title: Fluorine NMR-based screening on cell membrane extracts.
abstract: The possibility of measuring the action of inhibitors of specific enzymatic reactions in intact cells, cell lysates or membrane preparations represents a major advance in the lead discovery process. Despite the relevance of assaying in physiological conditions, only a small number of biophysical techniques, often requiring complex set-up, are applicable to these sample types. Here, we demonstrate the first application of n-fluorine atoms for biochemical screening (n-FABS), a homogeneous and versatile assay based on (19) F NMR spectroscopy, to the detection of high- and low-affinity inhibitors of a membrane enzyme in cell extracts and determination of their IC50 values. Our approach can allow the discovery of novel binding fragments against targets known to be difficult to purify or where membrane-association is required for activity. These results pave the way for future applications of the methodology to these relevant and complex biological systems.
authors: Department of Drug Discovery & Development, Fondazione Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genoa (Italy).
authors: Marina
authors: M
authors: Veronesi
authors: Elisa
authors: E
authors: Romeo
authors: Chiara
authors: C
authors: Lambruschini
authors: Daniele
authors: D
authors: Piomelli
authors: Tiziano
authors: T
authors: Bandiera
authors: Rita
authors: R
authors: Scarpelli
authors: Gianpiero
authors: G
authors: Garau
authors: Claudio
authors: C
authors: Dalvit
mesh_terms: D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology*; D055808:Drug Discovery / Q000379:methods*; D005461:Fluorine / Q000032:analysis; D006801:Humans; D008565:Membrane Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods
chemical_list: D008565:Membrane Proteins; D005461:Fluorine
keywords: 19F NMR spectroscopy; drug discovery; fragments; functional screening; inhibitors; membrane proteins