title: 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 (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.
authors.affiliation: Merck Frosst Center for Therapeutic Research, Montreal, Canada.
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.forename: Yeeman
authors.initials: Y
authors.lastname: Ramtohul
authors.forename: Sebastien
authors.initials: S
authors.lastname: Gagné
authors.forename: Angela
authors.initials: A
authors.lastname: Styhler
authors.forename: Hao
authors.initials: H
authors.lastname: Wang
authors.forename: Jocelyne
authors.initials: J
authors.lastname: Guay
authors.forename: Zheng
authors.initials: Z
authors.lastname: Huang
mesh_terms: D000123:Acetyltransferases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D000214:Acyl Coenzyme A / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D001681:Biological Assay; D002250:Carbon Radioisotopes; D000090943:Delta-5 Fatty Acid Desaturase; D044943:Fatty Acid Desaturases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D000081031:Fatty Acid Elongases; D056945:Hep G2 Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D050602:Linoleoyl-CoA Desaturase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D008954:Models, Biological; D008956:Models, Chemical
chemical_list: D000214:Acyl Coenzyme A; D002250:Carbon Radioisotopes; D000090943:Delta-5 Fatty Acid Desaturase; C017725:stearoyl-coenzyme A; D044943:Fatty Acid Desaturases; D050602:Linoleoyl-CoA Desaturase; D000123:Acetyltransferases; D000081031:Fatty Acid Elongases