Identification, validation and functional characterization of targets of myxobacterial compounds with potential for pharmacological cancer treatment

本项目侧重于通过基于化学蛋白质组学的方法进行目标识别和验证。在第一个资助期，我们利用功能化的预微溶素衍生物进行目标分析。利用细胞渗透性光探针的活性蛋白分析（ABPP）显示微管蛋白是靶蛋白，而固定化微管蛋白的亲和层析提供了与蛋白酶体亚基特异性结合的证据。特别是后一项调查结果出乎意料，将在下一个供资期间进一步证实。在新的资助期内，我们将利用我们建立的化学蛋白质组学平台对soraphen A和archazolid进行靶标表征。这两种功能化分子的设计将与P2和P9密切合作。为了获得定量数据，我们将在细胞培养中使用稳定同位素标记（SILAC）。该方法将允许根据其结合特异性立即对鉴定的蛋白命中进行排序，从而增加所获得结果的可信度。此外，我们将进一步完善ABPP方法，并实现无光连接子的天然富集策略，从而减少目标化合物的结构修饰程度。所有新颖的热门产品都将通过深入的生物学表征进行验证。基于计算和生化数据预测了阿古唑啉的假定靶点，并将在这些研究中得到验证。通过这一全面的靶标鉴定/验证策略，我们将为FOR化合物的作用机制提供重要的见解，从而有助于产生关于生物活性和作用方式的假设。

This project focuses on target identification and validation via chemical proteomic based methods. In the first funding period we utilized functionalized pretubulysin derivatives for target analysis. While activity based protein profiling (ABPP) with cell permeable photoprobes revealed tubulin as target protein, affinity chromatography with immobilized pretubulisin provided evidence for the specific binding to proteasomal subunits. Especially the latter finding was unexpected and will be further validated during the next funding period. In the new funding period we will use our established chemical proteomic platform for the target characterization of soraphen A and archazolid. The design of both functionalized molecules will be carried out in close collaboration with P2 and P9. In order to gain quantitative data we will use stable isotope labeling in cell culture (SILAC). This method will allow to immediately rank the identified protein hits according to their binding specificity and thus increase the confidence in the obtained results. In addition, we will further refine the method of ABPP and implement a photolinker free native enrichment strategy that would allow to reduce the extent of structural modifications on the target compound. All novel hits will be validated by in depth biological characterization. Putative targets for archazolid have been predicted based on computational and biochemical data and will be also validated within these studies. With this comprehensive target identification/validation strategy we will provide important insights into the mechanism of action of the FOR compounds and thus help to generate hypotheses on the biological activity and mode of action.