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. 2019 Nov 8;5(11):1952-1962.
doi: 10.1021/acsinfecdis.9b00284. Epub 2019 Sep 18.

A Novel Capsid Binding Inhibitor Displays Potent Antiviral Activity against Enterovirus D68

Chunlong Ma  1 , Yanmei Hu  1 , Jiantao Zhang  1 , Rami Musharrafieh  1 , Jun Wang  1
Affiliations

A Novel Capsid Binding Inhibitor Displays Potent Antiviral Activity against Enterovirus D68

Chunlong Ma et al. ACS Infect Dis. .

Abstract

Enterovirus D68 (EV-D68) is a respiratory viral pathogen that primarily infects children under the age of 8. Although EV-D68 infection typically leads to moderate to severe respiratory illnesses, recent years have seen increasing cases of EV-D68 triggered neurological complications such as acute flaccid myelitis (AFM). There is currently no vaccine or antiviral available for EV-D68; we therefore aimed to develop potent and specific small molecule antivirals against EV-D68. In this study, we report our discovery of a viral capsid inhibitor R856932 that inhibits multiple contemporary EV-D68 strains with single-digit to submicromolar efficacy. Mechanistic studies have shown that the tetrazole compound R856932 binds to the hydrophobic pocket of viral capsid protein VP1, thereby preventing viral uncoating and release of viral genome in the infected cells. The mechanism of action of R856932 was confirmed by time-of-addition, Western blot, RT-qPCR, viral heat inactivation, serial viral passage, and reverse genetics experiments. A single mutation located at VP1, A129V, confers resistance against R856932. However, a recombination virus encoding VP1-A129V appeared to have compromised fitness of replication compared to the wild-type EV-D68 virus as shown by the competition growth assay. Overall, the hit compound identified in this study, R856932, represents a promising starting point with a confirmed mechanism of action that can be further developed into EV-D68 antivirals.

Keywords: EV-D68; antiviral; capsid inhibitor; enterovirus; pleconaril.

