Identification of NPC1 as the target of U18666A, an inhibitor of lysosomal cholesterol export and Ebola infection

Elife. 2015 Dec 8;4:e12177. doi: 10.7554/eLife.12177.

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.

Keywords: Niemann-Pick C1; U18666A; biochemistry; cell biology; cholesterol transport; crosslinking; endosomes/lysosomes; human; sterol-sensing domain.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Androstenes / pharmacology*
  • Animals
  • Anticholesteremic Agents / pharmacology*
  • Antiviral Agents / pharmacology*
  • CHO Cells
  • Carrier Proteins / antagonists & inhibitors*
  • Cholesterol / metabolism*
  • Cricetulus
  • Ebolavirus / drug effects*
  • Enzyme Inhibitors / pharmacology*
  • Intracellular Signaling Peptides and Proteins
  • Membrane Glycoproteins / antagonists & inhibitors*
  • Virus Internalization / drug effects

Substances

  • Androstenes
  • Anticholesteremic Agents
  • Antiviral Agents
  • Carrier Proteins
  • Enzyme Inhibitors
  • Intracellular Signaling Peptides and Proteins
  • Membrane Glycoproteins
  • NPC1 protein, human
  • 3-beta-(2-(diethylamino)ethoxy)androst-5-en-17-one
  • Cholesterol