Chalcones have the characteristics of simple structure, easy synthesis and potent anti-tumor activity. Herein, a small library of fifty-five novel indole-chalcone derivatives were rationally designed and facilely synthesized. Consequently, their antiproliferative activity was systematically evaluated. Among which, compound 26 exhibited the most potent antiproliferative activity, with IC₅₀ value of 0.764 μM against MD-MBA-231 cells. Moreover, it displayed a 5-fold selectivity compared with normal human cells. Further investigation revealed that compound 26 bound at the colchicine binding site of tubulin, disrupted their fibrous structure, thereby blocking the progression of the cell cycle and inducing apoptosis. Molecular docking and cellular thermal shift assay (CETSA) experiments further demonstrated that compound 26 could specifically bind to hexokinase 2 (HK2) and inhibit its activity, leading to impaired mitochondrial function and hindered mitochondrial respiration. Based on the quantitative structure-activity relationship study, further structure modifications were performed. Employing biotin probe pull-down assays, we demonstrated that compound 26 exerted its antiproliferative activity through a dual targeting mechanism, which simultaneously disrupted microtubule function and inhibited HK2 activity. Taken together, these results highlighted that compound 26 might be a promising antiproliferative agent for human cancer therapy.

中文翻译：

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一种新型吲哚-吡啶查尔酮衍生物作为通过双靶向微管蛋白和 HK2 对抗肿瘤细胞的抗增殖剂的合成、生物学评价和机制研究


查耳酮具有结构简单、易合成、抗肿瘤活性强等特点。在此，一个包含 55 种新型吲哚-查尔酮衍生物的小型库被合理设计并轻松合成。因此，系统地评估了它们的抗增殖活性。其中，化合物 26 表现出最有效的抗增殖活性，对 MD-MBA-231 细胞的 IC₅₀ 值为 0.764 μM。此外，与正常人类细胞相比，它的选择性是正常人细胞的 5 倍。进一步研究显示，化合物 26 结合在微管蛋白的秋水仙碱结合位点，破坏了它们的纤维结构，从而阻断了细胞周期的进展并诱导细胞凋亡。分子对接和细胞热转移测定 （CETSA） 实验进一步表明，化合物 26 可与己糖激酶 2 （HK2） 特异性结合并抑制其活性，导致线粒体功能受损和线粒体呼吸受阻。基于定量构效关系研究，进行了进一步的结构修饰。采用生物素探针 pull-down 测定，我们证明化合物 26 通过双靶向机制发挥其抗增殖活性，同时破坏微管功能并抑制 HK2 活性。综上所述，这些结果强调化合物 26 可能是一种很有前途的人类癌症治疗抗增殖剂。