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. 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. The KEAP1 1The abbreviations used are:KEAP1kelch-like ECH-associated protein 1DCMdichloromethaneNMRnuclear magnetic resonanceRTroom temperatureACNacetonitrileIDinner diameterSPSsynchronous precursor selectionGAPDHglyceraldehyde-3-phosphate dehydrogenaseVSVGvescicular stomatitis virus glycoproteinCMVcytomegalovirusRABL5Rab-like protein 5HPCL1Hippocalcin-like protein 1PYRG2pyrimidine reductaseG2AREanti-oxidant response elementCETSAcellular thermal shift assayNADPHnicotinamide adenine dinucleotide phosphateNSCLCnon-small cell lung cancerTMTtandem mass tagsUTRuntranslated region./Nrf2 pathway is one of the most important cellular mechanisms to react and respond to oxidative stresses. Under normal physiological conditions, Nrf2 protein is maintained at low levels due to constitutive ubiquitination and degradation. This is achieved through recruitment to the Cul