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Inhibition of OSBP blocks retrograde trafficking by inducing partial Golgi degradation

Abstract

Sterol-binding proteins are important regulators of lipid homeostasis and membrane integrity; however, the discovery of selective modulators can be challenging due to structural similarities in the sterol-binding domains. We report the discovery of potent and selective inhibitors of oxysterol-binding protein (OSBP), which we term oxybipins. Sterol-containing chemical chimeras aimed at identifying new sterol-binding proteins by targeted degradation, led to a significant reduction in levels of Golgi-associated proteins. The degradation occurred in lysosomes, concomitant with changes in protein glycosylation, indicating that the degradation of Golgi proteins was a downstream effect. By establishing a sterol transport protein biophysical assay panel, we discovered that the oxybipins potently inhibited OSBP, resulting in blockage of retrograde trafficking and attenuating Shiga toxin toxicity. As the oxybipins do not target other sterol transporters and only stabilized OSBP in intact cells, we advocate their use as tools to study OSBP function and therapeutic relevance.

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Fig. 1: Structures and expression proteomics profiles of Chol-based degraders.
Fig. 2: Validation of GPP130 degradation by 1–4.
Fig. 3: Compounds 68 significantly degrade Golgi proteins.
Fig. 4: Oxybipin-1 and -2 selectively target OSBP.
Fig. 5: OSBP inhibition causes OSBP localization to the Golgi, trans-Golgi fragmentation and inhibition of STxB trafficking.
Fig. 6: Therapeutic potential and mechanism of action of OSBP inhibitors.

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Data availability

The data supporting the findings in this study are available within the paper and its supplementary files. Proteomics data can be found in Supplementary Dataset 1. Raw proteomics data on 18 and MnCl2 was uploaded to MASSIVE, with the accession number MSV000091726. Raw proteomics data on the iTSA of oxybipin-2, and the expression proteomics of 3 and OSW-1 can be found at MSV000093086. Source data are provided with this paper. Any other data can be requested and will be provided by the corresponding author.

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Acknowledgements

The Laraia Laboratory was supported by funding from the Novo Nordisk Foundation (grant nos. NNF19OC0055818, NNF19OC0058183, NNF21OC0067188), the Carlsberg Foundation (grant no. CF19-0072) and the Independent Research Fund Denmark (grant nos. 9041-00241B, 9041-00248B). CeMM and the Winter Laboratory are supported by the Austrian Academy of Sciences. The Winter laboratory is further supported by funding from the European Research Council under the European Union’s Horizon 2020 research and innovation program (grant agreement no. 851478), as well as by funding from the Austrian Science Fund (FWF, project nos. P32125, P31690 and P7909). A.F. is an F.R.S.-FNRS Chercheur Qualifié. The Forrester laboratory is funded by an Incentive for Scientific Research Grant (grant no. MIS: F.4518.23) from FNRS. We thank the Morph-Im platform and staff at the University of Namur for their microscopy support. The Gillet laboratory was funded by the French National Research Agency (ANR) under contract nos. LeishmaStop ANR-18-CE18-0016-01 and SMERSEC ANR-20-CE18-0016-01, by University Paris Saclay under contract nos. ReCoVEr and PIMVir, and the Joint ministerial program of R&D against CBRNe risks. We thank Ludger Johannes for helpful discussions and providing fluorescently labeled STxB. We also acknowledge the Cell and Tissue Imaging (PICT-IBiSA) and Nikon Imaging Centre, Institut Curie, a member of the French National Research Infrastructure France-BioImaging (grant no. ANR10-INBS-04).

