title: A simplified scintillation proximity assay for fatty acid synthase activity: development and comparison with other FAS activity assays.
abstract: Fatty acid synthase (FAS), an essential enzyme for de novo lipogenesis, has been implicated in a number of disease states, including obesity, dyslipidemia, and cancer. To identify small-molecule inhibitors of FAS, the authors developed a bead-based scintillation proximity assay (SPA) to detect the fatty acid products of FAS enzymatic activity. This homogeneous SPA assay discriminates between a radiolabeled hydrophilic substrate of FAS (acetyl-coenzyme A) and the labeled lipophilic products of FAS (fatty acids), generating signal only when labeled fatty acids are present. The assay requires a single addition of unmodified polystyrene imaging SPA beads and can be miniaturized to 384- or 1536-well density with appropriate assay statistics for high-throughput screening. High-potency FAS inhibitors were used to compare the sensitivity of the SPA bead assay with previously described assays that measure FAS reaction intermediates (CoA-SH and NADP+). The advantages and disadvantages of these different FAS assays in small-molecule inhibitor discovery are discussed.
authors.affiliation: Department of In Vitro Sciences, Merck Research Laboratories, Boston, Massachusetts, USA. nathan_bays@merck.com
authors.forename: Nathan W
authors.initials: NW
authors.lastname: Bays
authors.forename: Armetta D
authors.initials: AD
authors.lastname: Hill
authors.forename: Ilona
authors.initials: I
authors.lastname: Kariv
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism; D001681:Biological Assay / Q000379:methods*; D002460:Cell Line; D064429:Fatty Acid Synthases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D009249:NADP / Q000378:metabolism; D010168:Palmitates / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D017186:Titrimetry
chemical_list: D010168:Palmitates; D009249:NADP; D000105:Acetyl Coenzyme A; D064429:Fatty Acid Synthases