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

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.