Toxicology Graduate Program, Faculty of Science, Mahidol University, Bangkok, Thailand; Excellent Center for Drug Discovery (ECDD), Mahidol University, Bangkok, Thailand.
Department of Pharmacology, Faculty of Science, Mahidol University, Bangkok, Thailand.
Biomed Pharmacother. 2020 Jul;127:110149. doi: 10.1016/j.biopha.2020.110149. Epub 2020 Apr 25.
Topoisomerase IIα enzyme (Topo IIα) plays a critical function in DNA replication process and is considered to be a promising target of anti-cancer drugs. In the present study, we reported that the altholactone derivatives modified by adding a halogenated benzoate group showed greater inhibitory activity on Topo IIα enzyme in cell-free system concomitant with cytotoxicity against the CCA cell lines (KKU-M055 and KKU-M213) than those of the parent altholactone. However, the cytotoxic activities of four halogenated benzoate altholactone derivatives including iodo-, fluoro-, chloro-, and bromobenzoate derivatives (compound 1, 2, 3, and 4, respectively) were of equal potency. The fluorobenzoate derivative (compound 2) was chosen for investigating the underlying mechanism in CCA cells. Compound 2 arrested CCA cell cycle at sub G1 phase and induced apoptotic cell death. It markedly inhibited Topo IIα protein expression in both KKU-M055 and KKU-M213 cells, which was accompanied by DNA double-strand breaks demonstrated by an increase in phosphorylated H2A.X protein. Interestingly, KKU-M055 cells, which express higher Topo IIα mRNA compared to KKU-M213 cells, showed greater sensitivity to the compound, indicating the selectivity of the compound to Topo IIα enzyme. By computational docking analysis, the binding affinity of altholactone (-52.5 kcal/mol) and compound 2 (-56.7 kcal/mol) were similar to that of the Topo II poison salvicine (-53.7 kcal/mol). The aromatic moiety of both altholactones embedded in a hydrophobic pocket of Topo II ATPase domain. In addition, compound 2 induced the formation of linear DNA in Topo II-mediated cleavage assay. Collectively, our results demonstrate that the addition of fluorobenzoyl group to altholactone enhances potency and selectivity to inhibit type IIα topoisomerases. Atholactone and fluorobenzoate derivative act as Topo II cleavage complexes stabilizing compounds or Topo II poisons preferentially through binding at ATPase domain of Topo IIα, leading to DNA double-strand breaks and apoptosis induction. Such activity of 3-fluorobenzoate derivative of altholactone should be further explored for the development of an anti-cancer drug for CCA.
拓扑异构酶 IIα 酶（Topo IIα）在 DNA 复制过程中发挥着关键作用，被认为是抗癌药物的一个有前途的靶点。在本研究中，我们报道了通过添加卤代苯甲酸酯基团修饰的阿尔托内酯衍生物在无细胞体系中对拓扑异构酶 IIα 酶表现出更强的抑制活性，同时对 CCA 细胞系（KKU-M055 和 KKU-M213）具有细胞毒性，优于母体阿尔托内酯。然而，四种卤代苯甲酸酯阿尔托内酯衍生物的细胞毒性活性（碘代、氟代、氯代和溴代苯甲酸酯衍生物，分别为化合物 1、2、3 和 4）具有同等效力。选择氟代苯甲酸酯衍生物（化合物 2）来研究 CCA 细胞中的潜在机制。化合物 2 将 CCA 细胞周期阻滞在 sub G1 期，并诱导细胞凋亡。它显著抑制了 KKU-M055 和 KKU-M213 细胞中的拓扑异构酶 IIα 蛋白表达，这伴随着磷酸化 H2A.X 蛋白的增加，表明 DNA 双链断裂。有趣的是，与 KKU-M213 细胞相比，表达更高水平 Topo IIα mRNA 的 KKU-M055 细胞对该化合物更敏感，表明该化合物对 Topo IIα 酶的选择性。通过计算对接分析，阿尔托内酯（-52.5 kcal/mol）和化合物 2（-56.7 kcal/mol）的结合亲和力与拓扑异构酶 II 毒药 salvicine（-53.7 kcal/mol）相似。两种阿尔托内酯的芳香部分都嵌入拓扑异构酶 II ATP 酶结构域的疏水性口袋中。此外，化合物 2 在拓扑异构酶介导的切割实验中诱导了线性 DNA 的形成。总之，我们的研究结果表明，在阿尔托内酯中添加氟苯甲酰基可增强抑制 IIα 拓扑异构酶的效力和选择性。阿尔托内酯和氟苯甲酸酯衍生物作为拓扑异构酶切割复合物稳定剂或拓扑异构酶毒物，通过与拓扑异构酶 IIα 的 ATP 酶结构域结合，优先导致 DNA 双链断裂和诱导细胞凋亡。阿尔托内酯的 3-氟苯甲酸酯衍生物的这种活性应该进一步探索，以开发用于治疗 CCA 的抗癌药物。
