Development and validation of a fluorescence technology for both primary and secondary screening of kinases that facilitates compound selectivity and site-specific inhibitor determination
Development and validation of a fluorescence technology for both primary and secondary screening of kinases that facilitates compound selectivity and site-specific inhibitor determination
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DOI：
10.1089/154065804323056512
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发表时间：
2004-04-01
影响因子：
1.8
通讯作者：
Millis, SZ
中科院分区：
文献类型：
--
作者：
Morgan, AG;McCauley, TJ;Millis, SZ
IQ（R）技术是一种同质、通用的激酶和磷酸酶HTS检测平台。该技术是一种直接的、非竞争性的测定形式，不需要抗体或放射性试剂来测量磷酸化状态。使用荧光团标记的肽作为酶底物，通过直接测量底物的磷酸化状态来定量激酶或磷酸酶活性。磷酸化是通过专有的含铁化合物特异性结合肽上的磷酰基时发生的荧光强度变化来测量的。观察到的荧光的这种变化与荧光团标记的肽的磷酸化程度成比例。该技术提供了一种通用方法，可用于任何肽序列，对高浓度ATP不敏感。可以区分ATP结合位点与磷酸化位点的抑制，并使用与初步筛选相同的检测方法鉴定化合物选择性。该技术已针对反应混合物中发现的大量洗涤剂，有机物和其他试剂进行了测试，检测方法消除了与荧光和显色化合物相关的常见问题。该技术已被格式化为96孔、384孔和1，536孔微孔板格式，并且获得了0.7的代表性Z'值。使用该平台生成的IC50值与先前报告的值相关，并且进行小化合物文库的筛选以进一步评价测定。
The IQ(R) Technology has been developed to serve as a homogeneous, universal detection platform for HTS of kinases and phosphatases. The technology is a direct, noncompetitive assay format that does not require antibodies or radioactive reagents to measure phosphorylation state. Fluorophore-labeled peptides are used as enzyme substrates, and kinase or phosphatase activity is quantitated by direct measurement of the phosphorylation state of the substrate. Phosphorylation is measured by the change in fluorescence intensity that occurs when a proprietary iron-containing compound binds specifically to phosphoryl groups on peptides. This change in observed fluorescence is proportional to the extent of phosphorylation of the fluorophore-labeled peptide. The technology provides a universal method that can be used with any peptide sequence and is insensitive to high concentrations of ATP. Inhibition at the ATP-binding site versus the phosphorylation site can be differentiated and compound selectivity identified using the same detection method as in the primary screen. The technology has been tested against a large number of detergents, organics, and other reagents found in reaction mixtures, and the detection method eliminates common issues associated with fluorescent and chromogenic compounds. The technology has been formatted for 96-, 384-, and 1,536-well microplate formats, and a representative Z' value of 0.7 was obtained. IC50 values generated using this platform correlate with previously reported values, and screening of a small compound library was performed to evaluate the assay further.