Identification of Novel Compounds Inhibiting Chikungunya Virus-Induced Cell Death by High Throughput Screening of a Kinase Inhibitor Library
| Autor: | Camila T. da Silva, Joo Hwan No, Michael Adsetts Edberg Hansen, Olivier Schwartz, Deu John M. Cruz, Rafaela Milan Bonotto, Lucio H. Freitas-Junior, Juliana B. Taniguchi, Rafael G. B. Gomes, Benoit Lombardot |
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| Přispěvatelé: | Inst Pasteur Korea, Univ Feevale, Universidade Federal de Uberlândia (UFU), Univ Estadual Rio Grande do Sul, Universidade Estadual Paulista (Unesp), Inst Pasteur, Institut Pasteur Korea - Institut Pasteur de Corée, Réseau International des Instituts Pasteur (RIIP), Universidade Federal de Uberlândia, Universidade Estadual do Rio Grande do Sul, Universidade Estadual Paulista Júlio de Mesquita Filho = São Paulo State University (UNESP), Virus et Immunité - Virus and immunity, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), The authors would like to acknowledge Katja Fink for the gift of HuH-7 cells and Claudia N. D. dos-Santos for the gift of C6/36 cells., Universidade Estadual do Rio Grande do Sul (UERGS), Virus et Immunité - Virus and immunity (CNRS-UMR3569), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS) |
| Jazyk: | angličtina |
| Rok vydání: | 2013 |
| Předmět: |
MESH: Oxidation-Reduction
MESH: Cell Death Cell MESH: Chikungunya virus/physiology chemistry.chemical_compound MESH: Optical Imaging/methods Cytopathic effect MESH: Inhibitory Concentration 50 MESH: Alphavirus Infections/virology 0303 health sciences Cell Death lcsh:Public aspects of medicine Optical Imaging MESH: Protein Kinase Inhibitors/isolation & purification virus diseases 3. Good health MESH: Hepatocytes/drug effects medicine.anatomical_structure Infectious Diseases [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology MESH: Antiviral Agents/isolation & purification Chikungunya virus Oxidation-Reduction Research Article Programmed cell death lcsh:Arctic medicine. Tropical medicine Cell Survival lcsh:RC955-962 MESH: High-Throughput Screening Assays/methods Biology Antiviral Agents Virus Cell Line MESH: Protein Kinase Inhibitors/pharmacology 03 medical and health sciences Inhibitory Concentration 50 Oxazines medicine Humans Viability assay Protein Kinase Inhibitors 030304 developmental biology MESH: Cell Survival/drug effectsm MESH: Humans Staining and Labeling 030306 microbiology Alphavirus Infections Public Health Environmental and Occupational Health MESH: Hepatocytes/physiology MESH: Xanthenes/metabolism Resazurin lcsh:RA1-1270 Virology High-Throughput Screening Assays MESH: Cell Line chemistry Xanthenes Apoptosis Cell culture MESH: Alphavirus Infections/drug therapy MESH: Hepatocytes/virology Hepatocytes MESH: Staining and Labeling/methods MESH: Cell Survival/drug effects MESH: Oxazines/metabolism |
| Zdroj: | Web of Science Repositório Institucional da UNESP Universidade Estadual Paulista (UNESP) instacron:UNESP PLoS Neglected Tropical Diseases PLoS Neglected Tropical Diseases, Public Library of Science, 2012, 7 (10), pp.e2471. ⟨10.1371/journal.pntd.0002471⟩ PLoS Neglected Tropical Diseases, 2012, 7 (10), pp.e2471. ⟨10.1371/journal.pntd.0002471⟩ PLoS Neglected Tropical Diseases, Vol 7, Iss 10, p e2471 (2013) |
| ISSN: | 1935-2727 1935-2735 |
| DOI: | 10.1371/journal.pntd.0002471⟩ |
| Popis: | 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. Author Summary Recent outbreaks and expanding global distribution of Chikungunya virus (CHIKV) in different regions of Asia, Africa and Europe necessitates the development of effective therapeutic interventions. At present, only two antiviral compounds (chloroquine and ribavirin) that inhibit viral infection in vitro have been used in clinical cases of chikungunya infections. However, neither of these compounds have shown strong efficacy in vivo. Recent attempts to identify new antiviral candidates for CHIKV using cell-based phenotypic approach have been reported. In this study, we developed a simple cell-based high-throughput assay using resazurin to identify potential anti-CHIKV compounds. This high-throughput assay is based on the metabolic reduction of resazurin to the highly fluorescent resorufin by viable cells as an indicator of activity against CHIKV-induced CPE. We screened 4,000 small molecules belonging to the BioFocus kinase inhibitor chemical library and found a cluster of related molecules with antiviral activity against CHIKV. Finally, we characterized the putative mode of action of these active compounds using an image-based high content assay and conventional virological methods (i.e., virus yield reduction assay, microneutralization assay). |
| Databáze: | OpenAIRE |
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