title: FRET-based imaging of transbilayer movement of pepducin in living cells by novel intracellular bioreductively activatable fluorescent probes.
abstract: To elucidate the mechanisms of direct transmembrane penetration of pepducins, which are artificial lipopeptide G protein-coupled receptor (GPCR) modulators, we developed two types of FRET-based probes, Pep13-FL-SS-Dab (13) targeting the inner leaflet of the lipid bilayer and Pep13-Dab-SS-FL (14) targeting the cytosol, respectively. They are composed of a pepducin moiety and a fluorescent switch component consisting of 5(6)-carboxyfluorescein (FAM) as a fluorophore and dabcyl as a quencher connected through disulfide bond linkage. When they are internalized into the cytosol, intracellular glutathione can cleave the disulfide bond to release the quencher, which results in a turn-on fluorescence signal. Using these probes, we performed live cell imaging of transbilayer movements of pepducins on MCF-7 cells for the first time. The results suggested that the lipid moiety of the probes facilitated pepducin flipping across and tethering to the membrane. The present study raises the possibility of applying the probe architecture for direct intracellular drug delivery.
authors.affiliation: Laboratory of Pharmaceutical and Medicinal Chemistry, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu, 501-1196, Japan.
authors.forename: Mieko
authors.initials: M
authors.lastname: Tsuji
authors.forename: Satoshi
authors.initials: S
authors.lastname: Ueda
authors.forename: Tasuku
authors.initials: T
authors.lastname: Hirayama
authors.forename: Kensuke
authors.initials: K
authors.lastname: Okuda
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Sakaguchi
authors.forename: Aoi
authors.initials: A
authors.lastname: Isono
authors.forename: Hideko
authors.initials: H
authors.lastname: Nagasawa
mesh_terms: D000814:Aniline Compounds / Q000737:chemistry* / Q000378:metabolism; D001692:Biological Transport; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D005452:Fluoresceins / Q000737:chemistry* / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry; D006801:Humans; D008051:Lipid Bilayers / Q000378:metabolism; D018613:Microscopy, Confocal; D015394:Molecular Structure; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000814:Aniline Compounds; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008051:Lipid Bilayers; C583995:Pep13-Dab-SS-FL; C583994:Pep13-FL-SS-Dab; D010455:Peptides