Characterizing the Druggable Hotspots Targeted by Anti-Cancer Natural Product Withaferin A

这项建议使用了一种创新的化学蛋白质组学技术，称为同位素串联 正交蛋白水解激活的基于活性的蛋白质谱分析（isoTOP-ABPP）， 热点靶向的withaferin A，一种天然产物，来自Acnistus arborescens， 半胱氨酸反应性迈克尔受体和损害TNBC细胞活力。IsoTOP-ABPP使用基于反应性的 化学探针直接在复杂的蛋白质组中绘制全蛋白质组反应性，功能性和可药用热点 蛋白质组当以竞争方式使用时，共价作用的小分子可以被竞争， 与相应的基于反应性的探针进行比较以实现目标发现。使用isoTOP-ABPP平台， 该建议显示了鉴定醉茄素A靶向的多个半胱氨酸热点的初步数据， 包括PPP 2 R1 A的C377，主要肿瘤抑制蛋白磷酸酶2A的调节亚基 （PP2A）。初步数据显示，TNBC细胞中的醉茄素A处理损害了TNBC细胞中的磷酸化。 主要的PP 2A底物和致癌信号蛋白AKT，可能解释其有效的抗- 这些细胞中的癌细胞。使用基于片段的共价配体发现方法和isoTOP- 在ABPP平台上，半胱氨酸反应性丙烯酰胺先导物DKM 2-90也已被鉴定， 选择性靶向PP 2A，损害磷酸-AKT信号传导，并损害TNBC细胞活力。这项建议 假设醉茄素A通过靶向TNBC中的半胱氨酸热点而损害TNBC致病性， PPP 2 R1 A激活PP 2A活性并损害致癌信号通路如AKT。这 该提案将使用isoTOP-ABPP平台来研究醉茄素A的作用机制 在削弱TNBC致病性和使用共价配体发现方法来开发更多 针对醉茄素A靶标的可合成的有效和选择性调节剂。

This proposal uses an innovative chemoproteomic technology termed isotopic tandem orthogonal proteolysis-enabled activity-based protein profiling (isoTOP-ABPP) to map the druggable hotspots targeted by withaferin A, a natural product derived from Acnistus arborescens that bears a cysteine-reactive Michael acceptor and impairs TNBC cell viability. IsoTOP-ABPP uses reactivity-based chemical probes to map proteome-wide reactive, functional, and druggable hotspots directly in complex proteomes. When used in a competitive manner, covalently-acting small-molecules can be competed against corresponding reactivity-based probes to enable target discovery. Using isoTOP-ABPP platforms, this proposal shows preliminary data for identifying multiple cysteine hotspots targeted by withaferin A, including C377 of PPP2R1A, the regulatory subunit of the major tumor suppressor protein phosphatase 2A (PP2A). The preliminary data show that withaferin A treatment in TNBC cells impairs phosphorylation of the major PP2A substrate and oncogenic signaling protein AKT, potentially explaining its potent anti- cancer activity in these cells. Using fragment-based covalent ligand discovery methods and isoTOP- ABPP platforms, a cysteine-reactive acrylamide lead DKM 2-90 has also been identified that more selectively targets PP2A, impairs phospho-AKT signaling, and impairs TNBC cell viability. This proposal hypothesizes that withaferin A impairs TNBC pathogenicity through targeting a cysteine hotspot in PPP2R1A to activate PP2A activity and impair oncogenic signaling pathways such as AKT. This proposal will use isoTOP-ABPP platforms to investigate the mechanisms of action of withaferin A in impairing TNBC pathogenicity and use covalent ligand discovery approaches to develop more synthetically accessible potent and selective modulators against withaferin A targets.