| A miniaturized cell-based fluorescence resonance energy transfer assay for insulin-receptor activation. | |
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MedLine Citation:
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PMID: 16797469 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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This report describes the development, optimization, and implementation of a miniaturized cell-based assay for the identification of small-molecule insulin mimetics and potentiators. Cell-based assays are attractive formats for compound screening because they present the molecular targets in their cellular environment. A fluorescence resonance energy transfer (FRET) cell-based assay that measures the insulin-dependent colocalization of Akt2 fused with either cyan fluorescent protein or yellow fluorescent protein to the cellular membrane was developed. This ratiometric FRET assay was miniaturized into a robust, yet sensitive 3456-well nanoplate assay with Z' factors of approximately 0.6 despite a very small assay window (less than twofold full activation with insulin). The FRET assay was used for primary screening of a large compound collection for insulin-receptor agonists and potentiators. To prioritize compounds for further development, primary hits were tested in two additional assays, a biochemical time-resolved fluorescence resonance energy transfer assay to measure insulin-receptor phosphorylation and a translocation-based imaging assay. Results from the three assays were combined to yield 11 compounds as potential leads for the development of insulin mimetics or potentiators. |
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Authors:
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Shane Marine; Elize Zamiara; S Todd Smith; Erica M Stec; Jeremy McGarvey; Oleg Kornienko; Guoqiang Jiang; Kenny K Wong; Jeffrey H Stack; Bei B Zhang; Marc Ferrer; Berta Strulovici |
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Publication Detail:
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Type: Journal Article Date: 2006-06-09 |
Journal Detail:
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Title: Analytical biochemistry Volume: 355 ISSN: 0003-2697 ISO Abbreviation: Anal. Biochem. Publication Date: 2006 Aug |
Date Detail:
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Created Date: 2006-07-31 Completed Date: 2006-10-27 Revised Date: 2009-11-19 |
Medline Journal Info:
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Nlm Unique ID: 0370535 Medline TA: Anal Biochem Country: United States |
Other Details:
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Languages: eng Pagination: 267-77 Citation Subset: IM |
Affiliation:
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Department of Automated Biotechnology, Merck & Co., Inc., 502 Louise Lane, North Wales, PA 19454, USA. shane_marine@merck.com |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Active Transport, Cell Nucleus
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physiology Animals Bacterial Proteins / chemistry, metabolism Biological Assay / methods* CHO Cells Cell Membrane / metabolism* Cricetinae Fluorescence Resonance Energy Transfer / methods* Green Fluorescent Proteins / chemistry, metabolism Insulin / metabolism* Luminescent Proteins / chemistry, metabolism Nanotechnology Phosphorylation Proto-Oncogene Proteins c-akt / chemistry, metabolism Receptor, Insulin / analysis, metabolism* Time Factors |
| Chemical | |
Reg. No./Substance:
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0/Bacterial Proteins; 0/Cyan Fluorescent Protein; 0/Luminescent Proteins; 0/yellow fluorescent protein, Bacteria; 11061-68-0/Insulin; 147336-22-9/Green Fluorescent Proteins; EC 2.7.10.1/Receptor, Insulin; EC 2.7.11.1/Proto-Oncogene Proteins c-akt |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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