title: Application of Mass Spectrometry Profiling to Establish Brusatol as an Inhibitor of Global Protein Synthesis.
abstract: The KEAP1/Nrf2 pathway senses and responds to changes in intracellular oxidative stress. Mutations that result in constitutive activation of Nrf2 are present in several human tumors, especially non-small cell lung cancer. Therefore, compounds that inhibit Nrf2 activity might be beneficial in treating patients whose tumors show activation of this pathway. Recent reports suggest that the natural product brusatol can potently and selectively inhibit Nrf2 activity, resulting in cell cytotoxicity, and can be effectively combined with chemotherapeutic agents. Here, we analyzed the effects of brusatol on the cellular proteome in the KEAP1 mutant non-small cell lung cancer cell line A549. Brusatol was found to rapidly and potently decrease the expression of the majority of detected proteins, including Nrf2. The most dramatically decreased proteins are those that display a short half-life, like Nrf2. This effect was confirmed by restricting the analysis to newly synthesized proteins using a labeled methionine analogue. Moreover, brusatol increased the expression of multiple components of the ribosome, suggesting that it regulates the function of this macromolecular complex. Finally, we show that brusatol induces its potent cellular cytotoxicity effects on multiple cancer cell lines in a manner independent of KEAP1/Nrf2 activity and with a profile similar to the protein translation inhibitor silvestrol. In conclusion, our data show that the activity of brusatol is not restricted to Nrf2 but, rather, functions as a global protein synthesis inhibitor.
authors.affiliation: From the ‡Department of Discovery Oncology.
authors.forename: Steffan
authors.initials: S
authors.lastname: Vartanian
authors.affiliation: §Department of Protein Chemistry.
authors.forename: Taylur P
authors.initials: TP
authors.lastname: Ma
authors.affiliation: From the ‡Department of Discovery Oncology.
authors.forename: James
authors.initials: J
authors.lastname: Lee
authors.affiliation: ¶Department of Bioinformatics, Genentech Inc., 1 DNA Way, South San Francisco, California 94080.
authors.forename: Peter M
authors.initials: PM
authors.lastname: Haverty
authors.affiliation: §Department of Protein Chemistry.
authors.forename: Donald S
authors.identifier: http://orcid.org/0000-0003-3091-2804
authors.initials: DS
authors.lastname: Kirkpatrick
authors.affiliation: §Department of Protein Chemistry, yu.kebing@gene.com stokoe.david@gene.com.
authors.forename: Kebing
authors.initials: K
authors.lastname: Yu
authors.affiliation: From the ‡Department of Discovery Oncology, yu.kebing@gene.com stokoe.david@gene.com.
authors.forename: David
authors.initials: D
authors.lastname: Stokoe
mesh_terms: D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D000072019:Kelch-Like ECH-Associated Protein 1 / Q000235:genetics; D008175:Lung Neoplasms / Q000188:drug therapy / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods*; D009154:Mutation; D051267:NF-E2-Related Factor 2 / Q000378:metabolism*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D040901:Proteomics / Q000379:methods; D036702:Quassins / Q000494:pharmacology*
chemical_list: C431364:KEAP1 protein, human; D000072019:Kelch-Like ECH-Associated Protein 1; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D011500:Protein Synthesis Inhibitors; D036702:Quassins; C020237:brusatol