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Development of a Fluorescent Quenching Based High Throughput Assay to Screen for Calcineurin Inhibitors

Currently there is no effective treatment available for major neurodegenerative disorders associated to protein misfolding, including Alzheimer’s and Parkinson's disease. One of most promising therapeutic approaches under development focuses on inhibiting the misfolding and aggregation pathway....

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Autores principales: Mukherjee, Abhisek, Syeb, Kathleen, Concannon, John, Callegari, Keri, Soto, Claudio, Glicksman, Marcie A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4503349/
https://www.ncbi.nlm.nih.gov/pubmed/26176772
http://dx.doi.org/10.1371/journal.pone.0131297
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author Mukherjee, Abhisek
Syeb, Kathleen
Concannon, John
Callegari, Keri
Soto, Claudio
Glicksman, Marcie A.
author_facet Mukherjee, Abhisek
Syeb, Kathleen
Concannon, John
Callegari, Keri
Soto, Claudio
Glicksman, Marcie A.
author_sort Mukherjee, Abhisek
collection PubMed
description Currently there is no effective treatment available for major neurodegenerative disorders associated to protein misfolding, including Alzheimer’s and Parkinson's disease. One of most promising therapeutic approaches under development focuses on inhibiting the misfolding and aggregation pathway. However, it is likely that by the time clinical symptoms appear, there is a large accumulation of misfolded aggregates and a very substantial damage to the brain. Thus, it seems that at the clinical stage of the disease it is necessary also to develop strategies aiming to prevent the neuronal damage produced by already formed misfolded aggregates. Chronic activation of calcineurin (CaN), a type IIB phosphatase, has been implicated as a pivotal molecule connecting synaptic loss and neuronal damage to protein misfolding. The fact that the crystal structure of CaN is also well established makes it an ideal target for drug discovery. CaN activity assays for High Throughput Screening (HTS) reported so far are based on absorbance. In this article we report the development of a fluorescent quenching based CaN activity assay suitable for robotic screening of large chemical libraries to find novel inhibitors. The assay yielded a Z score of 0.84 with coefficient of variance ≤ 15%. Our results also show that this assay can be used to identify CaN inhibitors with a wide range of potencies.
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spelling pubmed-45033492015-07-17 Development of a Fluorescent Quenching Based High Throughput Assay to Screen for Calcineurin Inhibitors Mukherjee, Abhisek Syeb, Kathleen Concannon, John Callegari, Keri Soto, Claudio Glicksman, Marcie A. PLoS One Research Article Currently there is no effective treatment available for major neurodegenerative disorders associated to protein misfolding, including Alzheimer’s and Parkinson's disease. One of most promising therapeutic approaches under development focuses on inhibiting the misfolding and aggregation pathway. However, it is likely that by the time clinical symptoms appear, there is a large accumulation of misfolded aggregates and a very substantial damage to the brain. Thus, it seems that at the clinical stage of the disease it is necessary also to develop strategies aiming to prevent the neuronal damage produced by already formed misfolded aggregates. Chronic activation of calcineurin (CaN), a type IIB phosphatase, has been implicated as a pivotal molecule connecting synaptic loss and neuronal damage to protein misfolding. The fact that the crystal structure of CaN is also well established makes it an ideal target for drug discovery. CaN activity assays for High Throughput Screening (HTS) reported so far are based on absorbance. In this article we report the development of a fluorescent quenching based CaN activity assay suitable for robotic screening of large chemical libraries to find novel inhibitors. The assay yielded a Z score of 0.84 with coefficient of variance ≤ 15%. Our results also show that this assay can be used to identify CaN inhibitors with a wide range of potencies. Public Library of Science 2015-07-15 /pmc/articles/PMC4503349/ /pubmed/26176772 http://dx.doi.org/10.1371/journal.pone.0131297 Text en © 2015 Mukherjee 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
Mukherjee, Abhisek
Syeb, Kathleen
Concannon, John
Callegari, Keri
Soto, Claudio
Glicksman, Marcie A.
Development of a Fluorescent Quenching Based High Throughput Assay to Screen for Calcineurin Inhibitors
title Development of a Fluorescent Quenching Based High Throughput Assay to Screen for Calcineurin Inhibitors
title_full Development of a Fluorescent Quenching Based High Throughput Assay to Screen for Calcineurin Inhibitors
title_fullStr Development of a Fluorescent Quenching Based High Throughput Assay to Screen for Calcineurin Inhibitors
title_full_unstemmed Development of a Fluorescent Quenching Based High Throughput Assay to Screen for Calcineurin Inhibitors
title_short Development of a Fluorescent Quenching Based High Throughput Assay to Screen for Calcineurin Inhibitors
title_sort development of a fluorescent quenching based high throughput assay to screen for calcineurin inhibitors
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4503349/
https://www.ncbi.nlm.nih.gov/pubmed/26176772
http://dx.doi.org/10.1371/journal.pone.0131297
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