title: A miniaturized cell-based fluorescence resonance energy transfer assay for insulin-receptor activation.
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 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.affiliation: Department of Automated Biotechnology, Merck & Co., Inc., 502 Louise Lane, North Wales, PA 19454, USA. shane_marine@merck.com
authors.forename: Shane
authors.initials: S
authors.lastname: Marine
authors.forename: Elize
authors.initials: E
authors.lastname: Zamiara
authors.forename: S Todd
authors.initials: ST
authors.lastname: Smith
authors.forename: Erica M
authors.initials: EM
authors.lastname: Stec
authors.forename: Jeremy
authors.initials: J
authors.lastname: McGarvey
authors.forename: Oleg
authors.initials: O
authors.lastname: Kornienko
authors.forename: Guoqiang
authors.initials: G
authors.lastname: Jiang
authors.forename: Kenny K
authors.initials: KK
authors.lastname: Wong
authors.forename: Jeffrey H
authors.initials: JH
authors.lastname: Stack
authors.forename: Bei B
authors.initials: BB
authors.lastname: Zhang
authors.forename: Marc
authors.initials: M
authors.lastname: Ferrer
authors.forename: Berta
authors.initials: B
authors.lastname: Strulovici
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001681:Biological Assay / Q000379:methods*; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D007328:Insulin / Q000378:metabolism*; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism; D036103:Nanotechnology; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000737:chemistry / Q000378:metabolism; D011972:Receptor, Insulin / Q000032:analysis / Q000378:metabolism*; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D007328:Insulin; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D011972:Receptor, Insulin; D051057:Proto-Oncogene Proteins c-akt