Introduction

Enterovirus 71 (EV71) is a single-stranded, positive-sense, single-stranded RNA virus that belongs to the species Enterovirus alphacoxsackie of the family Picornaviridae. Coxsackieviruses A16 (CV-A16), CV-A6, and EV71 are the most common causative pathogens causing hand, foot, and mouth disease (HFMD) in infants and children younger than five years of age [52]. However, different viruses and subgenotypes predominate in different areas [5, 7, 22, 57]. The clinical presentation ranges from asymptomatic infection to mild HFMD, including fever, skin eruptions on the hands and feet, and vesicles in the mouth. However, EV71 infection has also been implicated in causing severe neurological complications, including meningitis, encephalitis, acute flaccid paralysis, cardiopulmonary failure, and even death [29, 68]. EV71 is a highly transmissible virus. Transmission of the virus from person to person occurs by direct contact with secretions such as saliva, fluid from blisters, or via the fecal-oral route. EV71 was first isolated from patients with neurological disease in California in 1969. Recently, outbreaks of EV71 infection have occurred periodically worldwide, particularly in the Asia-Pacific region [44]. According to a recent study conducted in Bangkok, Thailand, EV71 is the most common virus causing HMFD in kindergartens [57].

To date, three inactivated EV71 vaccines have been licensed in China, and several multivalent vaccines are under development [30]. However, there are no specific antiviral agents for EV71 treatment; only supportive therapy is used as the primary treatment in severe cases. Although several antiviral agents and small compounds, including lactoferrin, pleconaril, and rupintrivir, have been reported to have an inhibitory effect against EV71, none of them has been shown to be clinically efficacious [28, 34, 60, 73, 74]. Therefore, the development of effective antiviral agents targeting EV71 is still needed.

The use of repurposed drugs is an attractive antiviral strategy because their safety profiles are already available [16]. Several FDA-approved drugs with broad-spectrum antiviral activity have been selected to evaluate their inhibitory effect against EV71. In particular, drugs that showed inhibitory effects on positive-sense RNA viruses, including flaviviruses, togaviruses, and coronaviruses, can also inhibit EV71, which is also a positive-sense RNA virus [16, 31]. In this study, the anti-EV71 activity of several FDA-approved drugs was evaluated in vitro. Furthermore, to improve antiviral activity and possible efficacy, a drug combination approach was evaluated. Combining different drugs has been a successful strategy for the treatment of various viral infections, such as hepatitis C virus (HCV) and human immunodeficiency virus 1 (HIV-1) infections [1, 43, 62].

Materials and methods

Chemicals

Niclosamide (N3510, Sigma), nitazoxanide (N0290, Sigma), temsirolimus (PZ0020, Sigma), prochlorperazine (P9178, Sigma), itraconazole (I6657, Sigma), mycophenolic acid (M3536, Sigma), sofosbuvir (HY-15005, MCE), 2,4-dinitrophenol (34334, Sigma), paromomycin (P5057, Sigma), and triparanol (T5200, Sigma) were dissolved in dimethyl sulfoxide (DMSO; Sigma) to make stock solutions with a concentration of 10 mM. Metformin (HY-17471A, MCE) was dissolved in water at a concentration of 10 mM. All stock solutions were stored at -80°C.

Immediately before use, the stock solutions were diluted to their working concentrations in culture medium containing 2% fetal bovine serum (FBS) (2% FBS-MEM or 2% FBS-DMEM/F12 + Opti-MEM). The final concentration of DMSO in all experiments was 0.1%.

Cells and viruses

Vero cells (CCL-81, ATCC) were maintained in minimum essential medium (MEM; Gibco) supplemented with 10% heat-inactivated FBS (MEM-10%FBS) at 37°C with 5% CO2. The human neuroblastoma cell line SH-SY5Y (CRL-2266, ATCC) was maintained in Dulbecco’s modified Eagle medium/nutrient mixture F-12 (DMEM/F-12; 11320033, Gibco) and Opti-MEM reduced serum medium (11058021, Gibco) in a 1:1 ratio supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco) at 37°C with 5% CO2. The EV71 subgenotype C4 strain used in this study was kindly provided by Prof. Pilaipan Puthavathana [38] and propagated in Vero cells. Viral supernatants were harvested at 3 days postinfection, centrifuged to remove cell debris, and stored at -80°C. The virus titer of the stock virus was determined in Vero cells, using a plaque assay.

Enterovirus 71 infection

On the day before the experiment, Vero and SH-SY5Y cells were seeded in 96-well plates at a density of 3 × 104 and 2.5 × 104 cells/well, respectively. The next day, the culture medium was replaced with 2% FBS medium containing EV71 subgenotype C4 strain at the indicated multiplicity of infection (MOI) or 2% FBS medium without virus for mock infection for 1 hour at 37°C with 5% CO2. After incubation, the inoculum was removed and the cells were further cultured in 2% FBS-MEM or 2% FBS-DMEM/F12 + opti-MEM for Vero and SH-SY5Y cells, respectively.

