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Identification and characterization of inhibitors of hsFATP‐dependent fatty acid uptake in humanized yeast and human Caco‐2 cells
Author(s) -
SandovalAlvarez Angel,
Chokshi Aalap,
Singer Diane,
Black Paul N.,
DiRusso Concetta C.
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1040.3
Subject(s) - caco 2 , yeast , fatty acid , biochemistry , ec50 , cytotoxicity , cell culture , bodipy , viability assay , chemistry , in vitro , high throughput screening , biology , fluorescence , genetics , physics , quantum mechanics
We have employed a live‐cell high‐throughput method to screen for compounds that inhibit FATP‐mediated fatty acid uptake. Over 100,000 compounds from 3 chemically diverse compound libraries were screened. After preliminary selection using yeast expressing hsFATP2, secondary screens were performed to eliminate compounds that were autofluorescent, quenched fluorescence of the fatty acid analogue C1‐BODIPY‐C12 used in the primary screen, and permeabilized or were otherwise toxic to yeast. The initial set of selected hits included 145 compounds. Each potential hit was secondarily screened for inhibition of C1‐BODIPY‐C12 import into human Caco2 cells and this reduced the subset of potential hits to 22 compounds. Among these, six compounds were chosen as representative of a unique structural class for further characterization. Sigmoid dose‐response curves for C1‐BODIPY‐C12 uptake were generated in Caco‐2 cells and EC50 values were within the lower μM range. The compounds were determined to be non‐toxic to Caco‐2 cells using the MTT cytotoxicity assay. Teer tests confirmed that the integrity of the epithelial cell barrier of the intestinal cell line was not disrupted. As expected for a transport inhibitor, treatment with compound suppressed incorporation of radiolabelled fatty acids into all classes of lipids without suppressing acyl‐CoA synthetase activity. Supported by DK07076 from the NIH (CD and PNB).