Pim Kinase Inhibitors Evaluated with a Single-Molecule Engineered Nanopore Sensor
Pim Kinase Inhibitors Evaluated with a Single-Molecule Engineered Nanopore Sensor
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DOI：
10.1002/anie.201503141
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发表时间：
2015-07-06
影响因子：
16.6
通讯作者：
Bayley, Hagan
中科院分区：
文献类型：
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作者：
Harrington, Leon;Alexander, Leila T.;Bayley, Hagan
蛋白激酶是关键的治疗靶标。Pim激酶与几种白血病和癌症有关。在这里，我们利用Pim激酶的蛋白质纳米孔传感器，其具有通过增强的工程方法连接的假底物肽。通过观察通过单个纳米孔的离子电流的调节来测量与传感器肽结合的分析物。我们观察到MgATP和激酶与传感器的协同结合，该传感器用于开发一种上级方法来评估Pim激酶抑制剂，其特征在于抑制常数的无标记测定。该程序避免了当前检测中固有的许多偏倚或假阳性来源。例如，我们确定了差示扫描荧光法遗漏的有效抑制剂。该方法也适用于高通量芯片上的实现。
Protein kinases are critical therapeutic targets. Pim kinases are implicated in several leukaemias and cancers. Here, we exploit a protein nanopore sensor for Pim kinases that bears a pseudosubstrate peptide attached by an enhanced engineering approach. Analyte binding to the sensor peptide is measured through observation of the modulation of ionic current through a single nanopore. We observed synergistic binding of MgATP and kinase to the sensor, which was used to develop a superior method to evaluate Pim kinase inhibitors featuring label-free determination of inhibition constants. The procedure circumvents many sources of bias or false-positives inherent in current assays. For example, we identified a potent inhibitor missed by differential scanning fluorimetry. The approach is also amenable to implementation on high throughput chips.