High-throughput screening assay for inhibitors of heat-shock protein 90 ATPase activity
High-throughput screening assay for inhibitors of heat-shock protein 90 ATPase activity
复制标题
DOI：
10.1016/j.ab.2003.10.038
复制
发表时间：
2004-04-15
影响因子：
2.9
通讯作者：
Aherne, W
中科院分区：
文献类型：
--
作者：
Rowlands, MG;Newbatt, YM;Aherne, W
分子伴侣热休克蛋白90（HSP 90）通过稳定许多客户蛋白（其中许多是致癌的）在细胞中起着关键作用。HSP 90的内在ATP酶活性对这种活性是必不可少的。HSP 90是一种新的癌症药物靶点，因为抑制导致几个关键信号通路的同时中断，从而导致恶性肿瘤治疗的组合方法。已经描述了HSP 90 ATP酶活性的抑制剂，包括苯醌安莎霉素、格尔德霉素和17-烯丙基氨基-17-去甲氧基格尔德霉素以及根赤霉素。已经开发了高通量筛选以鉴定可以开发为具有改善的药理学性质的治疗剂的小分子抑制剂。无机磷酸盐的比色分析，基于形成磷钼酸盐络合物和随后与孔雀石绿色反应，用于测量酵母HSP 90的ATP酶活性。测定中测定的ATP的Km为510 +/- 70 μ M。已知的HSP 90抑制剂格尔德霉素和根赤霉素分别给出4.8和0.9 μ M的IC 50值，与使用常规偶联酶测定法发现的值相比。该测定法是稳健的和可重复的（2- 8%CV），并且用于在384孔格式中筛选类似于56，000种化合物的化合物集合，Z'因子在0.6和0.8之间。(C)2003年爱思唯尔公司All rights reserved.
The molecular chaperone heat-shock protein 90 (HSP90) plays a key role in the cell by stabilizing a number of client proteins, many of which are oncogenic. The intrinsic ATPase activity of HSP90 is essential to this activity. HSP90 is a new cancer drug target as inhibition results in simultaneous disruption of several key signaling pathways, leading to a combinatorial approach to the treatment of malignancy. Inhibitors of HSP90 ATPase activity including the benzoquinone ansamycins, geldanamycin and 17-allylamino-17-demethoxygeldanamycin, and radicicol have been described. A high-throughput screen has been developed to identify small-molecule inhibitors that could be developed as therapeutic agents with improved pharmacological properties. A colorimetric assay for inorganic phosphate, based on the formation of a phosphomolybdate complex and subsequent reaction with malachite green, was used to measure the ATPase activity of yeast HSP90. The K-m for ATP determined in the assay was 510 +/- 70 muM. The known HSP90 inhibitors geldanamycin and radicicol gave IC50 values of 4.8 and 0.9 muM respectively, which compare with values found using the conventional coupled-enzyme assay. The assay was robust and reproducible (2-8% CV) and used to screen a compound collection of similar to56,000 compounds in 384-well format with Z' factors between 0.6 and 0.8. (C) 2003 Elsevier Inc. All rights reserved.