Cell viability assay

Vero and SH-SY5Y cells were seeded in 96-well plates at a density of 3 × 104 and 2.5 × 104 cells/well, respectively. The cells were then treated with twofold serially diluted drugs in 2% FBS medium for 48 hours. Cell viability was determined using PrestoBlue Cell Viability Reagent (Invitrogen). After adding the cell-permeable resazurin-based (nonfluorescent) reagent, the reducing environment in viable cells converts resazurin to resorufin, which emits a red fluorescent signal that is detectable with an excitation wavelength of 560 nm and an emission wavelength of 590 nm. Alternatively, MTT dyes (Invitrogen) were also used. Live cells convert 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide to MTT formazan. MTT formazan can be detected by measuring absorbance at 570 nm after dissolving the sample in DMSO. Cells treated with 0.1% DMSO were used as untreated controls. GraphPad Prism 9 (GraphPad Software, Inc., CA) was used for non-linear regression analysis and determination of the 50% cytotoxic concentration (CC50).

Viral quantification using a plaque assay

Before infection, Vero cells were seeded in 24-well plates at a density that allowed them to grow to 100% confluency within 24 hours. The next day, the cells were inoculated with a tenfold serial dilution of viral samples for 1 hour at 37°C with 5% CO2. After incubation, the inoculum was removed, and the cells were overlaid with 1.2% microcrystalline cellulose (Avicel, RC-591) in 2% FBS-MEM and incubated at 37°C and 5% CO2 for three days. The overlayed Avicel was then removed, and the plates were fixed with 10% formalin (v/v) in phosphate-buffered saline (PBS) for 1–2 hours. The plates were then rinsed with tap water and stained for 5 minutes with 1% crystal violet (w/v) in 20% (v/v) ethanol. The plaques were counted to calculate the viral titers in plaque-forming units per milliliter (pfu/ml).

Drug treatments

Anti-EV71 activity screening

Eleven FDA-approved drug candidates were selected based on a review of the literature. Vero cells were seeded at a density of 3 × 104 cells/well in 96-well plates. Cells were pretreated with each drug at a concentration of 10 µM for 1 hour at 37°C with 5% CO2. The treated cells were then inoculated with EV71 subgenotype C4 at an MOI of 0.01 for 1 hour. The supernatant was then removed, and the cells were maintained in the medium with the drug for two days. The antiviral effect was determined based on the observed cytopathic effect (CPE).

Dose-response curves of single drug treatment

Prior to the day of drug treatments, Vero and SH-SY5Y cells were seeded in 96-well plates at a density of 3 × 104 and 2.5 × 104 cells/well, respectively. Each drug was serially diluted twofold and incubated with the cells for 1 hour. Subsequently, Vero and SH-SY5Y cells were infected with EV71 subgenotype C4 at an MOI of 0.01 and 2, respectively, for 1 hour. The inoculation supernatant was then removed, and the cells were further cultured in the medium containing the serially diluted drugs for two days. The virus titers were determined using a plaque assay. The half-maximal inhibitory concentration (IC50) was calculated from the dose-response curves.

Dose-response curves of two-drug combinations

Cells were pretreated with a series of serial twofold dilutions of the two-drug combinations for 1 hour at 37°C with 5% CO2, followed by infection with EV71 as described above. At 2 days postinfection, virus supernatants were collected and virus titers were measured by plaque assay.

Evaluation of two-drug combination treatments

The day before the experiment, Vero and SH-SY5Y cells were seeded in 96-well plates at a density of 3 × 104 and 2.5 × 104 cells/well, respectively. The cells were then pretreated with 16 or 20 different pairwise combinations of two drugs for 1 hour. The concentrations of each drug were 2, 1, 0.5, 0.25, and 0.125 times its IC50 value. Vero and SH-SY5Y cells were then infected for 1 hour with EV71 subgenotype C4 at an MOI of 0.01 and 2, respectively. The virus inoculum was then removed, and the cells were cultured further in the presence of the drugs for two days. The virus titers were determined by plaque assay.

Analysis of the interaction between two drugs

Drug interaction was analyzed using the SynergyFinder web application (https://synergyfinder.fimm.fi). We used four different reference models: the Loewe additivity (Loewe), zero independence potency (ZIP), highest single agent (HSA), and Bliss independence models [67]. Synergy scores less than − 10 indicated an antagonistic interaction, scores between − 10 and 10 indicated an additive effect, and scores greater than 10 indicated a synergistic effect between two drugs. The synergy score for each pair is presented as the mean and confidence interval.

