Abstract

ORPphilins are bioactive natural products that strongly and selectively inhibit the growth of some cancer cell lines and 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 phosphatidylinositol 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 OSBP levels. By taking advantage of the intrinsic fluorescence of SWG, we followed its fate in cell cultures and show that its incorporation at the trans-Golgi network depends on cellular abundance of OSBP. Using in vitro membrane reconstitution systems and cellular imaging approaches, we also report that SWG inhibits specifically the lipid transfer activity of OSBP. As a consequence, post-Golgi trafficking, membrane cholesterol levels, and PI(4)P turnover were affected. Finally, using intermolecular FRET analysis, we demonstrate that SWG directly binds to the lipid-binding cavity of OSBP. Collectively these results describe SWG as a specific and intrinsically fluorescent pharmacological tool for dissecting OSBP properties at the cellular and molecular levels. Our findings indicate that SWG binds OSBP with nanomolar affinity, that this binding is sensitive to the membrane environment, and that SWG inhibits the OSBP-catalyzed lipid exchange cycle.

Highlights

  • ORPphilins are small natural bioactive molecules that have strong inhibitory activities on the growth of several human cancer cell lines

  • We report the nanomolar affinity of schweinfurthin G (SWG) for oxysterol-binding protein (OSBP), its sensitivity to membrane interfacial environment, and provide evidence that SWG is able to inhibit the entire lipid exchange cycle catalyzed by OSBP by entering its OSBPrelated domain (ORD) domain

  • OSBP expression level correlates with SWG sensitivity The specific cytotoxicity of schweinfurthins for a subset of NCI-60 cancer cell lines is not explained so far

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Summary

Introduction

ORPphilins are small natural bioactive molecules that have strong inhibitory activities on the growth of several human cancer cell lines. Results OSBP expression level correlates with SWG sensitivity The specific cytotoxicity of schweinfurthins for a subset of NCI-60 cancer cell lines is not explained so far.

Results
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