Evaluation of broad-spectrum antiviral compounds against chikungunya infection using a phenotypic screening strategy

Cells

Huh-7 hepatocellular carcinoma cells were cultivated in DMEM F-12 medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) (Life Technologies), 100 units/ml Penicillin (GIBCO) and 100 µg/ml Streptomycin (GIBCO) at 37°C, 5% CO₂. The Vero cell line derived from the kidney of an African green monkey were cultivated in DMEM high glucose (GIBCO) supplemented with 10% FBS (Life Technologies), 100 U/ml penicillin (GIBCO) and 100 μg/ml streptomycin (GIBCO) at 37°C, 5% CO₂. Cells were kindly provided by Professor Amilcar Tanuri (UFRJ-Brazil).

Virus

The CHIKV 181/25 vaccine strain was used in the present study¹⁴. The strain was obtained from the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA) of the University of Texas Medical Branch. The viral stock was propagated in Vero cells. Supernatant of infected tissue cultures was harvested and titrated by plaque assay¹⁵. Briefly, Vero cells were seeded in 24-well plate and incubated at 37°C, 5% CO₂ for 24 hours. Virus suspension was diluted 10-fold in DEMEN high glucose, and 0.2 µl from each virus dilution was added to infect Vero cells. After 1 hour in 37°C, 5% CO_2, inoculum was removed and cells were washed twice with Dulbecco’s Phosphate-buffer saline (DPBS pH 7.4, Sigma-Aldrich). An overlay was added with High-glucose DMEM (GIBCO), 10% FBS (Life Technologies) and 3.5% carboxymethylcellulose (CMC, Sigma-Aldrich) prepared in distillated water. At 3 days after, the overlay was removed. Then, cells were fixed with 4% paraformaldehyde (PFA) diluted in DPBS and stained with 0.5% crystal violet to enable plaque visualization and counting. Virus titers were expressed as plaque forming units (PFU) per milliliter.

Production of mouse hyperimmune sera (MHS)

Mouse hyperimmune sera was obtained from previously prepared stocks¹⁶. Briefly, to prepare these stocks mice (Mus musculus) received 4 weekly inoculations of 0.2 ml of brain macerate suspensions from newborn mice infected with CHIKV in PBS, by the intraperitoneal route. At 5 days after the last immunization the animals were anesthetized and underwent intracardiac puncture for blood collection. CHIKV-MHS was obtained from this blood.

Compounds and commercial library

The compounds 6-azauridine (CAS #54251), interferon α2A (CAS #H6041), bafilomycin A1 (CAS # 88899552), chloroquine (CAS #50635), 5-fluorouracil (CAS #51218) and the Library of Pharmacologically Active Compounds (LOPAC), containing 1,280 compounds, were purchased from Sigma-Aldrich. Sofosbuvir and daclatasvir were kindly provided by Microbiológica Química e Farmacêutica (Brazil) and ledispavir was donated by MedChemExpress (USA).

Assay development

Huh-7 cells were seeded in black polystyrene 384-well assay plates (Greiner Bio-One) at 3,000 cells/well in 40 µl DMEM-F12 supplemented with 10% FBS and incubated overnight. Cells were infected with 10 µl of inoculum of CHIKV 181/25 at different multiplicities of infection (MOIs) of 0.5, 0.05 and 0.01. Plates were fixed at different periods of time (36, 48 and 72 hours) and submitted to the immunofluorescence assay (described below) and images are acquired using an InCell Analyzer 2200 (GE Life Sciences).

