波洛样激酶1（Plk1）被认为是一个有吸引力的抗癌疗法靶点。多年来，对于Plk1非催化波洛盒结构域（PBD）的研究提高了生成具有高度特异性的蛋白质相互作用抑制剂的期望。然而，规范的波洛盒依赖性相互作用的分子性质，需要与其磷酸锚定体通过广泛的水网络介导的相互作用，阻碍了寻找适用于Plk1 PBD药物发现的小分子的努力。本研究报告了第一个Plk1 PBD的非同址抑制剂的鉴定，名为Allopole，它是一种前药，能够破坏PBD与其同源磷酸体之间的细胞内相互作用，使Plk1从中心体和着丝粒解离，并引起有丝分裂阻滞和癌细胞杀伤。在结构水平上，它的解脱活性形式，Allopole-A，结合到一个被类似闩锁状环所遮盖的深部色氨酸-苯丙氨酸缝隙，并且需要其相邻区域来安全地保持与磷酸结合沟槽上锚定的配体。Allopole-A结合完全拆散了L2环，这一事件似乎足以触发磷酸配体的解离并在有丝分裂期间抑制PBD依赖性的Plk1功能。考虑到Allopole的高特异性和抑制增殖的功效，本研究有望为开发Plk1 PBD特异抗癌治疗药物开辟一条未开发的途径。

Polo-like kinase 1 (Plk1) is considered an attractive target for anticancer therapy. Over the years, studies on the noncatalytic polo-box domain (PBD) of Plk1 have raised the expectation of generating highly specific protein-protein interaction inhibitors. However, the molecular nature of the canonical PBD-dependent interaction, which requires extensive water network-mediated interactions with its phospholigands, has hampered efforts to identify small molecules suitable for Plk1 PBD drug discovery. Here, we report the identification of the first allosteric inhibitor of Plk1 PBD, called Allopole, a prodrug that can disrupt intracellular interactions between PBD and its cognate phospholigands, delocalize Plk1 from centrosomes and kinetochores, and induce mitotic block and cancer cell killing. At the structural level, its unmasked active form, Allopole-A, bound to a deep Trp-Phe-lined pocket occluded by a latch-like loop, whose adjoining region was required for securely retaining a ligand anchored to the phospho-binding cleft. Allopole-A binding completely dislodged the L2 loop, an event that appeared sufficient to trigger the dissociation of a phospholigand and inhibit PBD-dependent Plk1 function during mitosis. Given Allopole's high specificity and antiproliferative potency, this study is expected to open an unexplored avenue for developing Plk1 PBD-specific anticancer therapeutic agents.