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- Title
A Fluorescence-Based Thiol Quantification Assay for Ultra-High-Throughput Screening for Inhibitors of Coenzyme A Production.
- Authors
Christine C. Chung; Kenji Ohwaki; Jonathan E. Schneeweis; Erica Stec; Jeffrey P. Varnerin; Paul N. Goudreau; Amy Chang; Jason Cassaday; Lihu Yang; Takeru Yamakawa; Oleg Kornienko; Peter Hodder; James Inglese; Marc Ferrer; Berta Strulovici; Jun Kusunoki; Michael R. Tota; Toshimitsu Takagi
- Abstract
Abstract:Here we report the development and miniaturization of a cell-free enzyme assay for ultra-high-throughput screening (uHTS) for inhibitors of two potential drug targets for obesity and cancer: fatty acid synthase (FAS) and acetyl-coenzyme A (CoA) carboxylase (ACC) 2. This assay detects CoA, a product of the FAS-catalyzed condensation of malonyl-CoA and acetyl-CoA. The free thiol of CoA can react with 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin (CPM), a profluorescent coumarin maleimide derivative that becomes fluorescent upon reaction with thiols. FAS produces long-chain fatty acid and CoA from the condensation of malonyl-CoA and acetyl-CoA. In our FAS assay, CoA released in the FAS reaction forms a fluorescence adduct with CPM that emits at 530 nm when excited at 405 nm. Using this detection method for CoA, we measured the activity of sequential enzymes in the fatty acid synthesis pathway to develop an ACC2/FAS-coupled assay where ACC2 produces malonyl-CoA from acetyl-CoA. We miniaturized the FAS and ACC2/FAS assays to 3,456- and 1,536-well plate format, respectively, and completed uHTSs for small molecule inhibitors of this enzyme system. This report shows the results of assay development, miniaturization, and inhibitor screening for these potential drug targets.
- Subjects
ENZYMES; TARGETED drug delivery; OBESITY; CANCER; FATTY acids
- Publication
Assay & Drug Development Technologies, 2008, Vol 6, Issue 3, p361
- ISSN
1540-658X
- Publication type
Article
- DOI
10.1089/adt.2007.105