Discovery of novel poly(ADP-ribose) glycohydrolase inhibitors by a quantitative assay system using dot-blot with anti-poly(ADP-ribose)
Discovery of novel poly(ADP-ribose) glycohydrolase inhibitors by a quantitative assay system using dot-blot with anti-poly(ADP-ribose)
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DOI：
10.1016/j.bbrc.2010.01.044
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发表时间：
2010-02-19
影响因子：
3.1
通讯作者：
Tanuma, Sei-ichi
中科院分区：
文献类型：
--
作者：
Okita, Naoyuki;Ashizawa, Daisuke;Tanuma, Sei-ichi
聚腺苷二磷酸核糖基化是一种独特的蛋白质修饰，主要由聚腺苷二磷酸核糖聚合酶(PARP)和聚腺苷二磷酸核糖水解酶(PARG)调控，参与细胞的DNA修复和复制等反应。PARG对PARP合成的聚(ADP-核糖)的糖苷键进行水解，释放出ADP-核糖残基。最近的研究表明，PARG的抑制剂能够成为有效的抗癌药物。为了发现PARG的有效和特异的抑制剂，需要一种定量和高通量的筛选检测系统。然而，以前的PARG检测系统不适合高通量筛选，因为PARG活性是通过放射性同位素(RI)标记的聚(ADP-核糖)释放的ADP-核糖残基的放射性来衡量的。在这项研究中，我们开发了一种基于抗ADP-核糖和硝酸纤维素膜的斑点印迹分析的无RI的PARG活性定量检测系统。用我们的方法测得PARG反应的最大速度(V(Max))和米氏常数(k(M))分别为4.46u M和128.33 m o l/m in/mg。此外，被称为非竞争性PARG抑制剂的腺苷二磷酸(羟甲基)吡咯烷二醇(ADP-HPD)的IC50为0.66微米，这些动力学数值与传统的PARG测定结果相似。通过使用我们的检测系统，我们发现了两个新的PARG抑制剂，它们都是以xanthene为骨架的。因此，我们的定量和简便的方法对于高通量筛选PARG特异性抑制剂是有用的。(C)2010 Elsevier Inc.保留所有权利。
Poly(ADP-ribosyl)ation, which is mainly regulated by poly(ADP-ribose) polymerase (PARP) and poly (ADP-ribose) glycohydrolase (PARG), is a unique protein modification involved in cellular responses such as DNA repair and replication. PARG hydrolyzes glycosidic linkages of poly(ADP-ribose) synthesized by PARP and liberates ADP-ribose residues. Recent studies have suggested that inhibitors of PARG are able to be potent anti-cancer drug. In order to discover the potent and specific Inhibitors of PARG, a quantitative and high-throughput screening assay system is required. However, previous PARG assay systems are not appropriate for high-throughput screening because PARG activity is measured by radioactivities of ADP-ribose residues released from radioisotope (RI)-labeled poly(ADP-ribose). In this study, we developed a non-RI and quantitative assay system for PARG activity based on dot-blot assay using anti-poly (ADP-ribose) and nitrocellulose membrane. By our method, the maximum velocity (V(max)) and the michaelis constant (k(m)) of PARG reaction were 4.46 mu M and 128.33 mu mol/min/mg, respectively. Furthermore, the IC50 of adenosine diphosphate (hydroxymethyl) pyrrolidinediol (ADP-HPD), known as a non-competitive PARG inhibitor, was 0.66 mu M. These kinetics values were similar to those obtained by traditional PARG assays. By using our assay system, we discovered two novel PARG inhibitors that have xanthene scaffold. Thus, our quantitative and convenient method is useful for a high-throughput screening of PARG specific inhibitors. (C) 2010 Elsevier Inc. All rights reserved.