Abstract

The superfamily of the protein kinase C (PKC) comprises ten isozymes and is widely known for its key role in signal transduction. Protein kinase Cε (PKCε) is known to play key roles in tumor suppression. PKCε requires activation to interact with RACK2, and the adaptor protein then translocates activated PKCε to subcellular sites within the proximity of their substrates. An EAVSLKPT peptide interferes with the interaction of PKCε and its adaptor protein RACK2. Since signaling in the malignant cells are sufficiently changed then the scope and limitations of PKCe as a anticancer drug target has to be estimated more clearly. Acquiring isozyme-selective inhibitors is a difficult task due to the high sequence similarity within the ten PKCs. Small molecule-disruptors of the PKCε/RACK2 protein–protein interaction could suppress PKCε signaling and reduce malignant properties. The EAVSLKPT peptide was used as a base of a pharmacophore model. Thieno[2,3-b]quinolines as a wide cluster of specific small-molecule inhibitors of the PKCε/RACK2 protein–protein interaction and PKCε signaling were revealed. The structural features of active thieno[2,3-b]quinolines were expanded on the basis of this pharmacophore model. The interaction between PKCε and RACK2 was measured using an ELISA-based assay. It was found that N-(4-acetylphenyl)-3-amino-6,7-ethelendioxy-thieno[2,3-b]quinoline-2-carboxamide (1b) shows promising inhibitory activities on the interaction of PKCε with its adaptor protein, the receptor for activated C-kinase 2 (RACK2), hence interfering with PKCε signaling. Both 1a and 1b did not show some cytotoxic properties on susceptible PC-3 cell line but both active compounds showed a significant antisprouting activity. The quinolines without thiophene ring as “open” analogs of 1b were inactive in primary assays. A structural isomer of (1a meta-acetyl), compound (1b para-acetyl) was found to exhibit, in addition to strong inhibitory activity on PKCε signaling with an IC₅₀ of 4.25 µM, also anti-angiogenic activities. Thus thieno[2,3-b]quinolines 1a and 1b could be reliable and selective biochemical tools to investigate of PKCe/RACK2 effects.

中文翻译：

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噻吩并喹啉作为 PKCε/RACK2 蛋白质-蛋白质相互作用破坏剂的新生物学评价

摘要

蛋白激酶 C (PKC) 的超家族包含十种同工酶，并且因其在信号转导中的关键作用而广为人知。已知蛋白激酶 Cε (PKCε) 在肿瘤抑制中发挥关键作用。PKCε 需要激活才能与 RACK2 相互作用，然后衔接蛋白将激活的 PKCε 转移到其底物附近的亚细胞位点。EAVSLKPT 肽会干扰 PKCε 及其衔接蛋白 RACK2 的相互作用。由于恶性细胞中的信号发生了充分的变化，因此必须更清楚地估计 PKCe 作为抗癌药物靶标的范围和局限性。由于 10 个 PKC 中序列的高度相似性，获得同工酶选择性抑制剂是一项艰巨的任务。PKCε/RACK2 蛋白质-蛋白质相互作用的小分子干扰物可以抑制 PKCε 信号并减少恶性特性。EAVSLKPT 肽用作药效团模型的基础。噻吩并 [2,3-b] 喹啉作为 PKCε/RACK2 蛋白质-蛋白质相互作用和 PKCε 信号转导的一大类特异性小分子抑制剂被揭示出来。在此药效团模型的基础上扩展了活性噻吩并[2,3-b]喹啉的结构特征。使用基于 ELISA 的测定法测量 PKCε 和 RACK2 之间的相互作用。结果发现 在此药效团模型的基础上扩展了活性噻吩并[2,3-b]喹啉的结构特征。使用基于 ELISA 的测定法测量 PKCε 和 RACK2 之间的相互作用。结果发现 在此药效团模型的基础上扩展了活性噻吩并[2,3-b]喹啉的结构特征。使用基于 ELISA 的测定法测量 PKCε 和 RACK2 之间的相互作用。结果发现N -(4-acetylphenyl)-3-amino-6,7-ethelendioxy-thieno[2,3-b]quinoline-2-carboxamide ( 1b ) 显示出对 PKCε 与其衔接蛋白（受体）相互作用的有希望的抑制活性对于激活的 C 激酶 2 (RACK2)，因此会干扰 PKCε 信号。1a和1b均未显示出对易感 PC-3 细胞系的某些细胞毒性，但两种活性化合物均显示出显着的抗萌芽活性。没有噻吩环的喹啉作为1b的“开放”类似物在初级测定中没有活性。( 1a间位乙酰基)的结构异构体，化合物( 1b对位 -acetyl) 被发现除了对 PKCε 信号具有强抑制活性外，IC ₅₀为 4.25 µM，还具有抗血管生成活性。因此，噻吩并 [2,3-b] 喹啉1a和1b可能是研究 PKCe/RACK2 效应的可靠和选择性生化工具。