A High-Throughput Method for Measuring Drug Residence Time Using the Transcreener ADP Assay
A High-Throughput Method for Measuring Drug Residence Time Using the Transcreener ADP Assay
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DOI：
10.1177/2472555217695080
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发表时间：
2017-08-01
期刊：
影响因子：
3.1
通讯作者：
Lowery, Robert G.
中科院分区：
文献类型：
--
作者：
Kumar, Meera;Lowery, Robert G.
在药物开发过程中分析药物-靶标停留时间可以提高疗效，增加治疗窗口并减少副作用。停留时间可以估计为抑制剂与其靶标的解离速率（k（off））的倒数。传统的测量k（off）的方法需要合成标记配体或低通量的无标记方法。为了提供一种更适合于自动化高通量筛选（HTS）环境的替代方法，我们采用了经典的“跳跃稀释”催化测定方法来测定激酶抑制剂药物的k（off）值。我们使用Transcreener ADP（2）激酶试验作为一种通用的、均相的方法来监测药物从预先形成的激酶复合物中解离时激酶活性的恢复。我们测量了结合表皮生长因子受体（EGFR），ABL 1和Aurora激酶的几种药物的停留时间，发现抑制剂k（of）f值的排序与使用配体结合试验确定的文献值相关。此外，使用具有荧光偏振（FP）、荧光强度（FI）和时间分辨福斯特共振能量转移（TR-FRET）检测模式的Transcreener测定获得了非常相似的结果。这种HTS兼容的通用测定方法应有助于使用停留时间作为激酶药物发现计划早期化合物优先级和优化的参数。
Analysis of drug-target residence times during drug development can result in improved efficacy, increased therapeutic window, and reduced side effects. Residence time can be estimated as the reciprocal of the dissociation rate (k(off)) of an inhibitor from its target. The traditional methods for measuring k(off) require synthesis of labeled ligands or low-throughput label-free methods. To provide an alternative that is better suited to an automated high-throughput screening (HTS) environment, we adapted a classic "jump dilution" catalytic assay method for determination of k(off) values for kinase inhibitor drugs. We used the Transcreener ADP(2) Kinase assay as a universal, homogenous method to monitor the recovery of kinase activity as the drugs dissociated from preformed inhibitor-kinase complexes. We measured residence times for several drugs that bind the epidermal growth factor receptor (EGFR), ABL1, and Aurora kinases and found that the rank ordering of inhibitor k(of)f values correlated with literature values determined using ligand binding assays. Moreover, very similar results were obtained using the Transcreener assay with fluorescence polarization (FP), fluorescence intensity (FI), and time-resolved Forster resonance energy transfer (TR-FRET) detection modes. This HTS-compatible, generic assay method should facilitate the use of residence time as a parameter for compound prioritization and optimization early in kinase drug discovery programs.