Primary screening and assay validation

A library stock plate containing the aforementioned compounds at 2 mM in DMSO was used to prepare the intermediate plate by a 16.6-fold dilution in DPBS, to a concentration of 60 µM and 3% DMSO. Then, 10 µl of the intermediate plate content was transferred onto the cell-containing plate. The final concentration of library compounds in the assay plate was 10 µM, with 0.5% DMSO. Controls were placed in lateral columns in all plates. Positive controls were infected cells treated with 50 µM of 6-azauridine as well as non-infected cells treated with vehicle (0.5% DMSO in DPBS). Negative controls were infected cells treated with vehicle. Cells were infected by 10 µl of CHIKV 181/25 at MOI 0.05. Plates were incubated for 48 h at 37°C, 5% CO₂ under humidified atmosphere, and then fixed with 4% (w/v) PFA for 15 min at room temperature and washed twice with DPBS. Then, plates were incubated with CHIKV-MHS (mouse hyperimmune sera) diluted 1:1500 (v/v) prepared in blocking buffer (DPBS containing 5% FBS) for 30 min. Each plate was washed twice with DPBS, followed by incubation at room temperature for 30 min with the AlexaFluor488-conjugated goat anti-mouse IgG (Cat No. A-11001, Thermo-Scientific) diluted 1:2000 (v/v), and 5 µg/ml of 4’,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) in DPBS. Each plate was washed twice with DPBS. All plates were filled up with 50 µl of PBS/well.

Images were acquired using a confocal microscope High-Content System InCell Analyzer 2200 (GE Life Sciences) and processed by InCell Investigator v.1.6.1 software (GE, USA). Four different images were acquired from each well at x20 magnification. Automated image analysis was performed cell-by-cell through a defined mask based on fluorescence signal measurement and cell morphology. Cell segmentation parameters defined the analysis performed by the Investigator software. The nuclei were segmented from the DAPI staining images and each nucleus was determined as a minimum area of 50 µM. Total cells were filtered from the AlexaFlour488 channel and were defined as minimum area of 100 µM. The mean AlexaFluor488 florescence signal of infected cells were defined from the cytoplasm mask, with values based on means signal from fluorescence of wells from infected cells showing a six-times higher value compared to the mean of Alexa488 fluorescence signal of non-infected cells. Images were treated using image analysis program ImageJ v1.51 to set up colors and merge image channels to final visualization. The validation of screening was conducted in two independent experiment.

Data normalization

Infection ratio (IR) was defined as the ratio between (i) the total number of infected cells, and (ii) the total number of cells. Data were normalized with the negative (DMSO-treated, infected cells) and positive (infected cells treated with 50 µM 6-azauridine) controls. Normalized activity was calculated as described by Pascoalino et al.¹². Cell survival was expressed as the percentage of the total cell number from test sample divided by the average total cell number from the positive control wells: Cell number test sample/Avg. cell number of positive control) × 100. Normalized activity and cell survival values were processed with the GraphPad Prism software version 7. Representative graphs of sample distribution were obtained by plot data at TIBCO Spotfire 7.0 software. Plates were also submitted to quality control measurement of Z’-factor as described by Zhang et al.¹⁷.

Dose-response assay

For dose-response curves, drugs were prepared as described¹². The initial test concentrations were 100 nM for IFN-α2A, 50 nM for bafilomycin, 120 μM for chloroquine and mycophenolic acid, and 100 μM for sofosbuvir, daclatasvir, ledispavir and 5-fluorouracil. Dose response assay were calculated based on percentage of normalized activity and cells survival for each concentration tested. Data were plotted with GraphPad Prism software version 7. The sigmodal dose-response curve (variable slope) function were used to calculate the effective concentration that inhibited 50% of infection (EC₅₀), and the concentration of compound that presented a 50% reduction in cell number in comparison to the controls (CC₅₀). The ratio between CC₅₀ and EC₅₀ determines the selective index (SI).

Statistical analysis

Two-way analysis of variance (ANOVA) with Sidak’s test, a multiple comparison test, was conducted to calculate statistical significance (P < 0.05) of cell numbers from non-infected and infected CHIKV 181/25 at different multiplicity of infection and incubation time experiment. The coefficient of determination (R²) test were using to determinate statically coefficient of variation between screening replicates from normalize activity data. All data were plotted using GraphPad Prism Software version 7.