Western blot analysis

Infected cells were lysed in RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 1 mM sodium orthovanadate, and 1 mM NaF). Cell debris was removed by centrifugation at 12,000 × g for 10 min at 4°C. The cell lysate was then transferred to a new tube, and the protein concentration was measured by the Bradford method. The lysate was loaded onto a NuPAGE 10% Bis-Tris protein gel (1.0 mm) (NP0302BOX; Thermo Fisher Scientific), and after electrophoresis, the proteins were transferred to a nitrocellulose membrane (PALL) by diffusion blotting. The membrane was were then blocked with 3% (w/v) skim milk in 0.1% Tween 20 (v/v)-TBS for 1 hour at room temperature and then probed with a 1:1000 dilution of rabbit anti-Akt (#9272), anti-phospho-Akt (#4060), anti-phospho mTOR (#2971), anti-mTOR (#2983), and anti-EV71 (MAB979, Millipore) or a 1:10,000 dilution of antibody against 3-phosphate dehydrogenase (GAPDH, sc-32233; Santa Cruz Biotechnology, Inc.) at 4°C overnight with continuous rocking. The membrane was washed and incubated with a 1:2000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG or HRP-conjugated rabbit anti-mouse IgG for 2 hours at room temperature. Protein bands were visualized by incubating the membrane with Clarity Western ECL substrate (Bio-Rad Laboratories, Hercules, CA, USA) and photographed using ImageQuant LAS 4010 (GE Healthcare, Chicago, IL, USA). The protein band intensities were determined using ImageJ software (U.S. National Institutes of Health, Bethesda, MD, USA) and normalized to the GAPDH expression level. The ratios of p-AKT/AKT and p-mTOR/mTOR were calculated based on the GAPDH-normalized expression level.

Statistical analysis

The experiments were carried out in triplicate. Percent inhibition and protein band intensity values are shown as the mean ± SD. Comparisons between groups were made using an independent-sample t-test in SPSS Statistics software (SPSS, Inc., Chicago, IL, USA). A p-value ≤ 0.05 was considered statistically significant. Nonlinear regression analysis in GraphPad Prism 9 (GraphPad Software, Inc., CA) was used to determine IC50 and CC50 values.

Results

Antiviral activity of drug candidates against EV71 subgenotype C4 in Vero cells

FDA-approved drugs with antiviral activity were selected for the study of their antiviral activity against EV71. These included prochlorperazine [6, 17, 51], itraconazole [15, 53], niclosamide [23, 26, 33, 61, 64], nitazoxanide [20, 42, 46, 47, 50, 61], temsirolimus [13], mycophenolic acid [3, 10, 18], sofosbuvir [14, 48], metformin [8, 11, 45, 66], 2,4 dinitrophenol [55], paromomycin [19, 56], and triparanol [24].

The antiviral activity against EV71 of the selected drugs at a concentration of 10 µM was tested in Vero cells. A compound was judged to have an inhibitory effect if it resulted in a decrease in the virus-induced cytopathic effect (CPE). Using a 50% CPE reduction threshold, significant antiviral activity was observed only for prochlorperazine and itraconazole. Niclosamide at a concentration of 10 µM was toxic to the cells; therefore, the CPE assay could not be used to determine its antiviral activity. Therefore, the antiviral activity of prochlorperazine, itraconazole, and niclosamide was tested at lower concentrations.

The cytotoxicity and antiviral activity of the three drug candidates against the EV71 subgenotype C4 in Vero cells are shown in Fig. 1 and Table 1. The IC50 values for niclosamide, itraconazole, and prochlorperazine were 0.125, 0.82, and 0.22 µM, respectively. The CC50 values for niclosamide, itraconazole, and prochlorperazine were 0.26, > 10, and 18.18 µM, respectively. Treatment with itraconazole and prochlorperazine yielded favorable selectivity index (SI) values of > 12.20 and 82.41, respectively (Table 1).

Fig. 1
figure 1

Dose-response curves for single-drug treatment against EV71 subgenotype C4 in Vero cells. (A) niclosamide. (B) itraconazole. (C) prochlorperazine. Vero cells were pretreated with twofold serial dilutions of each drug for 1 hour and infected with EV71 at an MOI of 0.01. The infected cells were maintained for two days in medium containing serially diluted drugs or 0.1% DMSO without drugs. The virus production was detected using a plaque assay. The dose-response curves are expressed as percent inhibition relative to 0.1% DMSO-treated cells. The effect of drug treatment on cell viability was assessed relative to 0.1% DMSO-treated cells using PrestoBlue reagent. The data are presented as the mean ± standard deviation

