Molecular and Cellular Pharmacology of the Hypoxia-Activated Prodrug TH-302
Molecular and Cellular Pharmacology of the Hypoxia-Activated Prodrug TH-302
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DOI：
10.1158/1535-7163.mct-11-0634
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发表时间：
2012-03-01
影响因子：
5.7
通讯作者：
Hart, Charles P.
中科院分区：
文献类型：
--
作者：
Meng, Fanying;Evans, James W.;Hart, Charles P.
TH-302是一种2-硝基咪唑触发的溴异磷酰胺芥菜缺氧激活前药（HAP），目前正在进行临床评估。在这里，我们描述了TH-302的广谱活性、低氧选择性激活和作用机制。参照原型HAP替拉帕胺，考察TH-302活化的浓度和时间依赖性与氧浓度的函数关系，结果显示TH-302具有较好的氧抑制细胞毒性，且剂量效比替拉帕胺高得多。在32个人类癌细胞系中观察到缺氧条件下TH-302细胞毒性增强。利用过表达人NADPH的细胞证实了单电子还原酶依赖性；细胞色素P450氧化还原酶和辐射分解还原建立了TH-302断裂（活化）的单电子化学计量学。在考察TH-302活性的下游效应时，我们观察到缺氧依赖性诱导γ - H2AX磷酸化、DNA交联和细胞周期阻滞。我们使用中国仓鼠卵巢细胞DNA修复突变细胞系，发现同源依赖性修复缺陷细胞系，而碱基切除、核苷酸切除或非同源末端连接修复缺陷细胞系，在缺氧条件下对TH-302表现出明显的敏感性。与这一发现一致，在BRCA1、BRCA2和FANCA基因缺失的细胞系中也观察到对TH-302的敏感性增强。最后，我们在三维肿瘤球体和多细胞层模型中表征了TH-302的活性。TH-302在常氧条件下对H460球状细胞的效价明显高于H460单层细胞。由亲代HCT116细胞和表达氧不敏感细菌硝基还原酶的HCT116细胞混合组成的多细胞层表明，TH-302表现出显著的旁观者效应。巨蟹座；11 (3);740 - 51。(c) 2011年aacr。
TH-302 is a 2-nitroimidazole triggered hypoxia-activated prodrug (HAP) of bromo-isophosphoramide mustard currently undergoing clinical evaluation. Here, we describe broad-spectrum activity, hypoxia-selective activation, and mechanism of action of TH-302. The concentration and time dependence of TH-302 activation was examined as a function of oxygen concentration, with reference to the prototypic HAP tirapazamine, and showed superior oxygen inhibition of cytotoxicity and much improved dose potency relative to tirapazamine. Enhanced TH-302 cytotoxicity under hypoxia was observed across 32 human cancer cell lines. One-electron reductive enzyme dependence was confirmed using cells overexpressing human NADPH: cytochrome P450 oxidoreductase and radiolytic reduction established the single-electron stoichiometry of TH-302 fragmentation (activation). Examining downstream effects of TH-302 activity, we observed hypoxia-dependent induction of gamma H2AX phosphorylation, DNA cross-linking, and cell-cycle arrest. We used Chinese hamster ovary cell-based DNA repair mutant cell lines and established that lines deficient in homology-dependent repair, but not lines deficient in base excision, nucleotide excision, or nonhomologous end-joining repair, exhibited marked sensitivity to TH-302 under hypoxia. Consistent with this finding, enhanced sensitivity to TH-302 was also observed in lines deficient in BRCA1, BRCA2, and FANCA. Finally, we characterized TH-302 activity in the three-dimensional tumor spheroid and multicellular layer models. TH-302 showed much enhanced potency in H460 spheroids compared with H460 monolayer cells under normoxia. Multicellular layers composed of mixtures of parental HCT116 cells and HCT116 cells engineered to express an oxygen-insensitive bacterial nitroreductase showed that TH-302 exhibits a significant bystander effect. Mol Cancer Ther; 11(3); 740-51. (C) 2011 AACR.