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Harnessing the anti-cancer natural product nimbolide for targeted protein degradation

Abstract

Nimbolide, a terpenoid natural product derived from the Neem tree, impairs cancer pathogenicity; however, the direct targets and mechanisms by which nimbolide exerts its effects are poorly understood. Here, we used activity-based protein profiling (ABPP) chemoproteomic platforms to discover that nimbolide reacts with a novel functional cysteine crucial for substrate recognition in the E3 ubiquitin ligase RNF114. Nimbolide impairs breast cancer cell proliferation in-part by disrupting RNF114-substrate recognition, leading to inhibition of ubiquitination and degradation of tumor suppressors such as p21, resulting in their rapid stabilization. We further demonstrate that nimbolide can be harnessed to recruit RNF114 as an E3 ligase in targeted protein degradation applications and show that synthetically simpler scaffolds are also capable of accessing this unique reactive site. Our study highlights the use of ABPP platforms in uncovering unique druggable modalities accessed by natural products for cancer therapy and targeted protein degradation applications.

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Fig. 1: Nimbolide impairs breast cancer cell proliferation and survival.
Fig. 2: isoTOP-ABPP analysis of nimbolide in 231MFP breast cancer cell proteomes reveal RNF114 as a target.
Fig. 3: Nimbolide reacts covalently with C8 of RNF114.
Fig. 4: Nimbolide inhibits RNF114 activity through disrupting substrate recognition.
Fig. 5: Nimbolide can be used to recruit RNF114 for targeted protein degradation of BRD4.

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank the members of the Nomura Research Group, the Maimone laboratory and Novartis Institutes for BioMedical Research for critical reading of the manuscript. We acknowledge M. Moeller and A. Olding for assistance in nimbolide isolation studies. This work was supported by Novartis Institutes for BioMedical Research and the Novartis-Berkeley Center for Proteomics and Chemistry Technologies (NB-CPACT) for all listed authors. This work was also supported by grants from the National Institutes of Health (no. R01CA172667 for D.K.N., J.N.S., C.C.W. and L.O.; no. F31CA225173 for C.C.W.; no. F31CA239327 for J.N.S. and no. R01GM112948 for J.A.O.). This work was also supported by the Mark Foundation for Cancer Research ASPIRE award. J.A.O. is a Chan Zuckerberg Biohub investigator.

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Authors

Contributions

J.N.S., T.J.M. and D.K.N. conceived the project and wrote the paper. J.N.S., X.H., C.C.W., M.D.J., D.E.B., J.R.T., J.A.T., J.M.K., M.S., T.J.M. and D.K.N. provided intellectual contributions and insights into project direction. J.N.S., X.H., S.M.B., M.T., J.A.O., M.S., T.J.M. and D.K.N. designed the experiments and analyzed data. C.C.W. and M.D.J. developed bioinformatic methods and analyzed data for proteomics experiments. J.N.S., X.H., S.M.B., L.O., M.T., A.P., E.O., M.W. and D.K.N. performed experiments and analyzed data. A.P., E.O., M.W. and D.E.B. provided pure RNF114 protein. J.N.S., X.H. and T.J.M. designed and synthesized compounds. J.N.S., S.M.B., M.D.J., J.A.O., D.E.B., J.R.T., J.A.T., J.M.K., M.S., T.J.M. and D.K.N. edited the paper.

Corresponding authors

Correspondence to Thomas J. Maimone or Daniel K. Nomura.

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Competing interests

S.M.B., M.D.J., A.P., E.O., M.W., D.E.B., J.A.T., J.M.K. and M.S. are employees of Novartis Institutes for BioMedical Research. J.R.T. was an employee of Novartis Institutes for BioMedical Research when this study was submitted, but is now an employee of Vertex Pharmaceuticals. This study was funded by the Novartis Institutes for BioMedical Research and the Novartis-Berkeley Center for Proteomics and Chemistry Technologies. D.K.N. is a co-founder, share-holder and adviser for Artris Therapeutics and Frontier Medicines.

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Supplementary information

Supplementary Information

Supplementary Figures 1–13

Reporting Summary

Supplementary Note

Synthetic Procedures

Supplementary Dataset 1

IsoTOP-ABPP analysis of nimbolide treatment in situ in 231MFP breast cancer cells.

Supplementary Dataset 2

TMT-based quantitative proteomic analysis of proteins enriched by nimbolide-alkyne probe in situ treatment in 231MFP breast cancer cells.

Supplementary Dataset 3

TMT-based quantitative proteomic profiling of XH2 treatment in 231MFP breast cancer cells.

Supplementary Dataset 4

Structures of covalent ligands screened against RNF114.

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Spradlin, J.N., Hu, X., Ward, C.C. et al. Harnessing the anti-cancer natural product nimbolide for targeted protein degradation. Nat Chem Biol 15, 747–755 (2019). https://doi.org/10.1038/s41589-019-0304-8

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