Document Detail


A miniaturized cell-based fluorescence resonance energy transfer assay for insulin-receptor activation.
MedLine Citation:
PMID:  16797469     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
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:
Type:  Journal Article     Date:  2006-06-09
Journal Detail:
Title:  Analytical biochemistry     Volume:  355     ISSN:  0003-2697     ISO Abbreviation:  Anal. Biochem.     Publication Date:  2006 Aug 
Date Detail:
Created Date:  2006-07-31     Completed Date:  2006-10-27     Revised Date:  2009-11-19    
Medline Journal Info:
Nlm Unique ID:  0370535     Medline TA:  Anal Biochem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  267-77     Citation Subset:  IM    
Affiliation:
Department of Automated Biotechnology, Merck & Co., Inc., 502 Louise Lane, North Wales, PA 19454, USA. shane_marine@merck.com
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MeSH Terms
Descriptor/Qualifier:
Active Transport, Cell Nucleus / 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:
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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