Marinopyrrole A target elucidation by acyl dye transfer.
The targeting of marinopyrrole A to actin was identified using a fluorescent dye transfer strategy. The process began by appending a carboxylic acid terminal tag to a phenol in the natural product. The resulting probe was then studied in live cells to verify that it maintained activity comparable to marinopyrrole A. Two-color fluorescence microscopy confirmed that both unlabeled and labeled materials share comparable uptake and subcellular localization in HCT-116 cells. Subsequent immunoprecipitation studies identified actin as a putative target in HCT-116 cells, a result that was validated by mass spectral, affinity, and activity analyses on purified samples of actin. Further data analyses indicated that the dye in the marinopyrrole probe was selectively transferred to a single residue K(115), an event that did not occur with related acyl phenols and reactive labels. In this study, the combination of cell, protein, and amino acid analysis arose from a single sample of material, thereby, suggesting a means to streamline and reduce material requirements involved in mode of action studies.
Hughes
Chambers C
CC
Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, California 92093-0204, USA.
Yang
Yu-Liang
YL
Liu
Wei-Ting
WT
Dorrestein
Pieter C
PC
La Clair
James J
JJ
Fenical
William
W
D000199:Actins / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001688:Biological Products / Q000737:chemistry / Q000378:metabolism; D001692:Biological Transport; D005456:Fluorescent Dyes / Q000737:chemistry*; D045325:HCT116 Cells; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011758:Pyrroles / Q000737:chemistry* / Q000378:metabolism
D000199:Actins; D001688:Biological Products; D005456:Fluorescent Dyes; D011758:Pyrroles; C528298:marinopyrrole A