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Table 1 Single-drug treatment against EV71 subgenotype C4 in Vero and neuroblastoma cells (SH-SY5Y)
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Antiviral activity of two-drug combinations against EV71 subgenotype C4 in Vero cells

Dose-response curves of two-drug combinations

The dose-response curves of two-drug combination treatments and the IC50 values are shown in Fig. 2 and Table 2. In combination with itraconazole or prochlorperazine, the dose-response curve of niclosamide was shifted, resulting in a 15- and 7.14-fold reduction, respectively, in the IC50 value for niclosamide. The dose-response curve was also altered by the addition of niclosamide or prochlorperazine, with a 10- and 7.78-fold reduction, respectively, in the IC50 value of itraconazole. The dose-response curve of prochlorperazine was also altered by adding niclosamide or itraconazole. The IC50 value of prochlorperazine was reduced by 7.92- and 5.43-fold, respectively. The combinations of niclosamide-itraconazole and itraconazole-prochlorperazine showed a stronger antiviral effect than the combination of niclosamide-prochlorperazine.

Fig. 2
figure 2

Dose-response curves of two-drug combination treatment against EV71 subgenotype C4 in Vero cells. Vero cells were pretreated either with twofold serial dilutions of single drugs or two-drug combinations for 1 hour and infected with EV71 subgenotype C4 at an MOI of 0.01. Then the cells were maintained for two days in medium containing serial dilutions of a single drug or a two-drug combination. Viral titers were determined using a plaque assay. The dose-response curves are expressed as the percent inhibition relative to 0.1% DMSO-treated cells. The effect of drug treatment on cell viability was assessed relative to 0.1% DMSO-treated cells using an MTT assay. The two-drug combinations include niclosamide-itraconazole (NI), niclosamide-prochlorperazine (NP), and itraconazole-prochlorperazine (IP). The data are presented as the mean ± standard deviation. N, niclosamide; I, itraconazole; P, prochlorperazine

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Table 2 Two-drug combination treatment against EV71 subgenotype C4 in Vero cells
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Evaluation of two-drug combination treatment against EV71 subgenotype C4 in Vero cells

To identify drug combinations with high potential for EV71 treatment, the antiviral effects of two-drug combinations were investigated in Vero cells following the same infection methodology with each pairwise combination.

Niclosamide-itraconazole combination

The dose-response curve for niclosamide showed a strong shift in the presence of itraconazole (Fig. 3A). A similar change in the dose-response curve of itraconazole was also observed in the presence of niclosamide (Fig. 3B). The dose-response matrix shows the relationship between increasing inhibitory activity and increased concentrations of both drugs (Fig. 3C). The interaction between the two drugs was analyzed, and a mean Loewe synergy score of 12.01 was obtained, indicating a synergistic effect (score greater than 10), with a peak score of 24.29 (95% confidence interval, CI 10.68, 29.44) for the mixture of 0.2 µM itraconazole and 0.0625 µM niclosamide (Fig. 3D and E). Using other reference models, including ZIP, HSA, and Bliss independence, mean synergy scores of 12.89, 22.68, and 12.96, respectively, were obtained, confirming the synergistic effect between niclosamide and itraconazole. There was no significant cytotoxicity in any of the pairwise combinations (Fig. 3A and B).

Fig. 3
figure 3

Evaluation of the effect of niclosamide-itraconazole treatment on EV71 subgenotype C4 in Vero cells. Vero cells were pretreated for 1 hour with 16 different pairwise combinations of different concentrations of niclosamide and itraconazole. The cells were then infected with EV71 subgenotype C4 at an MOI of 0.01. After removing the inoculum, the cells were maintained for two days in the medium containing drugs. Virus titers were determined using a plaque assay. The percent inhibition relative to 0.1% DMSO-treated cells is shown in panel (A) for the treatment with twofold serial dilutions of niclosamide in the presence of different fixed concentrations of itraconazole and in panel (B) for the treatment with twofold serial dilutions of itraconazole in the presence of different fixed concentrations of niclosamide. The dose-response matrix of each drug treatment is shown in panel (C). The data are presented as the mean ± standard deviation. The interaction between pairs of drugs was analyzed using SynergyFinder. The synergy score of each pairwise combination is represented by the mean and the confidence interval (D). The 3D synergy plot is shown in panel (E). A synergy scores less than − 10 indicates antagonistic interaction between two drugs; a score between − 10 and 10 indicates an additive effect of the two drugs; and a score greater than 10 indicates a synergistic effect of the two drugs. N, niclosamide; I, itraconazole

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Niclosamide-prochlorperazine combination

