title: Automated microscopy screening for compounds that partially revert cholesterol accumulation in Niemann-Pick C cells.
abstract: 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 microM. 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 microM in reducing the cholesterol accumulation in cells with a NPC1 phenotype.
authors.affiliation: Department of Biochemistry, Weill Medical College of Cornell University, New York, NY 10021, USA.
authors.forename: Nina H
authors.initials: NH
authors.lastname: Pipalia
authors.forename: Amy
authors.initials: A
authors.lastname: Huang
authors.forename: Harold
authors.initials: H
authors.lastname: Ralph
authors.forename: Madalina
authors.initials: M
authors.lastname: Rujoi
authors.forename: Frederick R
authors.initials: FR
authors.lastname: Maxfield
mesh_terms: D000736:Androstenes / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D016466:CHO Cells; D002352:Carrier Proteins / Q000235:genetics; D002470:Cell Survival / Q000187:drug effects; D002784:Cholesterol / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D005372:Filipin / Q000737:chemistry; D006571:Heterocyclic Compounds / Q000494:pharmacology*; D006574:Heterocyclic Compounds, 2-Ring / Q000494:pharmacology; D006575:Heterocyclic Compounds, 3-Ring / Q000494:pharmacology; D006576:Heterocyclic Compounds, 4 or More Rings / Q000494:pharmacology; D006801:Humans; D007091:Image Processing, Computer-Assisted; D047908:Intracellular Signaling Peptides and Proteins; D008247:Lysosomes / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics; D008853:Microscopy / Q000379:methods*; D008856:Microscopy, Fluorescence; D015394:Molecular Structure; D009154:Mutation / Q000235:genetics; D000077296:Niemann-Pick C1 Protein; D009542:Niemann-Pick Diseases / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling
chemical_list: D000736:Androstenes; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D006571:Heterocyclic Compounds; D006574:Heterocyclic Compounds, 2-Ring; D006575:Heterocyclic Compounds, 3-Ring; D006576:Heterocyclic Compounds, 4 or More Rings; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; C106881:NPC1 protein, human; D000077296:Niemann-Pick C1 Protein; C006261:3-beta-(2-(diethylamino)ethoxy)androst-5-en-17-one; D005372:Filipin; D002784:Cholesterol