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.
Department of Chemistry, Duke University , Durham, North Carolina 27708-0346, United States.
M Ariel
MA
Geer Wallace
Department of Chemistry, Duke University , Durham, North Carolina 27708-0346, United States.
Do-Yeon
DY
Kwon
Douglas H
DH
Weitzel
Chen-Ting
CT
Lee
Department of Chemistry, Duke University , Durham, North Carolina 27708-0346, United States.
Tesia N
TN
Stephenson
Jen-Tsan
JT
Chi
Robert A
RA
Mook
Mark W
MW
Dewhirst
Department of Chemistry, Duke University , Durham, North Carolina 27708-0346, United States.
Jiyong
J
Hong
Department of Chemistry, Duke University , Durham, North Carolina 27708-0346, United States.
Michael C
MC
Fitzgerald
D000970:Antineoplastic Agents / Q000378:metabolism; D001688:Biological Products; D002478:Cells, Cultured; D064448:Filamins / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling; D008024:Ligands; D017705:Lignans / Q000378:metabolism*; D010084:Oxidation-Reduction; D020648:Peptide Elongation Factor 1 / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D055550:Protein Stability; D032134:Saururaceae / Q000737:chemistry
D000970:Antineoplastic Agents; D001688:Biological Products; D064448:Filamins; D008024:Ligands; D017705:Lignans; D020648:Peptide Elongation Factor 1; C054895:manassantin A
Filamin A; MDA-MB-231; Manassantin A; SILAC; SPROX; cancer; chemical denaturation; elongation factor 1α; iTRAQ; mass spectrometry; mode-of-action; proteomics; pulse proteolysis