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.

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Comprehensive libraries of plasmids for SARS‐CoV‐2 proteins with various tags (e.g. Strep, HA, Turbo) are now available. They enable the identification of numerous potential protein‐protein interactions between the SARS‐CoV‐2 virus and host proteins. To facilitate further cellular investigations, notably by imaging techniques, we present here a large library of SARS CoV‐2 protein constructs fused with green and red fluorescent proteins and their initial characterization in various human cell lines including lung epithelial cell models (A549, BEAS‐2B), as well as in budding yeast. The localization of a few SARS‐CoV‐2 proteins matches their proposed interactions with host proteins. These include the localization of Nsp13 to the centrosome, Orf3a to late endosomes, and Orf9b to mitochondria . This article is protected by copyright. All rights reserved