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Development and Validation of a Novel Leishmania donovani Screening Cascade for High-Throughput Screening Using a Novel Axenic Assay with High Predictivity of Leishmanicidal Intracellular Activity

Visceral leishmaniasis is an important parasitic disease of the developing world with a limited arsenal of drugs available for treatment. The existing drugs have significant deficiencies so there is an urgent need for new and improved drugs. In the human host, Leishmania are obligate intracellular p...

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Autores principales: Nühs, Andrea, De Rycker, Manu, Manthri, Sujatha, Comer, Eamon, Scherer, Christina A., Schreiber, Stuart L., Ioset, Jean-Robert, Gray, David W.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4583543/
https://www.ncbi.nlm.nih.gov/pubmed/26407168
http://dx.doi.org/10.1371/journal.pntd.0004094
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author Nühs, Andrea
De Rycker, Manu
Manthri, Sujatha
Comer, Eamon
Scherer, Christina A.
Schreiber, Stuart L.
Ioset, Jean-Robert
Gray, David W.
author_facet Nühs, Andrea
De Rycker, Manu
Manthri, Sujatha
Comer, Eamon
Scherer, Christina A.
Schreiber, Stuart L.
Ioset, Jean-Robert
Gray, David W.
author_sort Nühs, Andrea
collection PubMed
description Visceral leishmaniasis is an important parasitic disease of the developing world with a limited arsenal of drugs available for treatment. The existing drugs have significant deficiencies so there is an urgent need for new and improved drugs. In the human host, Leishmania are obligate intracellular parasites which poses particular challenges in terms of drug discovery. To achieve sufficient throughput and robustness, free-living parasites are often used in primary screening assays as a surrogate for the more complex intracellular assays. We and others have found that such axenic assays have a high false positive rate relative to the intracellular assays, and that this limits their usefulness as a primary platform for screening of large compound collections. While many different reasons could lie behind the poor translation from axenic parasite to intracellular parasite, we show here that a key factor is the identification of growth slowing and cytostatic compounds by axenic assays in addition to the more desirable cytocidal compounds. We present a screening cascade based on a novel cytocidal-only axenic amastigote assay, developed by increasing starting density of cells and lowering the limit of detection, and show that it has a much improved translation to the intracellular assay. We propose that this assay is an improved primary platform in a new Leishmania screening cascade designed for the screening of large compound collections. This cascade was employed to screen a diversity-oriented-synthesis library, and yielded two novel antileishmanial chemotypes. The approach we have taken may have broad relevance to anti-infective and anti-parasitic drug discovery.
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spelling pubmed-45835432015-10-02 Development and Validation of a Novel Leishmania donovani Screening Cascade for High-Throughput Screening Using a Novel Axenic Assay with High Predictivity of Leishmanicidal Intracellular Activity Nühs, Andrea De Rycker, Manu Manthri, Sujatha Comer, Eamon Scherer, Christina A. Schreiber, Stuart L. Ioset, Jean-Robert Gray, David W. PLoS Negl Trop Dis Research Article Visceral leishmaniasis is an important parasitic disease of the developing world with a limited arsenal of drugs available for treatment. The existing drugs have significant deficiencies so there is an urgent need for new and improved drugs. In the human host, Leishmania are obligate intracellular parasites which poses particular challenges in terms of drug discovery. To achieve sufficient throughput and robustness, free-living parasites are often used in primary screening assays as a surrogate for the more complex intracellular assays. We and others have found that such axenic assays have a high false positive rate relative to the intracellular assays, and that this limits their usefulness as a primary platform for screening of large compound collections. While many different reasons could lie behind the poor translation from axenic parasite to intracellular parasite, we show here that a key factor is the identification of growth slowing and cytostatic compounds by axenic assays in addition to the more desirable cytocidal compounds. We present a screening cascade based on a novel cytocidal-only axenic amastigote assay, developed by increasing starting density of cells and lowering the limit of detection, and show that it has a much improved translation to the intracellular assay. We propose that this assay is an improved primary platform in a new Leishmania screening cascade designed for the screening of large compound collections. This cascade was employed to screen a diversity-oriented-synthesis library, and yielded two novel antileishmanial chemotypes. The approach we have taken may have broad relevance to anti-infective and anti-parasitic drug discovery. Public Library of Science 2015-09-25 /pmc/articles/PMC4583543/ /pubmed/26407168 http://dx.doi.org/10.1371/journal.pntd.0004094 Text en © 2015 Nühs et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Nühs, Andrea
De Rycker, Manu
Manthri, Sujatha
Comer, Eamon
Scherer, Christina A.
Schreiber, Stuart L.
Ioset, Jean-Robert
Gray, David W.
Development and Validation of a Novel Leishmania donovani Screening Cascade for High-Throughput Screening Using a Novel Axenic Assay with High Predictivity of Leishmanicidal Intracellular Activity
title Development and Validation of a Novel Leishmania donovani Screening Cascade for High-Throughput Screening Using a Novel Axenic Assay with High Predictivity of Leishmanicidal Intracellular Activity
title_full Development and Validation of a Novel Leishmania donovani Screening Cascade for High-Throughput Screening Using a Novel Axenic Assay with High Predictivity of Leishmanicidal Intracellular Activity
title_fullStr Development and Validation of a Novel Leishmania donovani Screening Cascade for High-Throughput Screening Using a Novel Axenic Assay with High Predictivity of Leishmanicidal Intracellular Activity
title_full_unstemmed Development and Validation of a Novel Leishmania donovani Screening Cascade for High-Throughput Screening Using a Novel Axenic Assay with High Predictivity of Leishmanicidal Intracellular Activity
title_short Development and Validation of a Novel Leishmania donovani Screening Cascade for High-Throughput Screening Using a Novel Axenic Assay with High Predictivity of Leishmanicidal Intracellular Activity
title_sort development and validation of a novel leishmania donovani screening cascade for high-throughput screening using a novel axenic assay with high predictivity of leishmanicidal intracellular activity
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4583543/
https://www.ncbi.nlm.nih.gov/pubmed/26407168
http://dx.doi.org/10.1371/journal.pntd.0004094
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