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Figures

Figure 1.
Figure 1.
Chemical structures of R856932, pleconaril, and telaprevir. Pleconaril is a known EV-D68 viral VP1 capsid binding inhibitor, and telaprevir is a EV-D68 2A protease inhibitor.
Figure 2.
Figure 2.. Compound R856932 reduced the EV-D68 viral RNA and protein levels.
(A-D) R856932 reduced the EV-D68 viral RNA and protein levels. RD cells were infected with EV-D68 strain US/MO/14–18947 (MOI = 1) with treatment of the indicated compound. At 9 hpi, cells were harvested for viral RNA quantification by RT-qPCR (A and B) or viral protein quantification by western blot (C and D). Asterisks indicate a statistically significant difference in comparison with the DMSO control (one-way analysis of variance analysis by Prism 5, *** P < 0.001). The images were representatives of three independent experiments. (E) R856932 inhibited EV-D68 VP1 synthesis in immunofluorescence assay. RD cells infected with EV-D68 strain US/MO/14–18947 (MOI = 1) were fixed at 9 hpi and stained with anti-VP1 and DAPI for detecting VP1 and nucleus, respectively. Drug concentrations used were shown in the figure.
Figure 3.
Figure 3.. Time-of-addition experiments by quantify progeny virus yield in cell culture supernatant.
RD cells were infected with EV-D68 US/MO/14–18947 at MOI = 0.01 at 0h time-point. At 1 hpi, inoculant virus was removed and washed with PBS buffer, progeny viruses in cell culture supernatant were harvested at 14 hpi and were quantified with plaque assay. Red arrow represents the period of time that drug (compound R856932 or pleconaril) was present in the cell culture. The concentrations used for R856932 and pleconaril were 25 μM and 12.5 μM, respectively. Asterisks indicate a statistically significant difference in comparison with the DMSO control (one-way analysis of variance analysis by Prism 5, *** P < 0.001). The value is the mean of two independent experiments ± S.D.
Figure 4:
Figure 4:. Time-of-addition experiments by immunostaining VP1 protein inside the host cells.
RD cells were infected with EV-D68 US/MO/14–18947 at MOI = 1 at 0 h time-point, and at 1 hpi, inoculant virus was removed and washed with PBS buffer. A) Pleconaril or B) Compound R856932 was applied at −1, 0 or 2 hpi to 8 hpi; for the 0h–1h condition, Pleconaril or compound R856932 was only present during the infection stage for one hour, then pleconaril or compound R856932 was washed away and fresh media was applied. The concentrations used for R856932 and pleconaril were 50 μM and 12.5 μM, respectively. Cells were fixed at 8 hpi and viruses were detected via staining VP1 protein with anti-VP1. The images were representatives of three independent experiments.
Figure 5.
Figure 5.. R856932 reduced the EV-D68 viral attachment to host cells (A and B)
Pre-cooled RD cells were infected with EV-D68 strain US/MO/14–18947 (MOI of 30) and were incubated at 4 °C for 2 h. After removing unbound viruses by washing with ice-cold PBS, the attached viruses on cell surface were quantitated by real-time PCR (A) or visualized by immunostaining (B). Asterisks indicate a statistically significant difference in comparison with the DMSO control (one-way analysis of variance analysis by Prism 5, *** P < 0.001). The value is the mean of three independent experiments ± S.D. The images were representatives of three independent experiments. Drug concentrations used were shown in the figure.
Figure 6.
Figure 6.. Thermo-protection assay showing that R856932 prevented EV-D68 US/MO/14–18947 (A) and US/MO/14–18949 viruses (B) from heat inactivation.
Protective effects of pleconaril or compound R856932 on heat inactivation of EV-D68 US/MO/14–18947 (A) and US/MO/14–18949 viruses (B). 12.5 μM Pleconaril or 50 μM compound R856932 or DMSO was incubated with viruses (2×10^6 pfu/ml) for 30 minutes. The virus/compound mixture was then heated for 2 minutes at temperature ranging from 37 to 57 °C, followed by a rapid cool down to 4 °C. The resulting infectious virus was quantified via plaque assay using RD cells. The values are the mean ± standard deviation from three replicates.
Figure 7.
Figure 7.. Molecular docking of R856932 in EV-D68 viral capsid protein VP1.
(A) X-ray crystal structure of EV-D68 (Fermon CA62–1) capsid proteins in complex with pleconaril (PDB: 4WM7). VP1, VP2 and VP3 were colored as green, cyan, and grey, respectively. R856932-selected resistant mutants VP1-A129V, VP1-A45V, and VP2-T139A, as well as the pleconaril-resistant mutant VP1-V81A, were labeled and the side chains were shown as spheres. (B) Ligand interaction diagram of R856932 in the pleconaril binding site in VP1. The figure was generated in Schrödinger Glide. Note that A117 in the crystal structure corresponds to A129 in the conventional numbering. (C) Docking model of R856932 in VP1. VP1-A129 side chain was shown as spheres. (D) Overlay of R856932 and pleconaril in the canyon region of VP1. Docking was performed using Schrödinger Glide standard precision.
Figure 8.
Figure 8.. Representative plaque reduction assay with compound R856932 against recombinant rMO WT, rMO VP1-A129V and rMO VP2-T139A viruses.
Approximately 100 pfu/well of recombinant rMO WT, rMO VP1-A129V or rMO VP2-T139A viruses were applied to RD cell monolayers. 0.1 to 10 μM compound R856932, or 0.1 or 1.0 μM pleconaril was present in the 1.2% avicel overlay. RD cells were stained with crystal violet 3 days after infection. The images were representatives of three independent experiments.
Figure 9.
Figure 9.. Competition growth assay to assess the replication fitness of rMO VP1-A129V virus.
280 PFU rMO WT and 28,000 PFU rMO VP-A129V viruses (ratio = 1/100) were used to infect RD cells in a T25 flask. Every 2 days after infection, culture media supernatant was harvested and viruses were quantified with plaque assay, and 28,000 PFU progeny viruses were used for the next round of infection. The VP1 coding sequences were determined in each round of passage. The percentage of each viruses was estimated by measuring the height of the nucleotide sequence electropherogram peak as shown in the bar graph.
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