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Journal of Lipid Research, Vol. 49, 230-244, January 2008
Copyright © 2008 by American Society for Biochemistry and Molecular Biology

Methods

High-throughput screening for fatty acid uptake inhibitors in humanized yeast identifies atypical antipsychotic drugs that cause dyslipidemias

Hong Li, Paul N. Black, Aalap Chokshi, Angel Sandoval-Alvarez, Ravi Vatsyayan, Whitney Sealls and Concetta C. DiRusso¹

Center for Metabolic Disease, Ordway Research Institute, Inc., and Center for Cardiovascular Sciences, Albany Medical College, Albany, NY 12208

Published, JLR Papers in Press, October 10, 2007.

¹ To whom correspondence should be addressed. e-mail: cdirusso{at}ordwayresearch.org

Fatty acids are implicated in the development of dyslipidemias, leading to type 2 diabetes and cardiovascular disease. We used a standardized small compound library to screen humanized yeast to identify compounds that inhibit fatty acid transport protein (FATP)-mediated fatty acid uptake into cells. This screening procedure used live yeast cells expressing human FATP2 to identify small compounds that reduced the import of a fluorescent fatty acid analog, 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (C₁-BODIPY-C₁₂). The library used consisted of 2,080 compounds with known biological activities. Of these, 1.8% reduced cell-associated C₁-BODIPY-C₁₂ fluorescence and were selected as potential inhibitors of human FATP2-mediated fatty acid uptake. Based on secondary screens, 28 compounds were selected as potential fatty acid uptake inhibitors. Some compounds fell into four groups with similar structural features. The largest group was structurally related to a family of tricyclic, phenothiazine-derived drugs used to treat schizophrenia and related psychiatric disorders, which are also known to cause metabolic side effects, including hypertriglyceridemia. Potential hit compounds were studied for specificity of interaction with human FATP and efficacy in human Caco-2 cells. This study validates this screening system as useful to assess the impact of drugs in preclinical screening for fatty acid uptake.

Supplementary key words fatty acid transport protein • 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid • yeast • Caco-2

Abbreviations: ACSL, long-chain acyl-coenzyme A synthetase; AFU, arbitrary fluorescence unit; C₁-BODIPY-C₁₂, 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid; FAF BSA, fatty acid-free bovineserum albumin; FATP, fatty acid transport protein; HTS, high-throughput screening; K_i, inhibition constant; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; OD₆₀₀, optical density at 600 nm; YNBD, yeast minimal medium with dextrose; YPDA, yeast complete media with adenine

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