Development and validation of a high-throughput screening assay for human long-chain fatty acid transport proteins 4 and 5

Dietary long-chain fatty acid (LCFA) uptake across cell membranes is mediated principally by fatty acid transport proteins (FATPs). Six subtypes of this transporter are differentially expressed throughout the human and rodent body. To facilitate drugs discovery against FATP subtypes, the authors used mammalian cell lines stably expressing the recombinant human FATP4 and 5 and developed a high-throughput screening (HTS) assay using a 96-well fluorometric imaging plate reader (FLIPR). LCFA uptake signal-to-background ratios were between 3- and 5-fold. Two 4-aryl-dihydropyrimidinones, j3 and j5, produced inhibition of FATP4 with a half-maximal inhibitory concentration (IC(50)) of 0.21 and 0.63 microM, respectively, and displayed approximately 100-fold selectivity over FATP5. The US Drug Collection library was screened against the FATP5. A hit rate of around 0.4% was observed with a Z' factor of 0.6 +/- 0.2. Two confirmed hits are bile acids, chenodiol and ursodiol with an IC(50) of 2.4 and 0.22 microM, respectively. To increase throughput, a single time point measurement in a 384-well format was developed using the Analyst HT, and the results are comparable with the 96-well format. In conclusion, the FATP4 and 5 cell-based fluorescence assays are suitable for a primary drug screen, whereas differentiated cell lines are useful for a secondary drug screen.