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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The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
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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.
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 Figures 1–13
IsoTOP-ABPP analysis of nimbolide treatment in situ in 231MFP breast cancer cells.
TMT-based quantitative proteomic analysis of proteins enriched by nimbolide-alkyne probe in situ treatment in 231MFP breast cancer cells.
TMT-based quantitative proteomic profiling of XH2 treatment in 231MFP breast cancer cells.
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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