title: Identification of NPC1 as the target of U18666A, an inhibitor of lysosomal cholesterol export and Ebola infection.
abstract: Niemann-Pick C1 (NPC1) is a lysosomal membrane protein that exports cholesterol derived from receptor-mediated uptake of LDL, and it also mediates cellular entry of Ebola virus. Cholesterol export is inhibited by nanomolar concentrations of U18666A, a cationic sterol. To identify the target of U18666A, we synthesized U-X, a U18666A derivative with a benzophenone that permits ultraviolet-induced crosslinking. When added to CHO cells, U-X crosslinked to NPC1. Crosslinking was blocked by U18666A derivatives that block cholesterol export, but not derivatives lacking blocking activity. Crosslinking was prevented by point mutation in the sterol-sensing domain (SSD) of NPC1, but not by point mutation in the N-terminal domain (NTD). These data suggest that the SSD contains a U18666A-inhibitable site required for cholesterol export distinct from the cholesterol-binding site in the NTD. Inasmuch as inhibition of Ebola requires 100-fold higher concentrations of U18666A, the high affinity U16888A-binding site is likely not required for virus entry.
authors.affiliation: Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, United States.
authors.forename: Feiran
authors.initials: F
authors.lastname: Lu
authors.affiliation: Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, United States.
authors.forename: Qiren
authors.initials: Q
authors.lastname: Liang
authors.affiliation: Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, United States.
authors.forename: Lina
authors.initials: L
authors.lastname: Abi-Mosleh
authors.affiliation: Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, United States.
authors.forename: Akash
authors.initials: A
authors.lastname: Das
authors.affiliation: Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, United States.
authors.forename: Jef K
authors.initials: JK
authors.lastname: De Brabander
authors.affiliation: Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, United States.
authors.forename: Joseph L
authors.initials: JL
authors.lastname: Goldstein
authors.affiliation: Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, United States.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Brown
mesh_terms: D000736:Androstenes / Q000494:pharmacology*; D000818:Animals; D000924:Anticholesteremic Agents / Q000494:pharmacology*; D000998:Antiviral Agents / Q000494:pharmacology*; D016466:CHO Cells; D002352:Carrier Proteins / Q000037:antagonists & inhibitors*; D002784:Cholesterol / Q000378:metabolism*; D003412:Cricetulus; D029043:Ebolavirus / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors*; D000077296:Niemann-Pick C1 Protein; D053586:Virus Internalization / Q000187:drug effects
chemical_list: D000736:Androstenes; D000924:Anticholesteremic Agents; D000998:Antiviral Agents; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; 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; D002784:Cholesterol
keywords: Niemann-Pick C1; U18666A; biochemistry; cell biology; cholesterol transport; crosslinking; endosomes/lysosomes; human; sterol-sensing domain