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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

Chemical structures of the 6 hit compounds exhibiting anti-CHIKV activities.
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pntd-0002471-g004: Chemical structures of the 6 hit compounds exhibiting anti-CHIKV activities.

Mentions: The chemical structures of the 6 confirmed hit compounds are shown in Figure 4. Based on the structural analysis using the molecule-clustering module from Pipeline Pilot, 4 of the compounds - CND0335, CND0364, CND0366, CND0415 share a benzofuran scaffold, with a common substitution of 4-methoxy-2-methylphenyl at the 3- or 4- positions in the benzofuran core plus amide linker with hydrophobic groups at the 7- position. Two other compounds, CND0545 and CND3514, are singletons having a pyrrolopyridine or a thiazol-4-carboxamide base scaffold, respectively.


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)

Chemical structures of the 6 hit compounds exhibiting anti-CHIKV activities.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0002471-g004: Chemical structures of the 6 hit compounds exhibiting anti-CHIKV activities.
Mentions: The chemical structures of the 6 confirmed hit compounds are shown in Figure 4. Based on the structural analysis using the molecule-clustering module from Pipeline Pilot, 4 of the compounds - CND0335, CND0364, CND0366, CND0415 share a benzofuran scaffold, with a common substitution of 4-methoxy-2-methylphenyl at the 3- or 4- positions in the benzofuran core plus amide linker with hydrophobic groups at the 7- position. Two other compounds, CND0545 and CND3514, are singletons having a pyrrolopyridine or a thiazol-4-carboxamide base scaffold, respectively.

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