FRET-based calcium imaging: a tool for high-throughput/content phenotypic drug screening in Alzheimer disease.
Perturbed intracellular store calcium homeostasis is suggested to play a major role in the pathophysiology of Alzheimer disease (AD). A number of mechanisms have been suggested to underlie the impairment of endoplasmic reticulum calcium homeostasis associated with familial AD-linked presenilin 1 mutations (FAD-PS1). Without aiming at specifically targeting any of those pathophysiological mechanisms in particular, we rather performed a high-throughput phenotypic screen to identify compounds that can reverse the exaggerated agonist-evoked endoplasmic reticulum calcium release phenotype in HEK293 cells expressing FAD-PS1. For that purpose, we developed a fully automated high-throughput calcium imaging assay using a fluorescence resonance energy transfer-based calcium indicator at single-cell resolution. This novel robust assay offers a number of advantages compared with the conventional calcium measurement screening technologies. The assay was employed in a large-scale screen with a library of diverse compounds comprising 20,000 low-molecular-weight molecules, which resulted in the identification of 52 primary hits and 4 lead structures. In a secondary assay, several hits were found to alter the amyloid β (Aβ) production. In view of the recent failure of AD drug candidates identified by target-based approaches, such a phenotypic drug discovery paradigm may present an attractive alternative for the identification of novel AD therapeutics.
1Department of Translational Brain Research, DZNE-German Center for Neurodegenerative Diseases, Munich, Germany.
Kamran
K
Honarnejad
Achim K
AK
Kirsch
Alexander
A
Daschner
Aleksandra
A
Szybinska
Jacek
J
Kuznicki
Jochen
J
Herms
D000544:Alzheimer Disease / Q000188:drug therapy*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects*; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays / Q000379:methods; D006706:Homeostasis; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010641:Phenotype; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D002148:Calmodulin-Binding Proteins; C485182:Cyan Fluorescent Protein; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D002217:Carbachol
Alzheimer disease; FRET; calcium; high-throughput screening; image analysis; phenotypic drug discovery