Prochlorperazine at 0.4 µM caused a strong shift in the dose-response curve of niclosamide. Furthermore, prochlorperazine at concentrations of 0.2, 0.1, 0.05, and 0.025 µM decreased the IC50 value of niclosamide 5.67, 3.67, 1.86, and 1.44 times, respectively (Fig. 4A). The prochlorperazine dose-response curve also underwent a strong shift in the presence of 0.125 µM niclosamide. The IC50 value of prochlorperazine was decreased 3 and 1.29 times in the presence of 0.0625 and 0.03125 µM niclosamide, respectively (Fig. 4B). However, the dose-response curve of prochlorperazine was not affected by the presence of 0.015625 µM niclosamide. Only pairwise combinations containing high concentrations of both drugs showed an obvious increase in inhibitory activity (Fig. 4C). A mean Loewe synergy score of 3.25 indicated an additive effect (score greater than − 10 and less than 10) between niclosamide and prochlorperazine, with a maximum score of 26.71 [23.65, 30.48] (Fig. 4D and E). The mean synergy scores calculated by ZIP, HSA, and Bliss independence were − 7.02, 5.04, and − 8.61, respectively, which also indicated an additive effect between niclosamide and prochlorperazine. There was no significant cytotoxicity in any of the pairwise combinations (Fig. 3A and B).

Fig. 4
figure 4

Evaluation of the effect of niclosamide-prochlorperazine treatment on EV71 subgenotype C4 in Vero cells. Vero cells were pretreated for 1 hour with 20 different pairwise combinations of different concentrations of niclosamide and prochlorperazine. The cells were then infected with EV71 subgenotype C4 at an MOI of 0.01. After removing the inoculum, the cells were maintained for two days in the medium containing drugs. Virus titers were determined using a plaque assay. The percent inhibition relative to 0.1% DMSO-treated cells is shown in panel (A) for the treatment with twofold serial dilutions of niclosamide in the presence of different fixed concentrations of prochlorperazine and in panel (B) for the treatment with twofold serial dilutions of prochlorperazine in the presence of different fixed concentrations of niclosamide. The dose-response matrix of each drug treatment is shown in panel (C). The data are presented as the mean ± standard deviation. The interaction between pairs of drugs was analyzed using SynergyFinder. The synergy score of each pairwise combination is presented as the mean and the confidence interval (D). A 3D synergy plot is shown in panel (E). A synergy scores less than − 10 indicates an antagonistic interaction between the two drugs; a score between − 10 and 10 indicates an additive effect of the two drugs; and a score greater than 10 indicates a synergistic effect of the two drugs. N, niclosamide; P, prochlorperazine

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Itraconazole-prochlorperazine combination

The dose-response curve of itraconazole was strongly shifted by the addition of 0.4 µM prochlorperazine. Prochlorperazine at concentrations of 0.2 and 0.1 µM decreased the IC50 value of itraconazole by 2.84 and 1.64 times, respectively. However, the presence of 0.05 and 0.0025 µM prochlorperazine caused a smaller effect (Fig. 5A). In a similar pattern, the dose-response curves of prochlorperazine were strongly shifted by the presence of 0.8 µM itraconazole, and the IC50 value of prochlorperazine was decreased 7.95 and 2.90-fold by 0.4 and 0.2 µM itraconazole, respectively. The presence of 0.1 µM itraconazole did not cause a change in the IC50 value of prochlorperazine (Fig. 5B). Pairwise combinations with high concentrations of both drugs exhibited significant inhibitory effects (Fig. 5C). The mean Loewe synergy score of 3.77 indicated an additive effect (score greater than − 10 and less than 10) between itraconazole and prochlorperazine, with a peak score of 28.84 [20.54, 31.80] at the maximum concentration of both drugs (Fig. 5D and E). The mean synergy scores obtained using the ZIP and Bliss independence models were 1.47 and 1.41, respectively, which also indicated an additive effect of itraconazole and prochlorperazine. The HSA model yielded a mean synergy score of 10.3, suggesting a possible synergistic effect. There was no significant cytotoxicity in any of the pairwise combinations (Fig. 3A and B).

Fig. 5
figure 5

Evaluation of the effect of itraconazole-prochlorperazine treatment on EV71 subgenotype C4 in Vero cells. Vero cells were pretreated for 1 hour with 20 different pairwise combinations of different concentrations of itraconazole and prochlorperazine. The cells were then infected with EV71 subgenotype C4 at an MOI of 0.01. After removing the inoculum, the cells were maintained for two days in the medium containing drugs. Virus titers were determined using a plaque assay. The percent inhibition relative to 0.1% DMSO-treated cells is shown in panel (A) for the treatment with twofold serial dilutions of itraconazole in the presence of different fixed concentrations of prochlorperazine and in panel (B) for the treatment with twofold serial dilutions of prochlorperazine in the presence of different fixed concentrations of itraconazole. The dose-response matrix for each drug treatment is shown in panel (C). The data are represented as the mean ± standard deviation. The interaction between pairs of drugs was analyzed using SynergyFinder. The synergy score of each pairwise combination is presented as the mean and the confidence interval (D). A 3D synergy plot is shown in panel (E). A synergy score less than − 10 indicates an antagonistic interaction between two drugs; a score between − 10 and 10 indicates an additive effect of the two drugs; and a score greater than 10 indicates a synergistic effect of the two drugs. I, itraconazole; P, prochlorperazine

