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

该项目的重点是通过基于化学蛋白质组学的方法进行靶标识别和验证。在第一个资助期，我们利用功能化的管溶蛋白前体衍生物进行靶向分析。细胞可穿透性光探针的活性蛋白质谱(ABPP)显示微管蛋白是目的蛋白，而固定化前微管蛋白亲和层析为蛋白酶体亚基的特异性结合提供了证据。尤其是后一项调查结果是出乎意料的，将在下一个供资期间得到进一步验证。在新的资助期内，我们将使用我们已建立的化学蛋白质组学平台，对索拉芬A和古拉唑胺进行靶标鉴定。这两个功能化分子的设计将与P2和P9密切合作进行。为了获得定量数据，我们将使用细胞培养中的稳定同位素标记(SILAC)。这种方法将允许立即根据它们的结合特异性对识别的蛋白质命中进行排序，从而增加对所获得结果的置信度。此外，我们将进一步改进ABPP的方法，并实施不含PhotoLinker的天然富集化策略，以减少目标化合物的结构修改程度。所有新的命中将通过深入的生物学特征来验证。已经根据计算和生化数据预测了古拉唑胺的推定目标，并将在这些研究中得到验证。通过这一全面的目标识别/验证策略，我们将为For化合物的作用机制提供重要的见解，从而帮助生成关于生物活性和作用模式的假设。

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.