A Multiplexed Cell Assay in HepG2 Cells for the Identification of Delta-5, Delta-6, and Delta-9 Desaturase and Elongase Inhibitors

Abstract

A multiplexed cell assay has been optimized to measure the activities of fatty acyl-CoA elongase, delta-5 desaturase (Δ5D), delta-6 desaturase (Δ6D), and delta-9 desaturase (Δ9D) together using14C-labeled tracers in HepG2 cells, which express the human stearoyl-CoA desaturase-1 isoform (SCD1) exclusively. The Δ5 and Δ9 desaturase activities are indexed by the efficient conversion of [1-14C]-eicosatrienoic acid (C20:3, cis-8,11,14) to14C-arachidonic acid (C20:4, cis-5,8,11,14) and the conversion of [1-14C]-stearic acid to14C-oleic acid (C18:1, cis-9), respectively. CP-74006 potently blocks the Δ5D activity with an IC50value of 20 nM and simplifies the metabolism of [1-14C]-α-linolenate (C18:3, cis-9,12,15) by accumulating14C-eicosatetraenoic acid (C20:4, cis-8,11,14,17) as the major14C-eicosatrienoic acid (C20:3, cis-11,14,17) and14C-docosatetraenoic acid (C22:4, cis-10,13,16,19) as the minor metabolites through Δ6 desaturation and elongation. This simplified metabolite spectrum enables the delineation of the Δ6D activity by comparing the combined Δ6D/elongase activity index of the14C-(C20:4/C18:3) ratio with the corresponding elongation index of the14C-(C20:3/C18:3) ratio following compound treatment. SC-26196 and sterculic acid specifically inhibit the Δ6D and Δ9D activities with an IC50value of 0.1 µM and 0.9 µM, respectively. This medium-throughput cell assay provides an efficient tool in the identification of specific desaturase and elongase inhibitors.