A multiplexed cell assay in HepG2 cells for the identification of delta-5, delta-6, and delta-9 desaturase and elongase inhibitors

A multiplexed cell assay has been optimized to measure the activities of fatty acyl-CoA elongase, delta-5 desaturase (Delta5D), delta-6 desaturase (Delta6D), and delta-9 desaturase (Delta9D) together using (14)C-labeled tracers in HepG2 cells, which express the human stearoyl-CoA desaturase-1 isoform (SCD1) exclusively. The Delta5 and Delta9 desaturase activities are indexed by the efficient conversion of [1-(14)C]-eicosatrienoic acid (C20:3, cis-8,11,14) to (14)C-arachidonic acid (C20:4, cis-5,8,11,14) and the conversion of [1-(14)C]-stearic acid to (14)C-oleic acid (C18:1, cis-9), respectively. CP-74006 potently blocks the Delta5D activity with an IC(50) value of 20 nM and simplifies the metabolism of [1-(14)C]-alpha-linolenate (C18:3, cis-9,12,15) by accumulating (14)C-eicosatetraenoic acid (C20:4, cis-8,11,14,17) as the major (14)C-eicosatrienoic acid (C20:3, cis-11,14,17) and (14)C-docosatetraenoic acid (C22:4, cis-10,13,16,19) as the minor metabolites through Delta6 desaturation and elongation. This simplified metabolite spectrum enables the delineation of the Delta6D activity by comparing the combined Delta6D/elongase activity index of the (14)C-(C20:4/C18:3) ratio with the corresponding elongation index of the (14)C-(C20:3/C18:3) ratio following compound treatment. SC-26196 and sterculic acid specifically inhibit the Delta6D and Delta9D activities with an IC(50) value of 0.1 microM and 0.9 microM, respectively. This medium-throughput cell assay provides an efficient tool in the identification of specific desaturase and elongase inhibitors.