Identification of novel compounds inhibiting chikungunya virus-induced cell death by high throughput screening of a kinase inhibitor library.

International audienceChikungunya virus (CHIKV) is a mosquito-borne arthrogenic alphavirus that causes acute febrile illness in humans accompanied by joint pains and in many cases, persistent arthralgia lasting weeks to years. The re-emergence of CHIKV has resulted in numerous outbreaks in the eastern hemisphere, and threatens to expand in the foreseeable future. Unfortunately, no effective treatment is currently available. The present study reports the use of resazurin in a cell-based high-throughput assay, and an image-based high-content assay to identify and characterize inhibitors of CHIKV-infection in vitro. CHIKV is a highly cytopathic virus that rapidly kills infected cells. Thus, cell viability of HuH-7 cells infected with CHIKV in the presence of compounds was determined by measuring metabolic reduction of resazurin to identify inhibitors of CHIKV-associated cell death. A kinase inhibitor library of 4,000 compounds was screened against CHIKV infection of HuH-7 cells using the resazurin reduction assay, and the cell toxicity was also measured in non-infected cells. Seventy-two compounds showing ≥50% inhibition property against CHIKV at 10 µM were selected as primary hits. Four compounds having a benzofuran core scaffold (CND0335, CND0364, CND0366 and CND0415), one pyrrolopyridine (CND0545) and one thiazol-carboxamide (CND3514) inhibited CHIKV-associated cell death in a dose-dependent manner, with EC50 values between 2.2 µM and 7.1 µM. Based on image analysis, these 6 hit compounds did not inhibit CHIKV replication in the host cell. However, CHIKV-infected cells manifested less prominent apoptotic blebs typical of CHIKV cytopathic effect compared with the control infection. Moreover, treatment with these compounds reduced viral titers in the medium of CHIKV-infected cells by up to 100-fold. In conclusion, this cell-based high-throughput screening assay using resazurin, combined with the image-based high content assay approach identified compounds against CHIKV having a novel antiviral activity--inhibition of virus-induced CPE--likely by targeting kinases involved in apoptosis

CHIKV high-throughput screening of the BioFocus kinase inhibitor Library.

<p>Scatter plot distribution showing the results of the CHIKV high-throughput screening of the BioFocus kinase inhibitor library using resazurin reduction assay. Dots represent the normalized RFU of MOCK-infected HuH-7 with 0.5% DMSO vehicle (brown), CHIKV-infected HuH-7 with 0.5% DMSO vehicle (yellow), CHIKV-infected HuH-7 with 10 µM BioFocus compounds (red) or MOCK-infected HuH-7 with 50 µM CPZ (blue) (A). Histogram showing the distribution of MOCK-infected HuH-7 (brown), CHIKV-infected HuH-7 (yellow), CHIKV-infected HuH-7 with 10 µM BioFocus compounds (red) or MOCK-infected HuH-7 with 50 µM CPZ (blue) (B). The orange dotted lines represent 99.7% (μ±3σ) of the CHIKV-infected HuH-7 wells, while the green dotted lines represent the statistical cut-off (μ+4σ of the DMSO vehicle control) used for selecting primary hits (≥70% viability).</p

Virus neutralization assay.

<p>Confluent monolayer of HuH-7 cells were infected with 50 pfu CHIKV-118-GFP in the presence of compound CND0364 or CND0545 at various concentrations for 72 hrs, and then stained with 0.1% crystal violet solution to observe clearance of the cell monolayer caused by virus infection. Dotted boxes indicate concentration of compounds that exhibit protection against CHIKV-induced CPE. (DMSO – infection control, MOCK – negative control).</p

Correlation of CHIKV-infection with HuH-7 cell viability.

<p>DAPI-stained nuclei of MOCK-infected, CHIKV-infected and CPZ-treated HuH-7 cells observed using Operetta at 20× magnification (A). Average number of cells per field based on detected nuclei (B). RFU readout after resazurin treatment at 72 hpi (C). (false-color imaging were applied on panel A).</p

Quantification of CHIKV-118-GFP-infected cells by imaging.

<p>Acquired confocal images of CHIKV-118-GFP infection in HuH-7 cells analyzed using the customized plugin in the Image Mining platform. From the merged image (A), the regions of individual nuclei from the DAPI channel (B) are defined using a watershed algorithm (C). The resulting output shows each individual nucleus with distinct color (A color map is drawn in frame C). A positive signal from the GFP channel (D) is determined using a threshold value to generate the GFP foreground (E). CHIKV-infected cells are identified by the overlap between nuclei identified in C and GFP foreground in E (F) and the percentage of nuclei of infected cells is defined by the ratio between nuclei in F and C. (false-color imaging were applied on panels A, B, and C).</p

Activity profile of the 6 confirmed hit compounds against CHIKV based on resazurin reduction assay.

<p>Activity profile of the 6 confirmed hit compounds against CHIKV based on resazurin reduction assay.</p

Inhibition of CHIKV-associated apoptotic bodies.

<p>HuH-7 cells infected with CHIKV-118-GFP expressing GFP at 24 hpi. The number of observed apoptotic blebs associated with CHIKV infection (marked by white arrows) were significantly lower when treated with the reference compound MPA and hit compounds CND0364 and CND0545. In contrast, apoptotic blebs were still prominent in CHIKV-infected HuH-7 cells in the presence of CQ. (false-color imaging were applied).</p

Validation of the CHIKV high-throughput assay.

<p>Scatter plot and calculated Z'-factor of the resazurin reduction assay for CHIKV high-throughput screening. Dots represent wells with the following treatment: MOCK-infected HuH-7 (blue), CHIKV-infected HuH-7 with 0.5% DMSO vehicle (red), and 5 µM MPA (green). Area under the blue and orange dotted lines represent the variability of the measured percent inhibition for CHIKV-infection and MOCK infection controls, respectively. Arrow represents the degree of separation (Z'-factor) between MOCK and CHIKV infection controls (A). Dose-response curves of BAF, CQ, and MPA anti-CHIKV activity (red) and their effect on HuH-7 cell viability (blue) (B).</p

Chemical structures of the 6 hit compounds exhibiting anti-CHIKV activities.

<p>Chemical structures of the 6 hit compounds exhibiting anti-CHIKV activities.</p

Inhibition of virus infectivity.

<p>Image analysis on CHIKV-118-GFP infection of HuH-7 in the presence of 20 µM hit compounds, 50 µM MPA or 50 µM CQ. The percentages of infected cells are indicated on the lower right of each image (A). Production of infectious virus particles from the CHIKV-118-GFP-infected HuH-7 in the presence of 20 µM hit compounds, 50 µM MPA and 50 µM CQ (B). DMSO – CHIKV-118-GFP infection control, MOCK – non-infected control, MPA – mycophenolic acid, CQ – chloroquine. (false-color imaging were applied on panel A).</p