A Simplified Scintillation Proximity Assay for Fatty Acid Synthase Activity: Development and Comparison with Other FAS Activity Assays

Fatty acid synthase (F a S), an essential enzyme for de novo lipogenesis, has been implicated in a number of disease states, including obesity, dyslipidemia, and cancer. t o identify small-molecule inhibitors of F a S, the authors developed a bead- based scintillation proximity assay (SP a ) to detect the fatty acid products of F a S enzymatic activity. t his homogeneous SP a assay discriminates between a radiolabeled hydrophilic substrate of F a S (acetyl-coenzyme a ) and the labeled lipophilic products of F a S (fatty acids), generating signal only when labeled fatty acids are present. t he assay requires a single addition of unmodified polystyrene imaging SP beads and can be miniaturized to 384- or 1536-well density with appropriate assay statistics for high-throughput screening. h igh-potency F S inhibitors were used to compare the sensitivity of the SP a bead assay with previously described assays that measure F a S reaction intermediates ( c o a -S h and nad P + ). t he advantages and disadvantages of these different F a S assays in small-molecule inhibitor discovery are discussed. (Journal of Biomolecular Screening XXXX:xx-xx)