Abstract
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.
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📊 Figures
Figure 1
Application of acyl dye transfer to natural product mode of action studies. a) A dye (L) labeled natural product (NP) probe 1 binds to target protein 2 forming complex 3. Ligation-directed acyl transf...
Figure 2
Uptake and subcellular localization of probes 13 and 14 in HCT-116 cells. Confocal fluorescent images depicting the red fluorescence from the uptake of 6 in cells treated with a) 10 u03bcM 6 for 1 h, ...
Figure 3
Marinopyrrole probes target actin. a) A fluorescent gel showing cell lysate obtained from HCT-116 cells (10 8 cells) treated with 10 u03bcM 13 in L1 and 10 u03bcM 14 in L2 for 12 h. b) A Silver Blue s...
Figure 4
Marinopyrrole probe 13 transfers an IAF tag selectively to residue K 115 of actin. a) Structure of actin (green) noting the position of the targeted K 115 residue (blue) and other lysine residues (red...
Scheme 1
Structures of marinopyrrole A ( 6 ) and marinopyrrole B ( 7 ), IAF labels 9u201311, control 12 and the synthesis of bis -acetate 8, mono -labeled probe 13 and bis -labeled probe 14 .
Figure images are served from the NIH/NLM PubMed Central Open Access Subset or Europe PMC; copyright remains with the publishers and authors.
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