Author information

Authors and Affiliations

Authors

Contributions

N.H. and L.L. designed the project. N.H. carried out the compound synthesis, proteomics sample preparation and analysis, and cell biological validation of compounds. L.D. expressed and purified recombinant proteins and developed and performed all biophysical assays and cell viability measurements. C.R. designed, supervised, performed and analyzed proteomics experiments and supported cell biological validation. O.R.D. synthesized von Hippel Lindau probes and conducted their biological analysis. L.C. and A.F. performed and analyzed the Golgi integrity and retrograde trafficking experiments. M.C. performed the HiBit CETSA experiments and OSBP overexpression rescue experiments under supervision of G.E.W. H.P.B.-R. performed and analyzed docking experiments and performed FP experiments. M.M. performed the STx1 inhibition experiments under the supervision of J.B. and D.G. J.H. supported recombinant protein expression and synthesis. L.L. supervised the project. N.H., L.D. and L.L. wrote the paper with input from all authors.

Corresponding author

Correspondence to Luca Laraia.

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Competing interests

G.E.W. is scientific founder and shareholder of Proxygen and Solgate. The Winter laboratory received funding from Pfizer. The other authors declare no competing interests.

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Nature Chemical Biology thanks Minetaro Arita, Markus Schirle and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1

Synthesis of Cholesterol-bearing bifunctional molecules 1–4, 6 and 5. (a) Synthesis of cholenic acid (14). (b) Synthesis of linkers. (c) Synthesis of pomalidomide-conjugated linkers. (d) Synthesis of methylated negative control precursor (24). (e) Synthesis of bichols 1-4 and negative control. (f) Synthesis of N-methyl cholenamide.

Extended Data Fig. 2 Degradation of GPP130 by bifunctional molecules containing sterol and vHL recruiting moieties.

(a) Synthesis of VHL-recruiting PROTACs. (b) Effect of VHL-based PROTACs on GPP130 levels in HeLa cells for 18 hours (n = 3, representative experiment shown); (c) rescue of GPP130 degradation by lysosomal protease inhibitors Leupeptin (100 μg/mL) and Pepstatin (50 μg/mL); (n=2, representative experiment shown).

Source data

Extended Data Fig. 3

Synthesis of oxybipins. (a) Synthesis of oxybipin-2 (7). (b) Synthesis of oxybipin-1 (8).

Extended Data Fig. 4 3 and its derivatives do not bind to GPP130 directly and do not affect GPP130 N-glycosylation.

(a) Cellular thermal shift assay shows 3 exhibits a weak destabilization effect on GPP130 in intact HeLa cells (n = 2, representative experiment shown). (b) Isothermal dose response fingerprinting assay shows 3 failed to destabilize GPP130 in a dose-dependent manner at 54.3 °C (n = 3, representative experiment shown) and 63.4 °C (n = 1) in intact HeLa cells. (c) Effect of Tunicamycin (5 μg/mL), PNGase F, 3 and Oxybipin-1 (1 µM each) on the N-glycosylation of GPP130 in HeLa cells (n = 2, representative experiment shown).

Source data

Extended Data Fig. 5 Oxybipins selectively target OSBP.

(a) Binding curve of the FP titration of 25-NBD-cholesterol with the ORD of ORP1 and ORP2. Each data point represents the mean of a representative experiment (N=2), from three independent experiments (n=3) (b) Calculated Z′ factors using 22-NBD-cholesterol as tracer confirm the robustness of the competitive FP assay selectivity panel. Each data point represents the mean ± s.e.m of three independent experiments (N-2, n=3) (c) Screening of 8 (oxybipin-1), 7 (oxybipin-2), OSW-1 and 25-HC in the STP FP selectivity panel at 3 μM, using 22-NBD-Chol as a tracer. Each data point represents the mean of a representative experiment (N=2), from three independent experiments (n=3). (d) Effect on the stability of ORP2 and ORP9 by oxybipin-1 and -2 in intact KBM7 cells expressing HiBit tagged fusion proteins. Each data point represents mean ± s.e.m from three technical replicates of a representative experiment, from three independent experiments (N=3, n=3). (e) Chemical structures of 9 and 10. (f) Dose-dependent inhibition of GST-OSBP-ORD binding to 22-NBD-Chol by 9 and 10 as assessed by FP. Data are represented as mean ± s.e.m from three independent experiments (N=2, n=3). (h) 9 and 10 failed to enrich OSBP in pulldown experiments (n = 1). (g) Stabilization profile of oxybipin-2 at 10 μM, 3 μM and 0.3 μM. The destabilized proteins (blue dots) and stabilzed proteins (red dots) were plotted as log2 fold change (compound-treated /DMSO-treated) versus -log10 (p-value). P-values were determined by a Student’s two-tailed t test assuming equal variances from the data of three replicates.

