Polo-like kinase 4 (PLK4) is a serine/threonine protein kinase involved in regulating cell mitosis and centriole duplication, and has emerged as a therapeutic target for treating multiple cancers. At first, the design and in vitro validation of PLK4 inhibitors (12a-12e, 17a-17f, 22a-22e) bearing 1H-pyrazolo[3,4-b]pyridine scaffold was described and lead compound 22a (IC₅₀ = 0.106 μM) was identified. Then, selectivity- and activity-guided development of a series of potent and selective type-II PLK4 inhibitors using a homology model approach was carried out. Further structure-based optimization resulted in a potent type-II PLK4 inhibitor 29u (IC₅₀ = 0.026 μM), which exhibited outstanding selectivity in a panel of 47 kinases at a single concentration of 1.0 μM. Furthermore, compound 29u significantly inhibited the proliferation of breast cancer cell line MCF-7 with an IC₅₀ value of 1.52 μM, while it exhibited no inhibitory effect on normal cell lines (L02 and HUVECs). Meanwhile, the clone formation, senescence and migration abilities of compound 29u were evaluated using MCF-7 cells. The detailed biological evaluation revealed that compound 29u could arrest cell division in S/G2 phase by inhibiting PLK4, and then affect the expression of downstream signalling pathway proteins regulated by PLK4. Moreover, the in vitro preliminary evaluation of the drug-like properties of compound 29u exhibited outstanding plasma stability, moderate liver microsomal stability, and low risk of drug-drug interactions (DDIs). The current discovery will support the further development of compound 29u as a lead compound for PLK4-targeted anticancer drug discovery and as a useful chemical probe for the further biological research of PLK4.

Polo 样激酶 4 (PLK4) 是一种丝氨酸/苏氨酸蛋白激酶，参与调节细胞有丝分裂和中心粒复制，已成为治疗多种癌症的治疗靶点。首先，描述了带有 1 H-吡唑并[3,4- b ]吡啶支架的PLK4抑制剂（12a-12e、17a-17f、22a-22e ）和先导化合物22a（IC ₅₀ = 0.106 μM) 被鉴定。然后，使用同源模型方法进行了一系列有效和选择性的 II 型 PLK4 抑制剂的选择性和活性指导开发。进一步基于结构的优化产生了一种有效的 II 型 PLK4 抑制剂29u (IC ₅₀  = 0.026 μM)，在 1.0 μM 的单一浓度下，它在一组 47 种激酶中表现出出色的选择性。此外，化合物29u显着抑制乳腺癌细胞系MCF-7的增殖，IC ₅₀值为1.52 μM，而对正常细胞系（L02和HUVECs）没有抑制作用。同时，化合物29u的克隆形成、衰老和迁移能力使用 MCF-7 细胞进行评估。详细的生物学评价表明，化合物29u可以通过抑制PLK4在S/G2期阻止细胞分裂，进而影响PLK4调控的下游信号通路蛋白的表达。此外，对化合物29u的药物样特性的体外初步评估显示出出色的血浆稳定性、适度的肝微粒体稳定性和药物-药物相互作用 (DDI) 的低风险。目前的发现将支持化合物29u作为 PLK4 靶向抗癌药物发现的先导化合物的进一步开发，并作为 PLK4 进一步生物学研究的有用化学探针。