title: Development of a Medium-Throughput Targeted LCMS Assay to Detect Endogenous Cellular Levels of Malonyl-CoA to Screen Fatty Acid Synthase Inhibitors.
abstract: The fatty acid synthase (FAS) enzyme in mammalian cells is a large multidomain protein responsible for de novo synthesis of fatty acids. The steps catalyzed by FAS involve the condensation of acetyl-CoA and malonyl-CoA moieties in the presence of NADPH until palmitate is formed. Inhibition of FAS causes an accumulation of intracellular malonyl-CoA, as this metabolite is essentially committed to fatty acid synthesis once formed. Detection of intracellular metabolites for screening can be problematic due to a lack of appropriate tools, but here we describe a targeted liquid chromatography-mass spectroscopy (LCMS) method to directly measure endogenous levels of malonyl-CoA to drive a drug development structure-activity relationship (SAR) screening cascade. Our process involves preparation of samples at 96-well scale, normalization postpermeabilization via use of a whole-well imaging platform, and the LCMS detection methodology. The assay is amenable to multiplexing cellular endpoints, has a typical Z' of >0.6, and has high reproducibility of EC50 values.
authors.affiliation: Discovery Sciences, AstraZeneca, Cambridge, UK Philip.hopcroft@astrazeneca.com.
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Hopcroft
authors.affiliation: Discovery Sciences, AstraZeneca, Cambridge, UK.
authors.forename: David I
authors.initials: DI
authors.lastname: Fisher
mesh_terms: D001681:Biological Assay / Q000379:methods*; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry*; D064429:Fatty Acid Synthases / Q000037:antagonists & inhibitors*; D005227:Fatty Acids / Q000737:chemistry; D006801:Humans; D008316:Malonyl Coenzyme A / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D009249:NADP / Q000737:chemistry; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D005227:Fatty Acids; D008316:Malonyl Coenzyme A; D009249:NADP; D064429:Fatty Acid Synthases
keywords: LCMS; fatty acid synthase; malonyl-CoA; screening