Discovery of Manassantin A Protein Targets Using Large-Scale Protein Folding and Stability Measurements

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• 作者：M. Ariel Geer Wallace ; Do-Yeon Kwon ; Douglas H. Weitzel ; Chen-Ting Lee ; Tesia N. Stephenson ; Jen-Tsan Chi ; Robert A. Mook Jr. ; Mark W. Dewhirst ; Jiyong Hong ; Michael C. Fitzgerald
• 刊名：Journal of Proteome Research
• 出版年：2016
• 出版时间：August 5, 2016
• 年：2016
• 卷：15
• 期：8
• 页码：2688-2696
• 全文大小：411K
• 年卷期：0
• ISSN：1535-3907

文摘

Manassantin A is a natural product that has been shown to have anticancer activity in cell-based assays, but has a largely unknown mode-of-action. Described here is the use of two different energetics-based approaches to identify protein targets of manassantin A. Using the stability of proteins from rates of oxidation technique with an isobaric mass tagging strategy (iTRAQ-SPROX) and the pulse proteolysis technique with a stable isotope labeling with amino acids in cell culture strategy (SILAC-PP), over 1000 proteins in a MDA-MB-231 cell lysate grown under hypoxic conditions were assayed for manassantin A interactions (both direct and indirect). A total of 28 protein hits were identified with manassantin A-induced thermodynamic stability changes. Two of the protein hits (filamin A and elongation factor 1α) were identified using both experimental approaches. The remaining 26 hit proteins were only assayed in either the iTRAQ-SPROX or the SILAC-PP experiment. The 28 potential protein targets of manassantin A identified here provide new experimental avenues along which to explore the molecular basis of manassantin A’s mode of action. The current work also represents the first application iTRAQ-SPROX and SILAC-PP to the large-scale analysis of protein–ligand binding interactions involving a potential anticancer drug with an unknown mode-of-action.