Source data

Extended Data Fig. 6 Expression proteomics of HeLa cells treated with 3 and OSW-1.

(a) Expression proteomics profile of HeLa cells treated with 3 (1 µM) for 18 h. The down-regulated proteins (blue dots) and up-regulated proteins (red dots) were plotted as log2 fold change (compound-treated /DMSO-treated) versus -log10 (p-value). P-values were determined by a Student’s two-tailed t test assuming equal variances from the data of three replicates. The dotted horizontal line marks the significance threshold of p = 0.05 and dotted vertical lines represent the threshold of an absolute log2 fold change of 0.8. (b) Expression proteomics profile of HeLa cells treated with OSW-1 (1 nM) for 18 hours. (c) The down-regulated proteins of 3 can also be classified into Golgi proteins (Red) and proteins involved in N-Glycan biosynthesis (Blue). (d) Effect of OSW-1 and oxybipins on GPP130 degradation. HeLa cells were treated with indicated concentrations of OSW-1 and 1 µM oxybipins for 18 hours (n = 2, representative experiment shown).

Source data

Extended Data Fig. 7 STP FP selectivity panel reveals lack of selectivity of published OSBP inhibitors.

(a) Chemical structures of reported OSBP inhibitors T-HEV2, T-HEV3, T-HEV4, Itraconazole and TTP-8307. (b) Screening of TTP-8307, Itraconazole, T-HEV2, T-HEV3 and T-HEV4 in the STP FP selectivity panel at 1 μM, with 22-NBD-Chol as a tracer. Each bar represents the mean of a representative experiment, from two independent experiments (N=2, n=2). (c) Fluorescence polarization measurements of dose-dependent inhibition of ORP1-ORD binding to 25-NBD-Chol. Data are represented as mean ± s.e.m from three independent experiments (N=2, n=3). (d) Fluorescence polarization measurements of dose-dependent inhibition of ORP2-ORD binding to 25-NBD-Chol. Each data point represents the mean of a representative experiment, from three independent experiments (n=3) (e) Fluorescence polarization measurements of dose-dependent inhibition of GST-OSBP-ORD binding to 22-NBD-Chol. Data are represented as mean ± s.e.m from three independent experiments (N=2, n=3). (f) DLS measurements of GST-OSBP-ORD and GST-OSBP-ORD incubated with different OSBP inhibitors. One representative experiment was shown from three replicates (n=3). (g) Inhibition of sterol transport by GST-OSBP(ORD) (500 nM) in liposomes using a FRET assay. OSW-1 was tested at 1 µM, while itraconazole and TTP-8307 were tested at 1 µM (solid lines) and 10 µM (dashed lines). One representative experiment was shown from two independent replicates (n=2). (h) IC50 values of TTP-8307, Itraconazole, T-HEV2, T-HEV3, T-HEV4, 5, oxybipin-1 and oxybipin-2 against STPs in FP experiments.

Source data

Extended Data Fig. 8 Predicted binding modes of OSBP inhibitors.

Docking poses of oxybipin-2 (a), Cholesterol (b) (Purple – crystal structure pose, Blue – Docking pose), 3 (c), oxybipin-1 (d), T-HEV2 (e), T-HEV3 (f), and T-HEV4 (g) into the crystal structure of OSBP’s cholesterol binding domain (PDB ID:7V62)44. Key binding residues are illustrated. Yellow dashes indicate H-bonds. Green dashes indicate cation-pi interactions. Grey mesh represents the exterior surface of the ORD at a 4 Å distance from the ligand.

