A p38 substrate-specific MK2-EGFP translocation assay for identification and validation of new p38 inhibitors in living cells: a comprising alternative for acquisition of cellular p38 inhibition data.
A p38 substrate-specific MK2-EGFP translocation assay for identification and validation of new p38 inhibitors in living cells: a comprising alternative for acquisition of cellular p38 inhibition data.
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DOI：
10.1371/journal.pone.0095641
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Rothbauer U
中科院分区：
文献类型：
--
作者：
Anton R;Bauer SM;Keck PR;Laufer S;Rothbauer U
p38丝裂原活化蛋白激酶（MAPKs）在炎症中的基本作用强调了它们作为各种炎症性疾病（包括感染性、血管性、神经生物学和自身免疫性疾病）治疗靶点的重要性。尽管几十年的研究已经产生了几种p38抑制剂，但由于缺乏体内选择性和有效性，大多数临床试验都失败了。这强调了持续筛选p38抑制剂的新结构和化学型的需要。在这里，我们报告了在半自动图像高含量分析（HCA）系统中优化的MK2-EGFP易位试验，以筛选具有广泛化学型变化的3362种专有化合物的组合文库。通过确定MK2-EGFP在Rac/p38通路激活和复合处理后的再分配水平，我们发现了新的候选基因，它们可以调节活细胞中的p38活性。基于对人全血TNFα释放量、生化激酶活性测定和JNK3选择性测试的综合分析，我们发现这种基于细胞的测定揭示了被测化合物的细胞功效、选择性和效力的高度重叠和可预测性。因此，我们揭示了在低纳摩尔范围内起作用的新型亚型抑制剂的综合短列表，并可能为进一步的铅优化提供基础。根据之前的报道，我们证明了MK2-EGFP易位测定是p38-MAPK药物开发的一种合适的初级筛选方法，并且与其他基于细胞的方法（包括测定hpbmc或全血中的细胞因子释放）相比，提供了一种有吸引力的节省劳动力和成本的替代方法。
The fundamental role of p38 mitogen-activated protein kinases (MAPKs) in inflammation underlines their importance as therapeutic targets for various inflammatory medical conditions, including infectious, vascular, neurobiological and autoimmune disease. Although decades of research have yielded several p38 inhibitors, most clinical trials have failed, due to lack of selectivity and efficacy in vivo. This underlines the continuous need to screen for novel structures and chemotypes of p38 inhibitors. Here we report an optimized MK2-EGFP translocation assay in a semi-automated image based High Content Analysis (HCA) system to screen a combinatorial library of 3362 proprietary compounds with extensive variations of chemotypes. By determining the levels of redistribution of MK2-EGFP upon activation of the Rac/p38 pathway in combination with compound treatment, new candidates were identified, which modulate p38 activity in living cells. Based on integrated analysis of TNFα release from human whole blood, biochemical kinase activity assays and JNK3 selectivity testing, we show that this cell based assay reveals a high overlap and predictability for cellular efficacy, selectivity and potency of tested compounds. As a result we disclose a new comprehensive short-list of subtype inhibitors which are functional in the low nanomolar range and might provide the basis for further lead-optimization. In accordance to previous reports, we demonstrate that the MK2-EGFP translocation assay is a suitable primary screening approach for p38-MAPK drug development and provide an attractive labor- and cost saving alternative to other cell based methods including determination of cytokine release from hPBMCs or whole blood.
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DOI：
10.1073/pnas.251194298
发表时间：
2001-11-20
影响因子：
11.1
作者：
Bennett, BL;Sasaki, DT;Anderson, DW
通讯作者：
Anderson, DW
DOI：
10.2174/138620708785204081
发表时间：
2008-08-01
影响因子：
1.8
作者：
Henriksen, Ulla;Fog, Jacob;Praestegaard, Morten
通讯作者：
Praestegaard, Morten
影响因子：
3.7
作者：
Anton R;Chatterjee SS;Simundza J;Cowin P;Dasgupta R
通讯作者：
Dasgupta R
影响因子：
7.3
作者：
Buehler, Stefanie;Goettert, Marcia;Laufer, Stefan A.
通讯作者：
Laufer, Stefan A.
影响因子：
7.3
作者：
Fischer, Stefan;Wentsch, Heike K.;Laufer, Stefan A.
通讯作者：
Laufer, Stefan A.