Assay development

A high-content screening assay was developed to evaluate compounds activity against CHIKV infection in vitro. The first step consisted on defining the cell model to support viral infection. A range of cell lines has been reported as being susceptible to CHIKV infection, such as Vero, human fetal lung fibroblast (MRC-5), baby hamster kidney (BHK), human embryonic kidney 293 (HEK-239T) and Huh-7^18,19. The Huh-7 cell line was selected as it has desirable features for high content imaging, such as adherent monolayer growth, and is human cell line, meaning it is a more representative in vitro model than would be cells of another species. The second step was determining the optimal multiplicity of infection (MOI) and the necessary period of time for the efficient viral infection in 384-well plates. Cells were infected at three different MOI (0.5, 0.05 and 0.01) and incubated for different periods of time (36, 48 and 72 hours). The total cell number and the IR were determined. When cells were plated and infected concomitantly, even the lowest MOI tested showed high cytopathic effect (data not shown). Thus, cells were plated 24 h before infection (Figure 1A). For the highest MOI, CHIKV infection decreased cell number by 70% at 48 hours and by almost 100% at 72 hours compared with non-infected cells. There was no significant difference in cell number between non-infected cells and infected cells for both 0.05 and 0.01 MOIs at 36 hours. Compared to non-infected cells, a decrease in cell number by 42% and 22% at MOIs 0.05 and 0.01, respectively, was observed at 48 hours (Figure 1A). After 36 hours of incubation, the IR showed an association with the MOI: 0.95±0.04 (MOI 0.5), 0.40±0.19 (MOI 0.05) and 0.12±0.20 (MOI 0.01) (Figure 1A), demonstrating that the assay endpoint was within the dynamic range of the infection. For 48 hours of incubation, the IR reached 0.99 for all MOIs, but the lowest MOI gave high variation in IR between replicate wells. Therefore, with the aim of testing drugs, a 0.05 MOI at 48 hours of incubation was selected for further experiments to achieve the longer time of exposure to drug treatment possible under these conditions, a high ratio of infection with minor variability, and a cell survival rate greater than 50% (compared with non-infected) (Figure 1A). Figure 1B describes the established general scheme of CHIKV high-content assay.

Figure 1. Development of CHIKV high content assay.

(A) Huh-7 cells were infected with different MOIs (0.5, 0.05 and 0.01) of CHIKV. The cell number and the infection ratio (defined as the ratio between total cell number and number of infected cells per sample), were evaluated after 36, 48 and 72 hours of infection. Error bars represent the standard deviation of 48 wells. Quantification of total cell number was comparable between non-infected and CHIKV 181/25-infected cells (p < 0.05). (B) General scheme of CHIKV high content assay. On day 1, Huh-7 cells were plated onto 384-well plates at 3000 cells/well. Then, after 24 hours (day 2), compounds were added, followed by addition of virus diluted at MOI 0.05. The plates were incubated for 48 hours up to immunofluorescence assay and high content analysis on day 4.

Assay validation

To validate the assay, high-content screening was run using a commercial library of compounds. Cell infection was determined by indirect CHIKV immunofluorescence detection. Figure 2A shows a raw image and software segmentation analysis of the same image. The 6-azauridine compound was previously reported to have activity against CHIKV²⁰, and was chosen as the reference compound in this assay. The activity of 6-azauridine was assessed using a dose-response curve (Figure 2B). The EC₅₀ of 0.65 µM 6-azauridine and EC₁₀₀ of 50 µM 6-azauridine were determined against CHIKV. In order to validate the assay reproducibility and robustness, a commercial library composed of 1,280 compounds was tested at a single concentration (10 µM). A good window between positive and negative controls was observed (Figure 2C). As a result, the mean for all plates Z’-factor values were 0.86±0.09, indicating that the established assay is reliable. Additionally, there was a high correlation coefficient between runs (Coefficient of determination R²: 0.86), which was determined using normalized activity of each single well between the first (R1) and the second (R2) screens, including compounds and controls (Figure 2D).

