Molecular and cellular dissection of the OSBP cycle through a fluorescent inhibitor

ORPphilins, natural molecules that strongly and selectively inhibit the growth of some cancer cell lines, are proposed to target intracellular lipid-transfer proteins of the Oxysterol-binding protein (OSBP) family. These conserved proteins exchange key lipids, such as cholesterol and phopsphatidylinositol-4-phosphate (PI(4)P), between organelle membranes. Among ORPphilins, molecules of the schweinfurthin family interfere with intracellular lipid distribution and metabolism, but their functioning at the molecular level is poorly understood. We report here that cell line sensitivity to schweinfurthin G (SWG) is inversely proportional to cellular level of OSBP. By taking advantage of the intrinsic fluorescence of SWG, we follow its fate in cell cultures and show that its incorporation at the TGN depends on OSBP cellular abundance. We report that SWG inhibits specifically the lipid exchange cycle of OSBP. As a consequence, post-Golgi trafficking, membrane cholesterol levels and PI(4)P turnover are affected. Finally, we demonstrate the direct binding of SWG into OSBP lipid-binding cavity by intermolecular FRET. Collectively these data describe for the first time a specific and intrinsically fluorescent pharmacological tool to dissect OSBP properties at the cellular and molecular levels.