Development of a Fluorescence Polarization Bead-Based Coupled Assay to Target Different Activity/Conformation States of a Protein Kinase
Development of a Fluorescence Polarization Bead-Based Coupled Assay to Target Different Activity/Conformation States of a Protein Kinase
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开发基于荧光偏振珠的偶联测定，以针对蛋白激酶的不同活性/构象状态
DOI：
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发表时间：
2004
影响因子：
--
通讯作者：
C. Kumar
中科院分区：
文献类型：
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作者：
Zhuomei Lu;Z. Yin;L. James;R. Syto;Jill M Stafford;S. Koseoglu;Todd W. Mayhood;J. Myers;W. Windsor;P. Kirschmeier;A. Samatar;Bruce Malcolm;T. Turek;C. Kumar
Most of the protein kinase inhibitors being developed are directed toward the adenosine triphosphate (ATP) binding site that is highly conserved in many kinases. A major issue with these inhibitors is the specificity for a given kinase. Structure determination of several kinases has shown that protein kinases adopt distinct conformations in their inactive state, in contrast to their strikingly similar conformations in their active states. Hence, alternative assay formats that can identify compounds targeting the inactive form of a protein kinase are desirable. The authors describe the development and optimization of an Immobilized Metal Assay for Phosphochemicals (IMAP™)-based couple™d assay using PDK1 and inactive Akt-2 enzymes. PDK1 phosphorylates Akt-2 at Thr 309 in the catalytic domain, leading to enzymatic activation. Activation of Akt by PDK1 is measured by quantitating the phosphorylation of Akt-specific substrate peptide using the IMAP assay format. This IMAP-coupled assay has been formatted in a 384-well microplate format with a Z′ of 0.73 suitable for high-throughput screening. This assay was evaluated by screening the biologically active sample set LOPAC™ and validated with the protein kinase C inhibitor staurosporine. The IC50 value generated was comparable to the value obtained by the radioactive 33P-γ-ATP flashplate transfer assay. This coupled assay has the potential to identify compounds that target the inactive form of Akt and prevent its activation by PDK1, in addition to finding inhibitors of PDK1 and activated Akt enzymes.
影响因子：
56.9
作者：
Schindler, T;Bornmann, W;Kuriyan, J
通讯作者：
Kuriyan, J
c-Abl 激酶结构域与小分子抑制剂 PD173955 和伊马替尼 (STI-571) 复合物的晶体结构。
影响因子：
11.2
作者：
Nagar,Bhushan;Bornmann,WilliamG;Pellicena,Patricia;Schindler,Thomas;Veach,DarrenR;Miller,WTodd;Clarkson,Bayard;Kuriyan,John
通讯作者：
Kuriyan,John