title: Development of a Fluorescent Quenching Based High Throughput Assay to Screen for Calcineurin Inhibitors.
abstract: 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.
authors.affiliation: Dept of Neurology, University of Texas Health Science Center, Houston, Texas, United States of America.
authors.forename: Abhisek
authors.initials: A
authors.lastname: Mukherjee
authors.affiliation: Laboratory for Drug Discovery in Neurodegeneration, Department of Neurology, Brigham and Women's Hospital and Harvard Medical School, Cambridge, Massachusetts, United States of America.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Syeb
authors.affiliation: Laboratory for Drug Discovery in Neurodegeneration, Department of Neurology, Brigham and Women's Hospital and Harvard Medical School, Cambridge, Massachusetts, United States of America.
authors.forename: John
authors.initials: J
authors.lastname: Concannon
authors.affiliation: Dept of Neurology, University of Texas Health Science Center, Houston, Texas, United States of America.
authors.forename: Keri
authors.initials: K
authors.lastname: Callegari
authors.affiliation: Dept of Neurology, University of Texas Health Science Center, Houston, Texas, United States of America.
authors.forename: Claudio
authors.initials: C
authors.lastname: Soto
authors.affiliation: Laboratory for Drug Discovery in Neurodegeneration, Department of Neurology, Brigham and Women's Hospital and Harvard Medical School, Cambridge, Massachusetts, United States of America.
authors.forename: Marcie A
authors.initials: MA
authors.lastname: Glicksman
mesh_terms: D065095:Calcineurin Inhibitors / Q000737:chemistry*; D004353:Drug Evaluation, Preclinical; D057075:Enzyme Assays; D005456:Fluorescent Dyes / Q000737:chemistry; D057166:High-Throughput Screening Assays; D006801:Humans; D007700:Kinetics; D012394:Rosaniline Dyes / Q000737:chemistry; D054852:Small Molecule Libraries / Q000737:chemistry; D013050:Spectrometry, Fluorescence
chemical_list: D065095:Calcineurin Inhibitors; D005456:Fluorescent Dyes; D012394:Rosaniline Dyes; D054852:Small Molecule Libraries; C005095:malachite green