Cargando…
Identifying rapidly parasiticidal anti-malarial drugs using a simple and reliable in vitro parasite viability fast assay
BACKGROUND: The emergence of Plasmodium falciparum resistance to artemisinins threatens to undermine the effectiveness of artemisinin-based combination anti-malarial therapy. Developing suitable drugs to replace artemisinins requires the identification of new compounds that display rapid parasite ki...
Autores principales: | , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2015
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4635989/ https://www.ncbi.nlm.nih.gov/pubmed/26542470 http://dx.doi.org/10.1186/s12936-015-0962-2 |
_version_ | 1782399586042642432 |
---|---|
author | Linares, María Viera, Sara Crespo, Benigno Franco, Virginia Gómez-Lorenzo, María G. Jiménez-Díaz, María Belén Angulo-Barturen, Íñigo Sanz, Laura María Gamo, Francisco-Javier |
author_facet | Linares, María Viera, Sara Crespo, Benigno Franco, Virginia Gómez-Lorenzo, María G. Jiménez-Díaz, María Belén Angulo-Barturen, Íñigo Sanz, Laura María Gamo, Francisco-Javier |
author_sort | Linares, María |
collection | PubMed |
description | BACKGROUND: The emergence of Plasmodium falciparum resistance to artemisinins threatens to undermine the effectiveness of artemisinin-based combination anti-malarial therapy. Developing suitable drugs to replace artemisinins requires the identification of new compounds that display rapid parasite killing kinetics. However, no current methods fully meet the requirements to screen large compound libraries for candidates with such properties. This study describes the development and validation of an in vitro parasite viability fast assay for identifying rapidly parasiticidal anti-malarial drugs. METHODS: Parasite killing kinetics were determined by first culturing unlabelled erythrocytes with P. falciparum in the presence of anti-malarial drugs for 24 or 48 h. After removing the drug, samples were added to erythrocytes pre-labelled with intracellular dye to allow their subsequent identification. The ability of viable parasites to re-establish infection in labelled erythrocytes could then be detected by two-colour flow cytometry after tagging of parasite DNA. Thus, double-stained erythrocytes (with the pre-labelled intracellular dye and the parasite DNA dye) result only after establishment of new infections by surviving parasites. The capacity of the test anti-malarial drugs to eliminate viable parasites within 24 or 48 h could, therefore, be determined. RESULTS: The parasite viability fast assay could be completed within 48 h following drug treatment and distinguished between rapidly parasiticidal anti-malarial drugs versus those acting more slowly. The assay was validated against ten standard anti-malarial agents with known properties and results correlated well with established methods. An abbreviated assay, suitable for adaption to medium–high throughput screening, was validated and applied against a set of 20 compounds retrieved from the publically available Medicines for Malaria Venture ‘Malaria Box’. CONCLUSION: The quantification of new infections to determine parasite viability offers important advantages over existing methods, and is amenable to medium–high throughput screening. In particular, the parasite viability fast assay allows discrimination of rapidly parasiticidal anti-malarial candidates. |
format | Online Article Text |
id | pubmed-4635989 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-46359892015-11-07 Identifying rapidly parasiticidal anti-malarial drugs using a simple and reliable in vitro parasite viability fast assay Linares, María Viera, Sara Crespo, Benigno Franco, Virginia Gómez-Lorenzo, María G. Jiménez-Díaz, María Belén Angulo-Barturen, Íñigo Sanz, Laura María Gamo, Francisco-Javier Malar J Methodology BACKGROUND: The emergence of Plasmodium falciparum resistance to artemisinins threatens to undermine the effectiveness of artemisinin-based combination anti-malarial therapy. Developing suitable drugs to replace artemisinins requires the identification of new compounds that display rapid parasite killing kinetics. However, no current methods fully meet the requirements to screen large compound libraries for candidates with such properties. This study describes the development and validation of an in vitro parasite