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

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Geer Wallace, MA; Kwon, D-Y; Weitzel, DH; Lee, C-T; Stephenson, TN; Chi, J-T; Mook, RA; Dewhirst, MW; Hong, J; Fitzgerald, MC

Published Date

  • August 2016

Published In

Volume / Issue

  • 15 / 8

Start / End Page

  • 2688 - 2696

PubMed ID

  • 27322910

Electronic International Standard Serial Number (EISSN)

  • 1535-3907

International Standard Serial Number (ISSN)

  • 1535-3893

Digital Object Identifier (DOI)

  • 10.1021/acs.jproteome.6b00237

Language

  • eng