The chromatin-associated protein WDR5 is a promising pharmacological target in cancer, with most drug discovery efforts directed against an arginine-binding cavity in WDR5 called the WIN site. Despite a clear expectation that WIN site inhibitors will alter the repertoire of WDR5 interaction partners, their impact on the WDR5 interactome remains unknown. Here, we use quantitative proteomics to delineate how the WDR5 interactome is changed by WIN site inhibition. We show that the WIN site inhibitor alters the interaction of WDR5 with dozens of proteins, including those linked to phosphatidylinositol 3-kinase (PI3K) signaling. As proof of concept, we demonstrate that the master kinase PDPK1 is a bona fide high-affinity WIN site binding protein that engages WDR5 to modulate transcription of genes expressed in the G2 phase of the cell cycle. This dataset expands our understanding of WDR5 and serves as a resource for deciphering the action of WIN site inhibitors.

染色质相关蛋白 WDR5 是癌症中一个有前途的药理学靶点，大多数药物发现工作都针对 WDR5 中称为 WIN 位点的精氨酸结合腔。尽管明确预期 WIN 位点抑制剂将改变 WDR5 相互作用伙伴的全部功能，但它们对 WDR5 相互作用组的影响仍然未知。在这里，我们使用定量蛋白质组学来描述 WDR5 相互作用组如何通过 WIN 位点抑制而改变。我们发现 WIN 位点抑制剂改变了 WDR5 与数十种蛋白质的相互作用，包括与磷脂酰肌醇 3 激酶 (PI3K) 信号传导相关的蛋白质。作为概念证明，我们证明主激酶 PDPK1 是一种真正的高亲和力 WIN 位点结合蛋白，它与 WDR5 结合来调节细胞周期 G2 期表达的基因的转录。该数据集扩展了我们对 WDR5 的理解，并可作为破译 WIN 位点抑制剂作用的资源。