viability fast assay for identifying rapidly parasiticidal anti-malarial drugs. METHODS: Parasite killing kinetics were determined by first culturing unlabelled erythrocytes with P. falciparum in the presence of anti-malarial drugs for 24 or 48 h. After removing the drug, samples were added to erythrocytes pre-labelled with intracellular dye to allow their subsequent identification. The ability of viable parasites to re-establish infection in labelled erythrocytes could then be detected by two-colour flow cytometry after tagging of parasite DNA. Thus, double-stained erythrocytes (with the pre-labelled intracellular dye and the parasite DNA dye) result only after establishment of new infections by surviving parasites. The capacity of the test anti-malarial drugs to eliminate viable parasites within 24 or 48 h could, therefore, be determined. RESULTS: The parasite viability fast assay could be completed within 48 h following drug treatment and distinguished between rapidly parasiticidal anti-malarial drugs versus those acting more slowly. The assay was validated against ten standard anti-malarial agents with known properties and results correlated well with established methods. An abbreviated assay, suitable for adaption to medium–high throughput screening, was validated and applied against a set of 20 compounds retrieved from the publically available Medicines for Malaria Venture ‘Malaria Box’. CONCLUSION: The quantification of new infections to determine parasite viability offers important advantages over existing methods, and is amenable to medium–high throughput screening. In particular, the parasite viability fast assay allows discrimination of rapidly parasiticidal anti-malarial candidates. BioMed Central 2015-11-05 /pmc/articles/PMC4635989/ /pubmed/26542470 http://dx.doi.org/10.1186/s12936-015-0962-2 Text en © Linares et al. 2015 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Methodology Linares, María Viera, Sara Crespo, Benigno Franco, Virginia Gómez-Lorenzo, María G. Jiménez-Díaz, María Belén Angulo-Barturen, Íñigo Sanz, Laura María Gamo, Francisco-Javier Identifying rapidly parasiticidal anti-malarial drugs using a simple and reliable in vitro parasite viability fast assay |
title | Identifying rapidly parasiticidal anti-malarial drugs using a simple and reliable in vitro parasite viability fast assay |
title_full | Identifying rapidly parasiticidal anti-malarial drugs using a simple and reliable in vitro parasite viability fast assay |
title_fullStr | Identifying rapidly parasiticidal anti-malarial drugs using a simple and reliable in vitro parasite viability fast assay |
title_full_unstemmed | Identifying rapidly parasiticidal anti-malarial drugs using a simple and reliable in vitro parasite viability fast assay |
title_short | Identifying rapidly parasiticidal anti-malarial drugs using a simple and reliable in vitro parasite viability fast assay |
title_sort | identifying rapidly parasiticidal anti-malarial drugs using a simple and reliable in vitro parasite viability fast assay |
topic | Methodology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4635989/ https://www.ncbi.nlm.nih.gov/pubmed/26542470 http://dx.doi.org/10.1186/s12936-015-0962-2 |
work_keys_str_mv | AT linaresmaria identifyingrapidlyparasiticidalantimalarialdrugsusingasimpleandreliableinvitroparasiteviabilityfastassay AT vierasara identifyingrapidlyparasiticidalantimalarialdrugsusingasimpleandreliableinvitroparasiteviabilityfastassay AT crespobenigno identifyingrapidlyparasiticidalantimalarialdrugsusingasimpleandreliableinvitroparasiteviabilityfastassay AT francovirginia identifyingrapidlyparasiticidalantimalarialdrugsusingasimpleandreliableinvitroparasiteviabilityfastassay AT gomezlorenzomariag identifyingrapidlyparasiticidalantimalarialdrugsusingasimpleandreliableinvitroparasiteviabilityfastassay AT jimenezdiazmariabelen identifyingrapidlyparasiticidalantimalarialdrugsusingasimpleandreliableinvitroparasiteviabilityfastassay AT angulobartureninigo identifyingrapidlyparasiticidalantimalarialdrugsusingasimpleandreliableinvitroparasiteviabilityfastassay AT sanzlauramaria identifyingrapidlyparasiticidalantimalarialdrugsusingasimpleandreliableinvitroparasiteviabilityfastassay AT gamofranciscojavier identifyingrapidlyparasiticidalantimalarialdrugsusingasimpleandreliableinvitroparasiteviabilityfastassay |