The growth of solid tumors depends on tumor vascularization and the endothelial cells (ECs) that line the lumen of blood vessels. ECs generate a large fraction of ATP through glycolysis, and elevation of their glycolytic activity is associated with angiogenic behavior in solid tumors. 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) positively regulates glycolysis via fructose-2/6-bisphosphate, the product of its kinase activity. Partial inhibition of glycolysis in tumor ECs by targeting PFKFB3 normalizes the otherwise abnormal tumor vessels, thereby reducing metastasis and improving the outcome of chemotherapy. Although a limited number of tool compounds exist, orally available PFKFB3 inhibitors are unavailable. In this study we conducted a high-throughput screening campaign against the kinase activity of PFKFB3, involving 250,240 chemical compounds. A total of 507 initial hits showing >50% inhibition at 20 µM were identified, 66 of them plus 1 analog from a similarity search consistently displayed low IC₅₀ values (<10 µM). In vitro experiments yielded 22 nontoxic hits that suppressed the tube formation of primary human umbilical vein ECs at 10 µM. Of them, 15 exhibited binding affinity to PFKFB3 in surface plasmon resonance assays, including 3 (WNN0403-E003, WNN1352-H007 and WNN1542-F004) that passed the pan-assay interference compounds screening without warning flags. This study provides potential leads to the development of new PFKFB3 inhibitors.

实体瘤的生长取决于肿瘤血管形成和排列在血管腔内的内皮细胞 (EC)。ECs 通过糖酵解产生大部分 ATP，其糖酵解活性的升高与实体瘤中的血管生成行为有关。6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶 3 (PFKFB3) 通过果糖-2/6-二磷酸（其激酶活性的产物）正向调节糖酵解。通过靶向 PFKFB3 部分抑制肿瘤 EC 中的糖酵解，使原本异常的肿瘤血管正常化，从而减少转移并改善化疗结果。尽管存在的工具化合物数量有限，但还没有口服可用的 PFKFB3 抑制剂。在这项研究中，我们针对 PFKFB3 的激酶活性进行了高通量筛选活动，涉及 250，240 种化合物。总共识别出 507 个在 20 µM 时显示 >50% 抑制的初始命中，其中 66 个加上来自相似性搜索的 1 个类似物始终显示低 IC_{50 个}值（<10 µM）。体外实验产生了 22 个无毒命中，这些命中在 10 µM 时抑制了原代人脐静脉 EC 的管形成。其中，15 个在表面等离子共振测定中表现出对 PFKFB3 的结合亲和力，其中 3 个（WNN0403-E003、WNN1352-H007 和 WNN1542-F004）通过了泛测定干扰化合物筛选，没有警告标志。该研究为开发新的 PFKFB3 抑制剂提供了潜在的线索。