Figure 2. Validation of CHIKV high content assay.

(A) Interface of image processing and analysis. (B) Dose-response curve for reference compound 6-azauridine. Left y-axis: Normalized activity values (black squares and curves); Right y-axis: Cell survival values in percentage (red dots and curves); x-axis: Log of molar compound concentrations. Data points are means, and error bars represent standard deviations from two independent experiments. (C) Left graphic: Representative scatter plot of the infection ratio showing controls separation. Dots represent each single tested well and colors represent different treatments, where: 0.5% DMSO negative control (yellow); non-infected cells (red); 50 µM 6-azauridine positive control (green). Z’-factor value of 0.87 was obtained from total data controls from two independent runs for screening validation, between inter-replicates and intra-replicates plates. The continuous line represents the mean of each control, the dotted line represents 3 standard deviations from the mean of the negative and positive controls. Right graphic: Data correlation (normalized activity) from two screening runs of a small library; dots represent each single tested well and colors represent different treatments, where: 0.5% DMSO negative control (yellow); non-infected cells (red); 50 µM 6-azauridine positive control (green); and tested compounds (gray). The coefficient of determination of R square (R² 0.86) was calculated using GraphPad Prism software.

Evaluation of known antivirals against CHIKV

A set of compounds with known antiviral activity were evaluated against CHIKV. A total of 9 compounds were tested in dose-response curves. Figure 3 lists the name, molecular structures and dose-response curve plots for all compounds. Interferon α2A (IFN-α2A) and mycophenolic acid have reported activity against CHIKV^19,20. The HCS assay confirmed their reported activity, demonstrated by EC₅₀ values of 0.7 nM and 0.8 μM and high SI of >14 and 8.25, for IFN-α2A and mycophenolic acid, respectively. In order to evaluate compounds with previously reported activity against CHIKV, bafilomycin A1²¹ and chloroquine²² were tested, giving an EC₅₀ of 0.01 μM and 21 μM, respectively; however, they were cytotoxic in Huh-7 cells, with low SI values (5 and 0.3, respectively). The antiviral activity of daclatasvir, an anti-hepatitis C virus (HCV) drug²³, against CHIKV was associated with high cytotoxicity, and it had a low SI value of 1.3. Ledispavir, also an anti-HCV compound²⁴, and 5-fluorouracil, which has reported activity against ZIKV¹², did not present anti-CHIKV activity in the HCS assay. Sofosbuvir is an FDA-approved compound against HCV, and has been recently described as an active compound against other flaviviruses, including dengue and Zika^25,26. Sofosbuvir demonstrated dose-dependent activity against CHIKV (EC₅₀, 11 μM) with no cytotoxicity in Huh-7 cells. Representative images of sofosbuvir activity, alongside 6-azauridine, are displayed in Figure 4.

Figure 3. Dose response and molecular-structure of antiviral drug.

Dose response curves of interferon α2A, sofosbuvir, daclatasvir, ledispavir, bafilomycin A1, chloroquine, 5-fluorouracil and mycophenolic acid as anti-CHIKV activity (black) or the effect on Huh-7 survival (Red). Values of EC₅₀ means effective concentration of 50% infection inhibition, and SI means selective index (SI) based on CC₅₀ concentration of compounds of 50% cytotoxicity (not showed). ND, non-determined values.

Figure 4. Sofosbuvir activity against CHIKV.

Panel of representative images. First line of images shows nuclei staining with DAPI. Second line of images shows immunofluorescence against CHIKV (AlexaFluor488). Third line of images is the merge between the lines one and two. First column displays non-infected cells, followed by infected cells treated with 0.5% DMSO, 50 µM 6-azauridine, 12.5 µM sofosbuvir and 20 µM sofosbuvir.