of three independent experiments. Error bars represent standard deviations. Each dark grey circle represents the individual data points. The VP0 fold-change values of (C) are representative of two independent experiments. Lane M is a protein marker. β-actin bands are used for protein normalization and internal loading control. Statistical analysis is the one-way analysis of variance (one-way ANOVA) with Tukey–Kramer pair-wise comparison and independent samples t-test as necessary. *, **, and *** represent a statistically significance difference at p-value < 0.05, p-value < 0.001, and p-value < 0.0001, respectively. ns indicates no significance.