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.
Zhou
Wei
W
Biosciences Division, SRI International, Menlo Park, CA 94063, USA.
Madrid
Peter
P
Fluitt
Amy
A
Stahl
Andreas
A
Xie
Xinmin Simon
XS
D000818:Animals; D002460:Cell Line; D050612:Fatty Acid Transport Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D051379:Mice; D015394:Molecular Structure; D011744:Pyrimidinones / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results
D050612:Fatty Acid Transport Proteins; D005456:Fluorescent Dyes; D011744:Pyrimidinones; C493571:SLC27A4 protein, human; C484819:SLC27A5 protein, human
Gastroenterology. 2006 Apr;130(4):1259-69
16618417
Metabolism. 2006 Jan;55(1):8-12
16324913
Biochim Biophys Acta. 2004 Nov 8;1686(1-2):1-6
15522816
Pflugers Arch. 2004 Feb;447(5):722-7
12856180
Am J Physiol Endocrinol Metab. 2002 Mar;282(3):E593-600
11832362
Curr Opin Lipidol. 2007 Jun;18(3):271-6
17495600
J Lipid Res. 2005 Mar;46(3):597-602
15547301
J Cell Biol. 2003 Jun 23;161(6):1105-15
12821645
Curr Opin Lipidol. 2005 Apr;16(2):173-8
15767857
J Lipid Res. 2008 Jan;49(1):230-44
17928635
J Biomol Screen. 1999;4(2):67-73
10838414
Anal Biochem. 2005 Jan 1;336(1):11-9
15582553
Arch Biochem Biophys. 2008 Sep 15;477(2):363-71
18601897
Bioorg Med Chem Lett. 2006 Jul 1;16(13):3504-9
16644217
Biochim Biophys Acta. 2005 Oct 1;1736(3):163-80
16198626
Prog Lipid Res. 2009 Jan;48(1):52-61
19041341
Eur J Med Res. 1996 Jan 19;1(4):176-80
9386266
Dev Cell. 2002 Apr;2(4):477-88
11970897
Trends Endocrinol Metab. 2001 Aug;12(6):266-73
11445444
Am J Physiol Endocrinol Metab. 2001 Jun;280(6):E1000-6
11350782
Am J Physiol. 1996 May;270(5 Pt 1):E873-81
8967477
Mol Cell. 1999 Sep;4(3):299-308
10518211
J Biol Chem. 2003 Dec 5;278(49):49512-6
14512415
Mol Cell Biochem. 2006 Mar;284(1-2):135-40
16477381
J Lipid Res. 2006 Mar;47(3):665-72
16357361
Gastroenterology. 2006 Apr;130(4):1245-58
16618416
Biochim Biophys Acta. 1998 Dec 22;1443(3):381-5
9878842
Front Biosci. 1997 May 15;2:d222-31
9206980
Diabetes Care. 2009 Nov;32 Suppl 2:S237-45
19875558
Proc Nutr Soc. 2004 May;63(2):259-62
15294040
J Biol Chem. 2003 May 2;278(18):16039-44
12556534
Assay Drug Dev Technol. 2007 Apr;5(2):191-203
17477828
Lipids. 2001 Sep;36(9):981-9
11724471