A live-cell high-throughput screening assay for identification of fatty acid uptake inhibitors.
We developed a live-cell high-throughput assay system using the baker's yeast Saccharomyces cerevisiae to screen for chemical compounds that will inhibit fatty acid uptake. The target for the inhibitors is a mammalian fatty acid transport protein (mmFATP2), which is involved in the fatty acid transport and activation pathway. The mmFATP2 was expressed in a S. cerevisiae mutant strain deficient in Fat1p-dependent fatty acid uptake and reduced in long-chain fatty acid activation, fat1Deltafaa1Delta. To detect fatty acid import, a fluorescent fatty acid analog, 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (C1-BODIPY-C12), was incubated with cells expressing FATP2 in a 96-well plate. The mmFATP2-dependent C1-BODIPY-C12 uptake was monitored by measuring intracellular C1-BODIPY-C12 fluorescence on a microtiter plate reader, whereas extracellular fluorescence was quenched by a cell viability dye, trypan blue. Using this high-throughput screening method, we demonstrate that the uptake of the fluorescent fatty acid ligand was effectively competed by the natural fatty acid oleate. Inhibition of uptake was also demonstrated to occur when cells were pretreated with sodium azide or Triacsin C. This yeast live-cell-based assay is rapid to execute, inexpensive to implement, and has adequate sensitivity for high-throughput screening. The assay basis and limitations are discussed.
Li
Hong
H
Center for Metabolic Diseases, Ordway Research Institute, Inc., Albany, NY 12208, USA.
Black
Paul N
PN
DiRusso
Concetta C
CC
D001692:Biological Transport / Q000187:drug effects; D001896:Boron Compounds; D003066:Coenzyme A Ligases / Q000378:metabolism; D005069:Evaluation Studies as Topic; D050612:Fatty Acid Transport Proteins; D005227:Fatty Acids / Q000032:analysis / Q000378:metabolism*; D005456:Fluorescent Dyes; D007850:Lauric Acids; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins / Q000037:antagonists & inhibitors*; D018613:Microscopy, Confocal; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D012680:Sensitivity and Specificity; D014343:Trypan Blue; D000098859:Long-Chain-Fatty-Acid-CoA Ligase
D001896:Boron Compounds; D050612:Fatty Acid Transport Proteins; D005227:Fatty Acids; D005456:Fluorescent Dyes; D007850:Lauric Acids; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins; C076936:4,4-difluoro-5-methyl-4-bora-3,4-diaza-3-indacenedodecanoic acid; D003066:Coenzyme A Ligases; D000098859:Long-Chain-Fatty-Acid-CoA Ligase; D014343:Trypan Blue