Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 7-194 MCB Building, 420 Washington Ave SE, Minneapolis, MN 55455 USA.
Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue Center for Cancer Research, Purdue University, West Lafayette, IN 47907 USA.
Sci Rep. 2016 Jul 18;6:28971. doi: 10.1038/srep28971.
Fluorescent drug screening assays are essential for tyrosine kinase inhibitor discovery. Here we demonstrate a flexible, antibody-free TR-LRET kinase assay strategy that is enabled by the combination of streptavidin-coated quantum dot (QD) acceptors and biotinylated, Tb(3+) sensitizing peptide donors. By exploiting the spectral features of Tb(3+) and QD, and the high binding affinity of the streptavidin-biotin interaction, we achieved multiplexed detection of kinase activity in a modular fashion without requiring additional covalent labeling of each peptide substrate. This strategy is compatible with high-throughput screening, and should be adaptable to the rapidly changing workflows and targets involved in kinase inhibitor discovery.
荧光药物筛选测定对于酪氨酸激酶抑制剂的发现至关重要。在这里，我们展示了一种灵活的、无需抗体的 TR-LRET 激酶测定策略，该策略得益于链霉亲和素包被的量子点（QD）受体和生物素化、铽（Tb(3+)）敏化肽供体的组合。通过利用 Tb(3+)和 QD 的光谱特性，以及链霉亲和素-生物素相互作用的高结合亲和力，我们实现了以模块化方式对激酶活性进行多重检测，而无需对每个肽底物进行额外的共价标记。该策略与高通量筛选兼容，并且应该适应于激酶抑制剂发现中涉及的快速变化的工作流程和靶标。
