A miniaturized cell-based fluorescence resonance energy transfer assay for insulin-receptor activation

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

Research output: Contribution to journalArticlepeer-review

10 Scopus citations

Abstract

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 ∼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.

Original languageEnglish
Pages (from-to)267-277
Number of pages11
JournalAnalytical Biochemistry
Volume355
Issue number2
DOIs
StatePublished - 15 Aug 2006

Keywords

  • CFP
  • FRET
  • HTS
  • Imaging
  • Insulin Receptor
  • YFP

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