Limits...
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.

Show MeSH

Related in: MedlinePlus

Correlation of CHIKV-infection with HuH-7 cell viability.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).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3814572&req=5

pntd-0002471-g001: Correlation of CHIKV-infection with HuH-7 cell viability.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).

Mentions: We developed a cell-based high-throughput assay system using the hepatocarcinoma HuH-7 cell line and resazurin to assess CHIKV-associated cell death. HuH-7 was selected as the target cells based on previous reports demonstrating its high susceptibility to CHIKV infection [43], [44]. Cell viability was determined by measuring the reduction of resazurin to the highly fluorescent resorufin by metabolically active and viable cells [33]. Using an initial seeding density for HuH-7 at 5×103 cells per well, the effect of CHIKV-118-GFP infection (M.O.I. of 0.5) on cell number and cell viability was measured at 24, 48, 72 and 96 hrs post-infection (hpi), with 50 µM CPZ as a control cytotoxic compound. Cell number was quantified by staining the nuclei with DAPI and analyzed using a customized plug-in of the Image Mining platform while cell viability was measured by resazurin reduction assay. Figure 1A and 1B show the number of detected nuclei at 24, 48, 72 and 96 hpi. CHIKV-118-GFP infection of HuH-7 cells started showing significant reduction in cell number beginning from 48 hpi compared with MOCK-infection (P<0.0001). The percent reduction in the average number of cells resulting from CHIKV-118-GFP infection was 65%, 90%, and 95% at 48, 72, and 96 hpi, respectively. In comparison, treatment with 50 µM CPZ dramatically reduced the cell number by 86% after 24 hrs, and >99.9% after 48, 72, and 96 hrs. Based on the resazurin reduction assay, CHIKV-infected and CPZ-treated HuH-7 cells showed significant reduction in cell viability after 72 hpi, as shown by their lower fluorescence readout (Figure 1C). Treatment of HuH-7 cells with 50 µM CPZ resulted in a measured fluorescence readout (240,842±14,730 RFU) that was nearly identical with EMPTY wells (227,447±2,446 RFU), suggesting complete abolition of metabolic activity. In comparison, CHIKV-infected HuH-7 showed higher fluorescence readout (398,579±13,294 RFU), but was still significantly lower than that of MOCK-infected HuH-7 (801,587±22,230 RFU). It has been reported previously that short-term resazurin reduction occurs in dying cells caused by the production of free and unpaired electrons [45] While a good fluorescent signal can be achieved from MOCK-infected cells, distinguishable from that of CPZ-treated cells and EMPTY wells, within 6 hours after treatment with resazurin, the 12-hour treatment was necessary to have a clear separation of fluorescent signals between MOCK-infected and CHIKV-infected cells (data not shown).


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)

Correlation of CHIKV-infection with HuH-7 cell viability.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).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3814572&req=5

pntd-0002471-g001: Correlation of CHIKV-infection with HuH-7 cell viability.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).
Mentions: We developed a cell-based high-throughput assay system using the hepatocarcinoma HuH-7 cell line and resazurin to assess CHIKV-associated cell death. HuH-7 was selected as the target cells based on previous reports demonstrating its high susceptibility to CHIKV infection [43], [44]. Cell viability was determined by measuring the reduction of resazurin to the highly fluorescent resorufin by metabolically active and viable cells [33]. Using an initial seeding density for HuH-7 at 5×103 cells per well, the effect of CHIKV-118-GFP infection (M.O.I. of 0.5) on cell number and cell viability was measured at 24, 48, 72 and 96 hrs post-infection (hpi), with 50 µM CPZ as a control cytotoxic compound. Cell number was quantified by staining the nuclei with DAPI and analyzed using a customized plug-in of the Image Mining platform while cell viability was measured by resazurin reduction assay. Figure 1A and 1B show the number of detected nuclei at 24, 48, 72 and 96 hpi. CHIKV-118-GFP infection of HuH-7 cells started showing significant reduction in cell number beginning from 48 hpi compared with MOCK-infection (P<0.0001). The percent reduction in the average number of cells resulting from CHIKV-118-GFP infection was 65%, 90%, and 95% at 48, 72, and 96 hpi, respectively. In comparison, treatment with 50 µM CPZ dramatically reduced the cell number by 86% after 24 hrs, and >99.9% after 48, 72, and 96 hrs. Based on the resazurin reduction assay, CHIKV-infected and CPZ-treated HuH-7 cells showed significant reduction in cell viability after 72 hpi, as shown by their lower fluorescence readout (Figure 1C). Treatment of HuH-7 cells with 50 µM CPZ resulted in a measured fluorescence readout (240,842±14,730 RFU) that was nearly identical with EMPTY wells (227,447±2,446 RFU), suggesting complete abolition of metabolic activity. In comparison, CHIKV-infected HuH-7 showed higher fluorescence readout (398,579±13,294 RFU), but was still significantly lower than that of MOCK-infected HuH-7 (801,587±22,230 RFU). It has been reported previously that short-term resazurin reduction occurs in dying cells caused by the production of free and unpaired electrons [45] While a good fluorescent signal can be achieved from MOCK-infected cells, distinguishable from that of CPZ-treated cells and EMPTY wells, within 6 hours after treatment with resazurin, the 12-hour treatment was necessary to have a clear separation of fluorescent signals between MOCK-infected and CHIKV-infected cells (data not shown).

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