Identification and characterization of inhibitors of human apurinic/apyrimidinic endonuclease APE1.
Identification and characterization of inhibitors of human apurinic/apyrimidinic endonuclease APE1.
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DOI：
10.1371/journal.pone.0005740
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发表时间：
2009-06-01
期刊：
影响因子：
3.7
通讯作者：
Wilson DM 3rd
中科院分区：
文献类型：
--
作者：
Simeonov A;Kulkarni A;Dorjsuren D;Jadhav A;Shen M;McNeill DR;Austin CP;Wilson DM 3rd
APE 1是从DNA上切除脱碱基（AP）位点和3′-梗阻性末端的主要核酸酶，是碱基切除修复（BER）途径的重要参与者。BER能力在决定对产生氧化或烷基化DNA损伤的试剂以及某些链终止核苷类似物和5-氟尿嘧啶的反应性方面起着重要作用。我们描述了一个强大的，1536孔自动化筛选试验，采用脱氧寡核苷酸基板的红移荧光光谱区域，以确定APE 1核酸内切酶抑制剂的发展。该AP位点切割试验用于药理学活性化合物库（LOPAC 1280）的基于滴定的高通量筛选，该化合物库是代表所有主要靶类别的良好表征的药物样分子的集合。通过两种高通量筛选试验-噻唑橙子荧光团-DNA置换试验和E.大肠杆菌核酸内切酶IV counterscreen -和一个传统的，基于凝胶的放射性示踪剂切口测定。经验证的顶级化合物，即6-羟基-DL-多巴、活性蓝2和杨梅素，显示出抑制来自HEK 293 T和HeLa细胞系的全细胞蛋白提取物的AP位点切割活性，并增强烷化剂甲基甲烷磺酸盐的细胞毒性和遗传毒性效力。本文的研究报告了新型小分子APE 1靶向生物活性抑制剂探针的鉴定，这些探针代表了潜在药物开发的初始化学型。
APE1 is the major nuclease for excising abasic (AP) sites and particular 3′-obstructive termini from DNA, and is an integral participant in the base excision repair (BER) pathway. BER capacity plays a prominent role in dictating responsiveness to agents that generate oxidative or alkylation DNA damage, as well as certain chain-terminating nucleoside analogs and 5-fluorouracil. We describe within the development of a robust, 1536-well automated screening assay that employs a deoxyoligonucleotide substrate operating in the red-shifted fluorescence spectral region to identify APE1 endonuclease inhibitors. This AP site incision assay was used in a titration-based high-throughput screen of the Library of Pharmacologically Active Compounds (LOPAC1280), a collection of well-characterized, drug-like molecules representing all major target classes. Prioritized hits were authenticated and characterized via two high-throughput screening assays – a Thiazole Orange fluorophore-DNA displacement test and an E. coli endonuclease IV counterscreen – and a conventional, gel-based radiotracer incision assay. The top, validated compounds, i.e. 6-hydroxy-DL-DOPA, Reactive Blue 2 and myricetin, were shown to inhibit AP site cleavage activity of whole cell protein extracts from HEK 293T and HeLa cell lines, and to enhance the cytotoxic and genotoxic potency of the alkylating agent methylmethane sulfonate. The studies herein report on the identification of novel, small molecule APE1-targeted bioactive inhibitor probes, which represent initial chemotypes towards the development of potential pharmaceuticals.
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影响因子：
6.1
作者：
Häkkinen, SH;Kärenlampi, SO;Törrönen, AR
通讯作者：
Törrönen, AR
影响因子：
5.6
作者：
Berquist, Brian R.;McNeill, Daniel R.;Wilson, David M., III
通讯作者：
Wilson, David M., III
影响因子：
3.8
作者：
Demple, B;Sung, JS
通讯作者：
Sung, JS
影响因子：
14.9
作者：
CHEN, DS;HERMAN, T;DEMPLE, B
通讯作者：
DEMPLE, B
影响因子：
4
作者：
Brown, J;Brown, CA
通讯作者：
Brown, CA