Marinopyrrole A Target Elucidation by Acyl Dye Transfer,Journal of the American Chemical Society

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Marinopyrrole A Target Elucidation by Acyl Dye Transfer
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2009-09-02 , DOI: 10.1021/ja903149u
Chambers C Hughes ₁ , Yu-Liang Yang , Wei-Ting Liu , Pieter C Dorrestein , James J La Clair , William Fenical

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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.

中文翻译：

通过酰基染料转移对 Marinopyrrole A 靶标进行阐明

使用荧光染料转移策略鉴定了 marinopyrrole A 靶向肌动蛋白的情况。该过程首先将羧酸末端标签附加到天然产物中的苯酚上。然后在活细胞中研究所得探针，以验证其保持与 marinopyrrole A 相当的活性。双色荧光显微镜证实，未标记和标记材料在 HCT-116 细胞中具有相当的摄取和亚细胞定位。随后的免疫沉淀研究将肌动蛋白确定为 HCT-116 细胞中的假定靶标，这一结果通过对肌动蛋白纯化样品的质谱、亲和力和活性分析进行了验证。进一步的数据分析表明，marinopyrrole 探针中的染料选择性转移到单个残基 K(115)，而相关酰基酚和反应性标记不会发生这种情况。在这项研究中，细胞、蛋白质和氨基酸分析的组合来自于单个材料样品，从而提出了一种简化和减少作用模式研究中涉及的材料需求的方法。

更新日期：2009-09-02

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