Reading dynamic kinase activity in living cells for high-throughput screening
Reading dynamic kinase activity in living cells for high-throughput screening
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DOI：
10.1021/cb600202f
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发表时间：
2006-01-01
影响因子：
4
通讯作者：
Zhang, Jin
中科院分区：
文献类型：
--
作者：
Allen, Michael D.;DiPilato, Lisa M.;Zhang, Jin
蛋白激酶作为重要的信号分子，代表了一类新兴的药物靶点，并且通过高通量筛选在活细胞中测定其活性的能力应该为药物发现以及化学和功能基因组学提供令人兴奋的机会。在这里，我们描述了一种通用的方法，用于高通量阅读的动态激酶活性使用比率荧光传感器，并展示了阅读细胞内活性的蛋白激酶A（PKA）和环磷酸腺苷（cAMP）/ PKA途径下游的许多G蛋白偶联受体（GPCR）的一个例子。我们进一步证明了基于化合物调节活细胞中动态激酶活性的能力的第一化合物筛选，并表明对临床化合物的收集的这种筛选已经成功地鉴定了GPCR/ cAMP/ PKA途径的调节剂。
Protein kinases, as crucial signaling molecules, represent an emerging class of drug targets, and the ability to assay their activities in living cells with high-throughput screening should provide exciting opportunities for drug discovery and chemical and functional genomics. Here, we describe a general method for high-throughput reading of dynamic kinase activities using ratiometric fluorescent sensors, and showcase an example of reading intracellular activities of protein kinase A ( PKA) and the cyclic adenosine monophosphate ( cAMP)/ PKA pathway downstream of many G-protein coupled receptors ( GPCRs). We further demonstrate the first compound screen based on the ability of compounds to modulate dynamic kinase activities in living cells and show that such screening of a collection of clinical compounds has successfully identified modulators of the GPCR/ cAMP/ PKA pathway.