Dudgeon Drew D, Shinde Sunita N, Shun Tong Ying, Lazo John S, Strock Christopher J, Giuliano Kenneth A, Taylor D Lansing, Johnston Patricia A, Johnston Paul A
Drug Discovery Institute, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
Assay Drug Dev Technol. 2010 Aug;8(4):437-58. doi: 10.1089/adt.2010.0281.
We present here the characterization and optimization of a novel imaging-based positional biosensor high-content screening (HCS) assay to identify disruptors of p53-hDM2 protein-protein interactions (PPIs). The chimeric proteins of the biosensor incorporated the N-terminal PPI domains of p53 and hDM2, protein targeting sequences (nuclear localization and nuclear export sequence), and fluorescent reporters, which when expressed in cells could be used to monitor p53-hDM2 PPIs through changes in the subcellular localization of the hDM2 component of the biosensor. Coinfection with the recombinant adenovirus biosensors was used to express the NH-terminal domains of p53 and hDM2, fused to green fluorescent protein and red fluorescent protein, respectively, in U-2 OS cells. We validated the p53-hDM2 PPI biosensor (PPIB) HCS assay with Nutlin-3, a compound that occupies the hydrophobic pocket on the surface of the N-terminus of hDM2 and blocks the binding interactions with the N-terminus of p53. Nutlin-3 disrupted the p53-hDM2 PPIB in a concentration-dependent manner and provided a robust, reproducible, and stable assay signal window that was compatible with HCS. The p53-hDM2 PPIB assay was readily implemented in HCS and we identified four (4) compounds in the 1,280-compound Library of Pharmacologically Active Compounds that activated the p53 signaling pathway and elicited biosensor signals that were clearly distinct from the responses of inactive compounds. Anthracycline (topoisomerase II inhibitors such as mitoxantrone and ellipticine) and camptothecin (topoisomerase I inhibitor) derivatives including topotecan induce DNA double strand breaks, which activate the p53 pathway through the ataxia telangiectasia mutated-checkpoint kinase 2 (ATM-CHK2) DNA damage response pathway. Although mitoxantrone, ellipticine, camptothecin, and topotecan all exhibited concentration-dependent disruption of the p53-hDM2 PPIB, they were much less potent than Nutlin-3. Further, their corresponding cellular images and quantitative HCS data did not completely match the Nutlin-3 phenotypic profile.
我们在此展示了一种基于成像的新型位置生物传感器高内涵筛选（HCS）分析方法的特性及优化，该方法用于识别p53-hDM2蛋白-蛋白相互作用（PPI）的干扰剂。生物传感器的嵌合蛋白包含p53和hDM2的N端PPI结构域、蛋白质靶向序列（核定位和核输出序列）以及荧光报告基因，当在细胞中表达时，可通过生物传感器中hDM2组分亚细胞定位的变化来监测p53-hDM2 PPI。用重组腺病毒生物传感器共感染，用于在U-2 OS细胞中分别表达与绿色荧光蛋白和红色荧光蛋白融合的p53和hDM2的NH端结构域。我们用Nutlin-3验证了p53-hDM2 PPI生物传感器（PPIB）HCS分析方法，Nutlin-3是一种占据hDM2 N端表面疏水口袋并阻断与p53 N端结合相互作用的化合物。Nutlin-3以浓度依赖的方式破坏p53-hDM2 PPIB，并提供了一个强大、可重复且稳定的分析信号窗口，该窗口与HCS兼容。p53-hDM2 PPIB分析方法可轻松在HCS中实施，我们在1280种具有药理活性化合物的文库中鉴定出4种化合物，它们激活了p53信号通路并引发了与无活性化合物反应明显不同的生物传感器信号。蒽环类药物（拓扑异构酶II抑制剂，如米托蒽醌和椭圆玫瑰树碱）和喜树碱（拓扑异构酶I抑制剂）衍生物（包括拓扑替康）会诱导DNA双链断裂，通过共济失调毛细血管扩张突变-检查点激酶2（ATM-CHK2）DNA损伤反应途径激活p53途径。尽管米托蒽醌、椭圆玫瑰树碱、喜树碱和拓扑替康均表现出对p53-hDM2 PPIB的浓度依赖性破坏，但它们的效力远低于Nutlin-3。此外，它们相应的细胞图像和定量HCS数据与Nutlin-3的表型特征并不完全匹配。
