Marinopyrrole A Target Elucidation by Acyl Dye Transfer

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 K115, 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.

Seriniquinone, a selective anticancer agent, induces cell death by autophagocytosis, targeting the cancer-protective protein dermcidin

“…mode of action | chemical biology | drug discovery | marine natural products | cancer O ver the last decade, we have found that integrating a streamlined transition between marine natural products discovery (1), detailed cell and molecular biological studies (2), and medicinal chemical optimization offers a rich forum to advance leads that escape industrialized high-throughput practices (3)(4)(5). One place where drug discovery continues to be challenged lies in the discovery of new agents, which are highly selective for specific cancers (6)(7)(8).…”

Elucidating the Mode of Action of Marine Natural Products through an Immunoaffinity Fluorescent (IAF) Approach

“…10,11 The second case study explores an extension of the IAF approach to monitor protein natural product interactions through the transfer of an IAF tag from the natural product to its protein target upon binding. 12 In this study, we demonstrate how this 'tag-transfer' concept works through studies on the marinopyrrole natural products. 13,14 The final two case studies on the seriniquinone 15 and chlorizidine 16,17 natural products define how further extension into downstream and cell cycle markers allows one to develop a streamlined connectivity between cellular processing, its associated molecular targets, and ultimate downstream effects.…”

“…With structures at hand, we then turned our effort to explore their target identification and associated MOA in HCT-116 cells. 12 While only moderately cytotoxic, we were interested in exploring if these materials targeted pathways that may not be directly related with cell death phenotypes.…”

“…Chambers Hughes in the Fenical laboratory began by preparing an IAF probe from marinopyrrole A. 12 Here, we used prior data that indicated the fully acetylated marinopyrrole A could be made and maintained activity, as a guide for our choice in IAF labeling. Using established synthetic protocols and detailed NMR characterization, 7,14 we were able to prepare both a mono-labeled and di-labeled probe, as shown in Figure 4.…”

Affinity purification in target identification: the specificity challenge

“…Parallel labeling experiments against the target protein carbonic anhydrase have shown that the moderately reactive epoxide crosslinker exhibits the optimal balance of selectivity and reactivity (Chen et al 2003). Hamachi, Fenical and their respective co-workers developed a series of novel traceless affinity labeling probes containing electrophilic organocatalysts such as acyl phenolate, (Hughes et al 2009) 4-dimethylaminopyridine (DMAP) (Koshi et al 2008;Sun et al 2011;Wang et al 2011), tosylate, (Tsukiji et al 2009a, b;Tamura et al 2012), and acyl imidazole (Fujishima et al 2012;Matsuo et al 2013;Miki et al 2014). By targeting specific nucleophilic groups in the ligand-binding pocket of the protein, these probes exhibited high target specificity (Fig.…”