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

Virus neutralization assay.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).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3814572&req=5

pntd-0002471-g007: Virus neutralization assay.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).

Mentions: The effect of the 6 hit compounds on CHIKV-118-GFP infection of HuH-7 cells was evaluated by quantifying GFP-expressing GFP and comparing cell morphology during infection. The inhibitory properties of two reference compounds and the 6 confirmed hit compounds against CHIKV-118-GFP were evaluated by image analysis using the customized plug-in on the Image Mining platform (see Figure 6A). Chloroquine, a lysosomotropic agent that blocks viral entry by preventing pH dependent fusion [49], inhibited CHIKV-118-GFP infection by 75.6% at 50 µM. Mycophenolic acid, an inhibitor of GMP synthesis that results in decreased synthesis of RNA and DNA [50], inhibited more than 92% of infection at 50 µM. In contrast to CQ and MPA, none of the 6 hit compounds exhibited significant inhibition of CHIKV-118-GFP infection in HuH-7. The benzofuran and thiazol-4-carboxamide compounds showed <10% inhibition at 20 µM, with the exception of CND0364 (19.7% inhibition at 20 µM). Also, the pyrrolopyridine compound CND0545 only inhibited 28.3% of infection at 20 µM. However, it was observed that in the presence of the 6 hit compounds, the morphology of CHIKV-infected cells showed less apoptotic blebs compared with those treated with the DMSO vehicle control. Furthermore, the culture supernatant of CHIKV-infected HuH-7 cells showed a 10- to 100-folds decrease (1.16–2.01 log titer reduction) in viral titer when treated with the hit compounds compared to the DMSO vehicle control (Figure 6B). Interestingly, the reduction in viral titers resulting from the treatment of 6 hit compounds was comparable with that of chloroquine treatment (1.88 log titer reduction). Compounds CND0364 and CND0545, which exhibited the highest reduction in viral titers (1.69 log and 2.01 log titer reduction, respectively), inhibited the clearing of the cell monolayer caused by CHIKV-induced CPE at 12.5 µM and 25 µM, respectively in the microneutralization assay (Figure 7).


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)

Virus neutralization assay.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).
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0002471-g007: Virus neutralization assay.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).
Mentions: The effect of the 6 hit compounds on CHIKV-118-GFP infection of HuH-7 cells was evaluated by quantifying GFP-expressing GFP and comparing cell morphology during infection. The inhibitory properties of two reference compounds and the 6 confirmed hit compounds against CHIKV-118-GFP were evaluated by image analysis using the customized plug-in on the Image Mining platform (see Figure 6A). Chloroquine, a lysosomotropic agent that blocks viral entry by preventing pH dependent fusion [49], inhibited CHIKV-118-GFP infection by 75.6% at 50 µM. Mycophenolic acid, an inhibitor of GMP synthesis that results in decreased synthesis of RNA and DNA [50], inhibited more than 92% of infection at 50 µM. In contrast to CQ and MPA, none of the 6 hit compounds exhibited significant inhibition of CHIKV-118-GFP infection in HuH-7. The benzofuran and thiazol-4-carboxamide compounds showed <10% inhibition at 20 µM, with the exception of CND0364 (19.7% inhibition at 20 µM). Also, the pyrrolopyridine compound CND0545 only inhibited 28.3% of infection at 20 µM. However, it was observed that in the presence of the 6 hit compounds, the morphology of CHIKV-infected cells showed less apoptotic blebs compared with those treated with the DMSO vehicle control. Furthermore, the culture supernatant of CHIKV-infected HuH-7 cells showed a 10- to 100-folds decrease (1.16–2.01 log titer reduction) in viral titer when treated with the hit compounds compared to the DMSO vehicle control (Figure 6B). Interestingly, the reduction in viral titers resulting from the treatment of 6 hit compounds was comparable with that of chloroquine treatment (1.88 log titer reduction). Compounds CND0364 and CND0545, which exhibited the highest reduction in viral titers (1.69 log and 2.01 log titer reduction, respectively), inhibited the clearing of the cell monolayer caused by CHIKV-induced CPE at 12.5 µM and 25 µM, respectively in the microneutralization assay (Figure 7).

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