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Antiviral activity of drug candidates against EV71 subgenotype C4 in neuroblastoma cells

The antiviral activity of repurposed drug candidates against EV71 was examined in human neuroblastoma cells (SH-SY5Y), since this virus can cause a number of serious neurological complications. The cytotoxicity and antiviral activity of niclosamide, itraconazole, and prochlorperazine against EV71 subgenotype C4 are shown in Table 1 and Fig. 6. The IC50 values for niclosamide, itraconazole, and prochlorperazine were 0.29, 0.41, and 1.02 µM, respectively. The CC50 values for niclosamide, itraconazole, and prochlorperazine were 0.39, > 10, and 13.13 µM, respectively. Niclosamide treatment resulted in an unfavorable selectivity index (SI) of 1.34. Treatment with itraconazole and prochlorperazine resulted in a more favorable SI of > 24.39 and 12.87, respectively.

Fig. 6
figure 6

Evaluation of antiviral activity of the drug candidates against EV71 subgenotype C4 in neuroblastoma cells. Dose-response curves for single-drug treatment against EV71 are shown. (A) niclosamide. (B) itraconazole. (C) prochlorperazine. The cells were treated with twofold serial dilutions of the drug for 1 hour and infected with EV71 subgenotype C4 at an MOI of 2. The infected cells were then maintained for two days in the medium containing each drug or 0.1% DMSO as a control. Virus titers were determined using a plaque assay. The dose-response curves show the percent inhibition relative to 0.1%-DMSO-treated cells. The effect of drug treatment on cell viability relative to 0.1%-DMSO-treated cells was determined using PrestoBlue reagent. The data are shown as the mean ± standard deviation

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Evaluation of two-drug combination treatment against EV71 in neuroblastoma cells

Itraconazole-prochlorperazine combination

Only the combination of itraconazole and prochlorperazine was tested in neuroblastoma cells due to their favorable SI. It was found that the presence of various concentrations of prochlorperazine induced a shift in the dose-response curve of itraconazole (Fig. 7A). The strongest inhibitory effect was observed with the combination of itraconazole and 0.2 µM prochlorperazine. Similarly, the presence of itraconazole induced a shift in the dose-response curve of prochlorperazine, with stronger inhibitory effects observed in the presence of 0.8, 0.4, and 0.1 µM itraconazole. However, the presence of 0.2 µM itraconazole gave an unusual result compared to the single drug treatment and the other combinations (Fig. 7B). The dose-response matrix demonstrated significant inhibitory activity in pairwise combinations of itraconazole and prochlorperazine (Fig. 7C). The mean Loewe synergy score of 7.68 indicates an additive effect (score greater than − 10 and less than 10) between itraconazole and prochlorperazine, with a maximum score of 23.17 [19.6, 26.58] (Fig. 7D and E). When alternative reference models were used for the calculation of the synergy score, the HSA model yielded a synergy score of 9.02, indicating an additive effect. However, the synergy scores obtained using the ZIP and Bliss independence models were − 12.47 and − 10.86, respectively, indicating an antagonistic effect between itraconazole and prochlorperazine. There was no significant cytotoxicity in any of the pairwise combinations (Fig. 3A and B).

Fig. 7
figure 7

Evaluation of the effect of itraconazole-prochlorperazine treatment on EV71 subgenotype C4 in neuroblastoma cells. Neuroblastoma cells (SH-SY5Y) were pretreated for 1 hour with 16 different pairwise combinations of different concentrations of itraconazole and prochlorperazine. The cells were then infected with EV71 subgenotype C4 at an MOI of 2. After removing the inoculum, the cells were maintained for two days in the medium containing drugs. Virus titers were determined using a plaque assay. The percent inhibition relative to 0.1%-DMSO-treated cells is shown in panel (A) for the treatment with twofold serial dilutions of itraconazole in the presence of different fixed concentrations of prochlorperazine and in panel (B) for the treatment with twofold serial dilutions of prochlorperazine in the presence of different fixed concentrations of itraconazole. The dose-response matrix for each drug treatment is shown in panel (C). The data are presented as the mean ± standard deviation. The interaction between pairs of drugs was analyzed using SynergyFinder. The synergy score of each pairwise combination is presented as the mean and the confidence interval (D). A 3D synergy plot is shown in panel (E). A synergy score less than − 10 indicates an antagonistic interaction between the two drugs; a score between − 10 and 10 indicates an additive effect of the two drugs; and a score greater than 10 indicates a synergistic effect of the two drugs. I, itraconazole; P, prochlorperazine

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Inhibition of AKT phosphorylation by prochlorperazine and itraconazole

EV71 infection induces apoptosis, which limits viral replication [70]. However, previous studies have demonstrated that EV71 infection also activates PI3K/Akt signaling, which is the pathway that promotes cell survival and growth, in an early stage of infection to complete its replication cycle [70, 75]. Therefore, agents that inhibit PI3K/Akt signaling activation may result in inhibition of EV71.

