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

这项提议使用了一种名为同位素串联的创新化学蛋白质组技术 支持蛋白质分解的基于活性的蛋白质图谱(IsoTOP-ABPP)用于定位可药物 With aferin A是一种从木立猕猴桃中提取的天然产物，具有 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 化学探针直接在复合体中定位蛋白质组范围内的反应性、功能性和可药物热点 蛋白质组。当以竞争的方式使用时，共价作用的小分子可以竞争 对照相应的基于反应性的探测器，以实现目标发现。使用等TOP-ABPP平台， 该提案显示了用于识别由黄素A靶向的多个半胱氨酸热点的初步数据， 包括主要肿瘤抑制蛋白磷酸酶2A的调节亚单位PPP2R1A的C377 (PP2A)。初步数据表明，在TNBC细胞中，With aferin A处理会损害其磷酸化。 主要的PP2A底物和致癌信号蛋白AKT，潜在地解释了其强大的抗 这些细胞中的癌症活动。使用基于片段的共价配体发现方法和IsoTOP- ABPP平台上，半胱氨酸型活性丙烯酰胺先导DKM 2-90也已被鉴定出更多 选择性地靶向PP2A，损害磷酸化AKT信号，并损害TNBC细胞的活力。这项建议 假设铁蛋白A通过靶向半胱氨酸热点而削弱TNBC的致病性 PPP2R1a激活PP2A活性，破坏AKT等致癌信号通路。这 Proposal将利用IsoTOP-ABPP平台研究With aferin A的作用机制 在削弱TNBC致病性方面，并使用共价配体发现方法开发更多 可合成的有效和选择性的调节剂，可对抗黄素A靶标。

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.