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Originally published In Press as doi:10.1194/jlr.M500388-JLR200 on November 15, 2005

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Journal of Lipid Research, Vol. 47, 284-301, February 2006
Copyright © 2006 by American Society for Biochemistry and Molecular Biology

Automated microscopy screening for compounds that partially revert cholesterol accumulation in Niemann-Pick C cells

Nina H. Pipalia1, Amy Huang1, Harold Ralph, Madalina Rujoi and Frederick R. Maxfield2

Department of Biochemistry, Weill Medical College of Cornell University, New York, NY 10021

Published, JLR Papers in Press, November 15, 2005.

1 N. H. Pipalia and A. Huang contributed equally to this work.

2 To whom correspondence should be addressed. e-mail: frmaxfie{at}med.cornell.edu

Niemann-Pick disease type C (NPC) is an autosomal recessive genetic disorder manifested by abnormal accumulation of unesterified cholesterol and other lipids. We screened combinatorially synthesized chemical libraries to identify compounds that would partially revert cholesterol accumulation. Cultured CHO cells with NPC phenotypes (CT60 and CT43) were used for screening along with normal CHO cells as a control. We developed an automated microscopy assay based on imaging of filipin fluorescence for estimating cholesterol accumulation in lysosomal storage organelles. Our primary screen of 14,956 compounds identified 14 hit compounds that caused significant reduction in cellular cholesterol accumulation at 10 µM. We then screened a secondary library of 3,962 compounds selected based on chemical similarity to the initial hits and identified 7 compounds that demonstrated greater efficacy and lower toxicity than the original hits. These compounds are effective at concentrations of 123 nM to 3 µM in reducing the cholesterol accumulation in cells with a NPC1 phenotype.

Supplementary key words high-content screening • filipin • lysosomal storage

Abbreviations: BMP, bis-(monoacylglycero)-phosphate; DiI-LDL, low density lipoprotein labeled with C18-dialkyl-indocarbocyanine; FC, free cholesterol; LBPA, lyso-bisphosphatidic acid; LDH, lactate dehydrogenase; LSO, lysosome-like storage organelle; NPC, Niemann-Pick disease type C; PFA, paraformaldehyde; SCAP, sterol-regulatory element binding protein cleavage-activating protein; U18666A, 3-ß-[2-diethylaminoethoxy]androst-5-17-one hydrochloride


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