Anticancer studies have shown that prochlorperazine and itraconazole inhibit Akt signaling [39, 58, 72]. Although these drugs are believed to inhibit viral replication through inhibition of endosomal acidification and cholesterol transport, respectively, inhibition of Akt signaling may also contribute to their antiviral effect. To investigate the effect of prochlorperazine and itraconazole on the PI3K/Akt signaling pathway, expression levels of the Akt protein were determined (Fig. 8). The results showed that EV71 infection activated the Akt signaling pathway by increasing phosphorylated Akt (p-Akt) in both Vero and neuroblastoma cells (Fig. 8A–D). Akt phosphorylation was reduced by treatment with prochlorperazine or itraconazole (Fig. 8B and D). One of the main cellular signaling pathways downstream of Akt is the mammalian rapamycin target (mTOR) pathway, which, upon activation, stimulates cell proliferation, protein synthesis, and de novo fatty acid synthesis, leading to an anti-apoptotic state that may favor viral replication [41, 71]. Therefore, mTOR activation was investigated in EV71-infected neuroblastoma cells. EV71 infection resulted in an increase in phosphorylated mTOR (p-mTOR) (Fig. 8C and E). Relatively lower expression of p-mTOR was observed in cells treated with prochlorperazine, and this correlated with the reduced level of EV71 replication in neuroblastoma cells.

Fig. 8
figure 8

Western blot analysis of the effect of itraconazole and prochlorperazine treatment on EV71 subgenotype C4 infection in Vero and neuroblastoma cells. Vero or neuroblastoma (SH-SY5Y) cells were pretreated for 1 hour with prochlorperazine, itraconazole, a mixture of both drugs, or 0.1% DMSO. The cells were then infected with EV71 at an MOI of 1 and 2 for Vero and SH-SY-5Y cells, respectively. After removing the inoculum, the cells were maintained in the medium containing drugs for 1 hour (Vero) or 24 hours (SH-SY5Y). The infected cells were then collected and subjected to western blot analysis. (A) Vero cells. (C) SH-SY5Y cells. The intensities of the protein bands were quantitated, and the relative expression was determined for Vero cells (B) and SH-SY5Y cells (D, E). The data are shown as the mean ± standard deviation (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001). PCZ, prochlorperazine; ITZ, itraconazole; (-), absent; (+), present

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Discussion

Viruses require cellular machinery of the host in every step of their replication. Therefore, these factors are potential targets of antiviral agents with broad antiviral activity. Recently, several studies demonstrated the antiviral activity of FDA-approved drugs through repurposed drug screenings. Many repurposed drugs showed broad-spectrum antiviral activity, targeting host cellular factors. Moreover, the use of repurposed drugs also expedites antiviral development due to their known safety profiles and the availability of pharmacokinetic data. In this study, several repurposed drugs with broad-spectrum antiviral activity were screened for their antiviral activity against EV71. Three drugs demonstrated antiviral activity against EV71, including prochlorperazine, niclosamide, and itraconazole. Itraconazole was reported previously to be a potent inhibitor of EV71 [53, 69]. Itraconazole was also found to inhibit other enteroviruses, including coxsackievirus A16, coxsackievirus B3, poliovirus 1, and enterovirus 68 [15, 53].

Upon infection, host cell factors are manipulated by viruses to create a favorable environment for viral replication. For entry of the virus, EV71 requires intact membrane cholesterol during endocytosis [35]. A previous study showed that lipid rafts are required for internalization of EV71 and are involved in activation of the PI3K/Akt signaling pathway in the early stage of infection [76]. It has been suggested that phosphorylation of PI3K/Akt signaling pathway components is a mechanism by which host cell apoptosis induced by EV71 infection is delayed [63, 70]. In particular, phosphorylation of Akt1 also activates mTOR, resulting in increased cell proliferation, protein synthesis, and de novo fatty acid synthesis [71].

