title: Triazoles inhibit cholesterol export from lysosomes by binding to NPC1.
abstract: Niemann-Pick C1 (NPC1), a membrane protein of lysosomes, is required for the export of cholesterol derived from receptor-mediated endocytosis of LDL. Lysosomal cholesterol export is reportedly inhibited by itraconazole, a triazole that is used as an antifungal drug [Xu et al. (2010) Proc Natl Acad Sci USA 107:4764-4769]. Here we show that posaconazole, another triazole, also blocks cholesterol export from lysosomes. We prepared P-X, a photoactivatable cross-linking derivative of posaconazole. P-X cross-linked to NPC1 when added to intact cells. Cross-linking was inhibited by itraconazole but not by ketoconazole, an imidazole that does not block cholesterol export. Cross-linking of P-X was also blocked by U18666A, a compound that has been shown to bind to NPC1 and inhibit cholesterol export. P-X also cross-linked to purified NPC1 that was incorporated into lipid bilayer nanodiscs. In this in vitro system, cross-linking of P-X was inhibited by itraconazole, but not by U18666A. P-X cross-linking was not prevented by deletion of the N-terminal domain of NPC1, which contains the initial binding site for cholesterol. In contrast, P-X cross-linking was reduced when NPC1 contained a point mutation (P691S) in its putative sterol-sensing domain. We hypothesize that the sterol-sensing domain has a binding site that can accommodate structurally different ligands.
authors.affiliation: Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390.
authors.forename: Michael N
authors.initials: MN
authors.lastname: Trinh
authors.affiliation: Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390.
authors.forename: Feiran
authors.initials: F
authors.lastname: Lu
authors.affiliation: Laboratory of Cell Biology, The Rockefeller University, New York, NY 10065.|Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10065.
authors.forename: Xiaochun
authors.initials: X
authors.lastname: Li
authors.affiliation: Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390.
authors.forename: Akash
authors.initials: A
authors.lastname: Das
authors.affiliation: Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390.
authors.forename: Qiren
authors.initials: Q
authors.lastname: Liang
authors.affiliation: Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390.
authors.forename: Jef K
authors.initials: JK
authors.lastname: De Brabander
authors.affiliation: Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390; mike.brown@utsouthwestern.edu joe.goldstein@utsouthwestern.edu.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Brown
authors.affiliation: Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390; mike.brown@utsouthwestern.edu joe.goldstein@utsouthwestern.edu.
authors.forename: Joseph L
authors.initials: JL
authors.lastname: Goldstein
mesh_terms: D000736:Androstenes / Q000494:pharmacology; D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology; D001665:Binding Sites / Q000235:genetics; D001692:Biological Transport / Q000235:genetics*; D016466:CHO Cells; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism*; D003412:Cricetulus; D004705:Endocytosis / Q000502:physiology; D017964:Itraconazole / Q000494:pharmacology; D007654:Ketoconazole / Q000494:pharmacology; D008247:Lysosomes / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D000072417:Protein Domains / Q000235:genetics; D014230:Triazoles / Q000494:pharmacology*
chemical_list: D000736:Androstenes; D000935:Antifungal Agents; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D014230:Triazoles; C006261:3-beta-(2-(diethylamino)ethoxy)androst-5-en-17-one; D017964:Itraconazole; C101425:posaconazole; D002784:Cholesterol; D007654:Ketoconazole
keywords: Niemann–Pick C disease; cholesterol transport; lipid nanodiscs; photoactivatable cross-linking; sterol-sensing domain