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

Cruz DJ, Bonotto RM, Gomes RG, da Silva CT, Taniguchi JB, No JH, Lombardot B, Schwartz O, Hansen MA, Freitas-Junior LH - PLoS Negl Trop Dis (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Center for Neglected Diseases Drug Discovery (CND3), Institut Pasteur Korea, Seongnam-si, Gyeonggi-do, South Korea.

ABSTRACT
Chikungunya 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.

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Related in: MedlinePlus

Validation of the CHIKV high-throughput assay.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).
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pntd-0002471-g002: Validation of the CHIKV high-throughput assay.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).

Mentions: The use of resazurin reduction assay for high-throughput screening of CHIKV inhibitors was evaluated using the Z'-factors for the “percent inhibition” against CHIKV-infection between the 0.5% DMSO vehicle and 5 µM MPA-treated groups and between 0.5% DMSO vehicle and MOCK-infected groups. Figure 2A shows the scatter-plot distribution of the percent inhibition for the 0.5% DMSO vehicle, 5 µM MPA and MOCK-infected groups. The average Z'-factor between the CHIKV-infected 0.5% DMSO vehicle and 5 µM MPA-treated groups in the 15 384-well test plates was 0.578±0.075 (Z'-factors ranged from 0.460 to 0.703) while the average Z'-factor between the CHIKV-infected 0.5% DMSO vehicle and MOCK-infected groups was 0.645±0.059 (Z' factors ranged from 0.544 to 0.724). The RFU of the 0.5% DMSO vehicle negative control group showed a higher degree of variability (CV = 13.9±3.3%) compared with those of the positive control groups −5 µM MPA-treated (CV = 8.1±1.6%) and MOCK-infected (CV = 5.1±1.3%). While the 0.5% DMSO vehicle control group showed a CV slightly higher than 10%, the Z'-factors between the MOCK-infected and 0.5% DMSO vehicle control groups were >0.5 in all 15 test plates, indicating a reasonable separation between positive and negative controls. These findings demonstrate the reliability of the resazurin reduction assay for use in the CHIKV high-throughput screening. Figure 2B shows the activity and toxicity of 3 reference compounds (bafilomycin A1, chloroquine and mycophenolic acid) against CHIKV infection in HuH-7. BAF showed an EC50 value of 56 nM. Since BAF did not exhibit >50% toxicity at the highest concentration tested (200 nM), a projected CC50 value 237 nM was extrapolated based on the trend of the percent viability curve. For CQ, the EC50 and CC50 values were determined at 29 µM and 90 µM, respectively. A bell-shaped curve was observed for the percent inhibition as the concentration of CQ approached 100 µM and coincided with the steep decline in percent viability, indicating high toxicity at 100 µM. MPA showed an EC50 value of 1.6 µM, with no observable cytotoxicity at the highest concentration tested (100 µM). The antiviral activity of MPA determined by the resazurin reduction assay was within the range of the 50% inhibitory concentration of MPA previously reported (1.5 µM–4.1 µM) [29], while the measured antiviral activity and toxicity of BAF and CQ were within 3-fold of the observed inhibitory property from previously published reports [46], [47]. Interestingly, MPA treatment of CHIKV-infected HuH-7 cells at concentrations ≥3.25 µM resulted in higher fluorescence readout compared with non-infected HuH-7 treated with the same amount of the compound. The underlying mechanism for this observed phenomenon has not been elucidated.


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

Cruz DJ, Bonotto RM, Gomes RG, da Silva CT, Taniguchi JB, No JH, Lombardot B, Schwartz O, Hansen MA, Freitas-Junior LH - PLoS Negl Trop Dis (2013)

Validation of the CHIKV high-throughput assay.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).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3814572&req=5

pntd-0002471-g002: Validation of the CHIKV high-throughput assay.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).
Mentions: The use of resazurin reduction assay for high-throughput screening of CHIKV inhibitors was evaluated using the Z'-factors for the “percent inhibition” against CHIKV-infection between the 0.5% DMSO vehicle and 5 µM MPA-treated groups and between 0.5% DMSO vehicle and MOCK-infected groups. Figure 2A shows the scatter-plot distribution of the percent inhibition for the 0.5% DMSO vehicle, 5 µM MPA and MOCK-infected groups. The average Z'-factor between the CHIKV-infected 0.5% DMSO vehicle and 5 µM MPA-treated groups in the 15 384-well test plates was 0.578±0.075 (Z'-factors ranged from 0.460 to 0.703) while the average Z'-factor between the CHIKV-infected 0.5% DMSO vehicle and MOCK-infected groups was 0.645±0.059 (Z' factors ranged from 0.544 to 0.724). The RFU of the 0.5% DMSO vehicle negative control group showed a higher degree of variability (CV = 13.9±3.3%) compared with those of the positive control groups −5 µM MPA-treated (CV = 8.1±1.6%) and MOCK-infected (CV = 5.1±1.3%). While the 0.5% DMSO vehicle control group showed a CV slightly higher than 10%, the Z'-factors between the MOCK-infected and 0.5% DMSO vehicle control groups were >0.5 in all 15 test plates, indicating a reasonable separation between positive and negative controls. These findings demonstrate the reliability of the resazurin reduction assay for use in the CHIKV high-throughput screening. Figure 2B shows the activity and toxicity of 3 reference compounds (bafilomycin A1, chloroquine and mycophenolic acid) against CHIKV infection in HuH-7. BAF showed an EC50 value of 56 nM. Since BAF did not exhibit >50% toxicity at the highest concentration tested (200 nM), a projected CC50 value 237 nM was extrapolated based on the trend of the percent viability curve. For CQ, the EC50 and CC50 values were determined at 29 µM and 90 µM, respectively. A bell-shaped curve was observed for the percent inhibition as the concentration of CQ approached 100 µM and coincided with the steep decline in percent viability, indicating high toxicity at 100 µM. MPA showed an EC50 value of 1.6 µM, with no observable cytotoxicity at the highest concentration tested (100 µM). The antiviral activity of MPA determined by the resazurin reduction assay was within the range of the 50% inhibitory concentration of MPA previously reported (1.5 µM–4.1 µM) [29], while the measured antiviral activity and toxicity of BAF and CQ were within 3-fold of the observed inhibitory property from previously published reports [46], [47]. Interestingly, MPA treatment of CHIKV-infected HuH-7 cells at concentrations ≥3.25 µM resulted in higher fluorescence readout compared with non-infected HuH-7 treated with the same amount of the compound. The underlying mechanism for this observed phenomenon has not been elucidated.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Center for Neglected Diseases Drug Discovery (CND3), Institut Pasteur Korea, Seongnam-si, Gyeonggi-do, South Korea.

ABSTRACT
Chikungunya 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.

Show MeSH
Related in: MedlinePlus