An ultra-high throughput cell-based screen for wee1 degradation inhibitors.
An ultra-high throughput cell-based screen for wee1 degradation inhibitors.
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DOI：
10.1177/1087057110375848
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发表时间：
2010-09
影响因子：
--
通讯作者：
Hodder P
中科院分区：
文献类型：
--
作者：
Madoux F;Simanski S;Chase P;Mishra JK;Roush WR;Ayad NG;Hodder P
酪氨酸激酶Wee 1是一种关键的细胞传感机制的一部分，它发出DNA复制完成的信号，确保进入有丝分裂的正确时机。Wee 1通过磷酸化细胞周期蛋白依赖性激酶CDK 1作为有丝分裂进入的抑制剂。Wee 1的活性主要是通过其磷酸化和随后的泛素蛋白酶体途径降解在蛋白质水平上调节。为了便于识别阻止Wee 1降解的小分子，使用瞬时转染Wee 1-荧光素酶融合蛋白的HeLa细胞开发了基于细胞的均相测定。为了确保uHTS相容性，将测定按比例缩放至1，536孔板格式，并将细胞批量转染并冷冻保存。该小型化均相测定证明了稳健的性能，计算的Z′因子为0.65±0.05。针对约218，000种化合物的公开可用文库筛选该测定法，以鉴定Wee 1稳定剂。非选择性，细胞毒性和混杂的化合物通过使用类似格式的counterscreen快速分类，该counterscreen测量N-细胞周期蛋白B-荧光素酶融合蛋白的稳定性，以及在亲本HeLa细胞系中执行活力评估。该筛选活动导致发现了四种不相关的细胞可渗透的小分子，其显示具有微摩尔效力的选择性Wee 1-荧光素酶稳定化。这些化合物之一，SID 4243143，显示出抑制细胞周期进程，强调了Wee 1降解对细胞周期的重要性。我们的研究结果表明，这种uHTS的方法是适合于确定选择性的化学探针，防止Wee 1降解，并普遍适用于发现抑制剂的泛素蛋白酶体途径。
The tyrosine kinase Wee1 is part of a key cellular sensing mechanism that signals completion of DNA replication, ensuring proper timing of entry into mitosis. Wee1 acts as an inhibitor of mitotic entry by phosphorylating cyclin-dependent kinase CDK1. Wee1 activity is mainly regulated at the protein level through its phosphorylation and subsequent degradation by the ubiquitin proteasome pathway. To facilitate identification of small molecules preventing Wee1 degradation, a homogeneous cell-based assay was developed using HeLa cells transiently transfected with a Wee1-Luciferase fusion protein. To insure uHTS compatibility, the assay was scaled to 1,536-well plate format and cells were transfected in bulk and cryopreserved. This miniaturized homogenous assay demonstrated robust performance, with a calculated Z′ factor of 0.65±0.05. The assay was screened against a publicly available library of ~218,000 compounds in order to identify Wee1 stabilizers. Nonselective, cytotoxic and promiscuous compounds were rapidly triaged through the use of a similarly formatted counterscreen that measured stabilization of a N-cyclin B-Luciferase fusion protein, as well as execution of viability assessment in the parental HeLa cell line. This screening campaign led to the discovery of four unrelated cell-permeable small molecules that showed selective Wee1-Luciferase stabilization with micromolar potency. One of these compounds, SID4243143, was shown to inhibit cell cycle progression, underscoring the importance of Wee1 degradation to the cell cycle. Our results suggest that this uHTS approach is suitable for identifying selective chemical probes that prevent Wee1 degradation, and generally applicable to discovering inhibitors of the ubiquitin proteasome pathway.
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DOI：
10.1073/pnas.0609299104
发表时间：
2007-04-24
影响因子：
11.1
作者：
Hallstrom, Taija M. Kiviharju-af;Jaamaa, Sari;Laiho, Marikki
通讯作者：
Laiho, Marikki
DOI：
10.1073/pnas.0307700101
发表时间：
2004-03-30
影响因子：
11.1
作者：
Watanabe, N;Arai, H;Osada, H
通讯作者：
Osada, H
影响因子：
3.6
作者：
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通讯作者：
Hodder, Peter
DOI：
10.1073/pnas.0500410102
发表时间：
2005-08-16
影响因子：
11.1
作者：
Watanabe, N;Arai, H;Osada, H
通讯作者：
Osada, H
影响因子：
9.2
作者：
Lenart, Peter;Petronczki, Mark;Peters, Jan-Michael
通讯作者：
Peters, Jan-Michael