Extended Data Fig. 9 Effect of OSBP inhibitors on Golgi integrity and OSBP relocalization.

(a) Scanning confocal microscopy of HeLa cells treated with DMSO, 1 μM Itraconazole or TPP-8307 for 4 h. Cells were immunolabelled with TGN46 (647) and OSBP (488); nuclei labelled with Hoechst. Scale bar = 10 μm, dotted line indicates cell outline. b) Scanning confocal microscopy of HeLa cells treated with DMSO, 100 nM or 1 μM oxybipin-2 for 8 h. Cells were immunolabelled with TGN46 (647) and OSBP (488), nuclei labelled with Hoechst. Scale bar = 10 μm, dotted line indicates cell outline. (c,d) Graphs show one point per cell, independent experimental replicates (n=3) for a total of 47 (DMSO), 48 (1 μM oxybipin-2), 32 (100 nM oxybipin-2, n=2). c) Quantification of OSBP localization to the Golgi, as identified by TGN46. OSBP fluorescence intensity was measured in TGN46 ROI and normalised to total cellular OSBP. One-way ANOVA was performed (P < 0.0001) with Dunnett’s multiple comparisons test: **** P < 0.0001. (d) Quantification of TGN46 fragmentation measured by number of spots per cell, line represents median. One-way ANOVA was performed on log10 transformed data (P < 0.0001) with Dunnett’s multiple comparison test: ns = 0.00828, **** P < 0.0001. (e-f) STxB trafficking is inhibited by OSBP inhibition but not control compounds. (e) Scanning confocal images of STxB-Cy3 trafficking in HeLa cells treated for 4 h with DMSO vehicle, 10 µM Itraconazole, TPP-8307, THEV2, THEV3 or THEV4, or for 2 h with 20 nM OSW1. Cells were immunolabelled with transferrin receptor (TfR, 488), giantin (647) and nuclei stained with Hoechst. Scale bar = 20 µm. (f) Quantification of STxB trafficking measured by number of spots per cell. Graph shows one point per cell, shape represents each independent experimental replicate (n=3) for a total of 132 (DMSO), 124 (OSW1), 102 (Itraconazole), 98 (TPP-8307), 91 (THEV2), 141 (THEV3), 124 (THEV4), line indicates mean. One-way ANOVA P < 0.0001, Dunnett’s multiple comparison test: **** P < 0.0001, ns=0.6790 (TPP-8307), ns=0.3904 (THEV2), ns>0.9999 (THEV3), ns=0.9968 (THEV4).

Source data

Extended Data Fig. 10 Effects of oxybipins on cancer cell viability.

(a) Effect of 3 and oxybipin-1 on HeLa cell viability at 24 h. Each data point represents mean± s.e.m from two technical replicates, two independent experiments. (N=2, n=2) (b-e) Effect of oxybipins and control compounds on the viability of cancer cells at 72 h. All data in mean ± s.e.m for three replicates (N=2, n=3). (f-l) Effect of oxybipins and control compounds on the viability of KBM7 wildtype cells and KBM7 cells overexpressing OSBP(wt)-2HA at 72 h. Each data point represents mean ± s.e.m from three technical replicates of a representative experiment, from three independent experiments (N=3, n=3).

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–4, Tables 1–3 and Synthesis of compounds 1–37.

Reporting Summary

Supplementary Table 4

Raw and analyzed proteomics data.

Supplementary Data 1

Source data for Supplementary Fig. 1.

Source data

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Unprocessed western blots.

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Unprocessed western blots.

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Unprocessed western blots.

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Unprocessed western blots.

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Statistical source data.

Source Data Extended Data Fig. 4

Unprocessed western blots.

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Statistical source data.

Source Data Extended Data Fig. 5

Unprocessed western blots.

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Unprocessed western blots.

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Statistical source data.

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He, N., Depta, L., Rossetti, C. et al. Inhibition of OSBP blocks retrograde trafficking by inducing partial Golgi degradation. Nat Chem Biol (2024). https://doi.org/10.1038/s41589-024-01653-x

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