Development and evaluation of human AP endonuclease inhibitors in melanoma and glioma cell lines.
Development and evaluation of human AP endonuclease inhibitors in melanoma and glioma cell lines.
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DOI：
10.1038/sj.bjc.6606058
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发表时间：
2011-02-15
影响因子：
8.8
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DNA碱基切除修复（BER）的调节有可能增强对化疗的反应并改善肿瘤（如黑色素瘤和神经胶质瘤）的结局。APE 1是BER中的一个关键蛋白，处理潜在的细胞毒性脱碱基位点（AP位点），是一个有前途的癌症新靶点。在本研究中，我们旨在开发用于癌症治疗的APE 1小分子抑制剂。采用工业标准的高通量虚拟筛选策略。Sybyl8.0（Tripos，圣刘易斯，密苏里州，美国）分子建模软件套件用于构建抑制剂模板。然后使用ROCS 2.3（Open Eye Scientific，Santa Fe，NM，USA）应用相似性搜索策略，从260万种化合物的化学多样性数据库中提取化合物的药理学相关子集。使用基于遗传算法的程序GOLD 2.7（CCDC，剑桥，UK），对这些子集中的化合物进行与APE 1模型活性位点的对接。预测的配位体的姿势进行排名的基础上，几个评分功能。使用基于荧光的APE 1切割测定法对具有有希望的药物特性的顶级虚拟命中进行详细的体外分析，并使用核酸内切酶IV切割测定法、荧光猝灭测定法和放射性标记的寡核苷酸测定法进行反筛选。然后对生化APE 1抑制剂进行详细的细胞毒性分析。通过这种方法分离出几种特异性APE 1抑制剂。APE 1抑制的IC50范围为30 nM至50 μM。我们证明，APE 1抑制剂导致AP位点在基因组DNA中的积累，并增强烷化剂在黑色素瘤和神经胶质瘤细胞系中的细胞毒性。我们的研究提供了证据，APE 1是一个新兴的药物靶点，可能在黑色素瘤和胶质瘤患者中具有治疗应用。
Modulation of DNA base excision repair (BER) has the potential to enhance response to chemotherapy and improve outcomes in tumours such as melanoma and glioma. APE1, a critical protein in BER that processes potentially cytotoxic abasic sites (AP sites), is a promising new target in cancer. In the current study, we aimed to develop small molecule inhibitors of APE1 for cancer therapy. An industry-standard high throughput virtual screening strategy was adopted. The Sybyl8.0 (Tripos, St Louis, MO, USA) molecular modelling software suite was used to build inhibitor templates. Similarity searching strategies were then applied using ROCS 2.3 (Open Eye Scientific, Santa Fe, NM, USA) to extract pharmacophorically related subsets of compounds from a chemically diverse database of 2.6 million compounds. The compounds in these subsets were subjected to docking against the active site of the APE1 model, using the genetic algorithm-based programme GOLD2.7 (CCDC, Cambridge, UK). Predicted ligand poses were ranked on the basis of several scoring functions. The top virtual hits with promising pharmaceutical properties underwent detailed in vitro analyses using fluorescence-based APE1 cleavage assays and counter screened using endonuclease IV cleavage assays, fluorescence quenching assays and radiolabelled oligonucleotide assays. Biochemical APE1 inhibitors were then subjected to detailed cytotoxicity analyses. Several specific APE1 inhibitors were isolated by this approach. The IC50 for APE1 inhibition ranged between 30 nM and 50 μM. We demonstrated that APE1 inhibitors lead to accumulation of AP sites in genomic DNA and potentiated the cytotoxicity of alkylating agents in melanoma and glioma cell lines. Our study provides evidence that APE1 is an emerging drug target and could have therapeutic application in patients with melanoma and glioma.