title: A fluorescence-based thiol quantification assay for ultra-high-throughput screening for inhibitors of coenzyme A production.
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.
authors.affiliation: Department of Metabolic Disorders, Merck Research Laboratories, Merck & Co., Inc., Rahway, NJ, USA.
authors.forename: Christine C
authors.initials: CC
authors.lastname: Chung
authors.forename: Kenji
authors.initials: K
authors.lastname: Ohwaki
authors.forename: Jonathan E
authors.initials: JE
authors.lastname: Schneeweis
authors.forename: Erica
authors.initials: E
authors.lastname: Stec
authors.forename: Jeffrey P
authors.initials: JP
authors.lastname: Varnerin
authors.forename: Paul N
authors.initials: PN
authors.lastname: Goudreau
authors.forename: Amy
authors.initials: A
authors.lastname: Chang
authors.forename: Jason
authors.initials: J
authors.lastname: Cassaday
authors.forename: Lihu
authors.initials: L
authors.lastname: Yang
authors.forename: Takeru
authors.initials: T
authors.lastname: Yamakawa
authors.forename: Oleg
authors.initials: O
authors.lastname: Kornienko
authors.forename: Peter
authors.initials: P
authors.lastname: Hodder
authors.forename: James
authors.initials: J
authors.lastname: Inglese
authors.forename: Marc
authors.initials: M
authors.lastname: Ferrer
authors.forename: Berta
authors.initials: B
authors.lastname: Strulovici
authors.forename: Jun
authors.initials: J
authors.lastname: Kusunoki
authors.forename: Michael R
authors.initials: MR
authors.lastname: Tota
authors.forename: Toshimitsu
authors.initials: T
authors.lastname: Takagi
mesh_terms: D000103:Acetyl-CoA Carboxylase / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D000818:Animals; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D054890:Fatty Acid Synthase, Type I / Q000037:antagonists & inhibitors* / Q000378:metabolism; D005453:Fluorescence; D006801:Humans; D051381:Rats; D013438:Sulfhydryl Compounds / Q000032:analysis*
chemical_list: D004791:Enzyme Inhibitors; D013438:Sulfhydryl Compounds; D054890:Fatty Acid Synthase, Type I; D000103:Acetyl-CoA Carboxylase