Itraconazole is a triazole that is used in antifungal treatment. It is known to decrease ergosterol synthesis, which is required for fungal cell membrane integrity, via the inhibition of 14α-demethylase [40, 59]. In the early stage of enterovirus infection, Akt phosphorylation activates mTOR and its downstream products, resulting in increased lipid synthesis. Itraconazole decreases Akt activity, thus inhibiting Akt/mTOR signaling and downregulating lipogenic gene expression [71]. Furthermore, previous studies have demonstrated that itraconazole targets OSBP and OSBP-related protein 4 (ORP4), which are localized in enterovirus replication organelles and disrupt their function in cholesterol transport, thus inhibiting enterovirus replication [53]. Itraconazole also targets the viral NS3A protein, thus disrupting its function in PI4KB recruitment [15]. Our results showed a potent antiviral activity of itraconazole in both Vero cells and a neuroblastoma cell line, with a favorable selectivity index.

Prochlorperazine is a piperazine phenothiazine that is used to treat nausea, schizophrenia, migraines, and anxiety. Previous studies have demonstrated the antiviral activity of prochlorperazine in dengue virus (DENV), Zika virus (ZIKV), and hepatitis C virus (HCV) infections. This drug inhibits DENV infection by blocking viral binding via the D2 dopamine receptor and viral entry through clathrin-mediated endocytosis [21, 51]. Furthermore, a previous study showed that prochlorperazine can bind to the non-structural protein 3 (NS3) of DENV and ZIKV [49]. Prochlorperazine also inhibits HCV entry in the virus-host membrane fusion step by increasing the fluidity of the host membrane [6, 17]. Our study demonstrated potent in vitro anti-EV71 activity of prochlorperazine (Vero, IC50 = 0.22 µM; SH-SY5Y, IC50 = 1.02 µM). Prochlorperazine has been shown to interfere with Akt and mTORC1 activation, resulting in a decrease in proliferation signaling in cancer cells [4]. In this study, we demonstrated that prochlorperazine and itraconazole both inhibit EV71 infection through inhibition of Akt phosphorylation in the PI3K/Akt signaling pathway.

Niclosamide is an anthelmintic drug that is used to treat infestations of tapeworms. It is capable of regulating multiple cellular signaling pathways, including mTORC1, STAT3, NF-κB, Notch, and Wnt/β-catenin [9, 32, 65]. In several repurposed drug tests, niclosamide was shown to inhibit infection with several uses, including DENV [27], Japanese encephalitis virus (JEV) [12], HCV [36], Epstein-Barr virus (EBV) [23], HIV-1 [37], and SARS-CoV-2 [2, 24]. Niclosamide prevents DENV infection by inhibiting endosomal acidification and interfering with viral particle maturation [25, 27]. It also prevents mTORC1 activation, which inhibits EBV and HIV-1 [23, 37]. Our study demonstrated that niclosamide can potentially inhibit EV71 (Vero, IC50 = 0.125 µM; SH-SY5Y, IC50 = 0.29 µM), which is consistent with a recent study on screening of antiviral compounds from an FDA-approved drug library [54]. EV71 has been shown to enter cells through pH-dependent endocytosis [35], and therefore, niclosamide possibly inhibits EV71 infection by blocking endosomal acidification, as was observed with DENV.

The combination of drugs is a strategy used to maximize drug efficiency and to minimize drug resistance by using drugs that act on different cellular targets or viral proteins. In this study, two-drug combinations were tested in Vero cells. Niclosamide and itraconazole showed the best synergistic antiviral activity against EV71. This two-drug combination reduced the IC50 values of both drugs, suggesting that they are more effective when used in combination. The remaining combinations, niclosamide-prochlorperazine and itraconazole-prochlorperazine, showed additive effects. Both niclosamide and prochlorperazine inhibit endosomal acidification and mTOR activation, while both itraconazole and prochlorperazine inhibit the Akt/mTOR signaling pathway. These two-drug combinations act on the same cellular targets, resulting in additive rather than synergistic effects.

We also assessed the antiviral activity of two-drug combinations in neuroblastoma cells. However, niclosamide caused high cytotoxicity in this cell line, which is reflected in its unfavorable selectivity index of 1.34. Therefore, only the combination of itraconazole and prochlorperazine was tested in neuroblastoma cells, and this combination was found to have an additive effect.

In summary, we have identified prochlorperazine as a repurposed drug with antiviral activity against EV71. Importantly, prochlorperazine was also found to inhibit Akt phosphorylation in cells infected with EV71. The combination of niclosamide and itraconazole demonstrated a synergistic effect in Vero cells, but the combination of itraconazole and prochlorperazine showed only an additive effect in Vero and neuroblastoma cells. Different antiviral activities of these drugs, either alone or in combination, were observed in different cell lines. Thus, they still need to be evaluated in animal models. Our results indicate that it is feasible to develop therapeutic treatments of EV71 infection using repurposed drugs, alone or in two-drug combinations.