Translational DNA Repair Group, Laboratory of Molecular Oncology, Academic Unit of Oncology, School of Molecular Medical Sciences, Nottingham University Hospitals, University of Nottingham, Nottingham, UK.
Br J Cancer. 2011 Feb 15;104(4):653-63. doi: 10.1038/sj.bjc.6606058. Epub 2011 Jan 25.
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 IC(50) 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.
调节 DNA 碱基切除修复（BER）有可能增强化疗反应，并改善黑色素瘤和神经胶质瘤等肿瘤的预后。APE1 是 BER 中一种关键的蛋白质，可处理潜在细胞毒性的脱碱基位点（AP 位点），是癌症治疗中一个很有前途的新靶点。在本研究中，我们旨在开发用于癌症治疗的 APE1 小分子抑制剂。
采用了一种行业标准的高通量虚拟筛选策略。使用 Sybyl8.0（Tripos，圣路易斯，密苏里州，美国）分子建模软件套件构建抑制剂模板。然后使用 ROCS 2.3（Open Eye Scientific，圣达菲，新墨西哥州，美国）的相似性搜索策略，从包含 260 万种化合物的化学多样性数据库中提取具有药理学相关性的化合物亚组。将这些亚组中的化合物与 APE1 模型的活性位点进行对接，使用基于遗传算法的程序 GOLD2.7（CCDC，剑桥，英国）。根据几个评分函数对预测的配体构象进行排序。具有良好药物性质的顶级虚拟命中物通过基于荧光的 APE1 切割测定法进行详细的体外分析，并通过内切核酸酶 IV 切割测定法、荧光猝灭测定法和放射性标记寡核苷酸测定法进行反向筛选。然后对生化 APE1 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定的 APE1 抑制剂。APE1 抑制的 IC50 范围在 30 nM 至 50 μM 之间。我们证明 APE1 抑制剂会导致基因组 DNA 中 AP 位点的积累，并增强黑色素瘤和神经胶质瘤细胞系中烷化剂的细胞毒性。
我们的研究提供了证据表明 APE1 是一个新兴的药物靶点，并可能在患有黑色素瘤和神经胶质瘤的患者中有治疗应用。
