Development of a mechanism-based high-throughput screen assay for leucine-rich repeat kinase 2--discovery of LRRK2 inhibitors.
Development of a mechanism-based high-throughput screen assay for leucine-rich repeat kinase 2--discovery of LRRK2 inhibitors.
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开发基于机制的富含亮氨酸重复激酶 2 的高通量筛选测定——LRRK2 抑制剂的发现。
DOI：
10.1016/j.ab.2010.05.033
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发表时间：
2010
影响因子：
2.9
通讯作者：
Yue,Zhenyu
中科院分区：
文献类型：
--
作者：
Liu,Min;Poulose,Shibu;Schuman,Eli;Zaitsev,AlexandraD;Dobson,Brittany;Auerbach,Ken;Seyb,Kathleen;Cuny,GregoryD;Glicksman,MarcieA;Stein,RossL;Yue,Zhenyu
LRRK 2是一种大而复杂的蛋白质，具有激酶和GT3活性，并可能通过毒性功能获得机制成为PD发病机制中最相关的参与者。激酶活性是LRRK 2功能的关键组成部分，代表了药物发现的可行靶标。我们现在报告的发展机制为基础的TR-FRET检测LRRK 2激酶活性使用全长LRRK 2。在该测定中，选择PLK-肽作为磷酰基受体。使用稳态动力学研究和计算机模拟的组合来计算ATP和PLK-肽的初始浓度，以产生有利于识别ATP非竞争性抑制剂的稳态情况。还在不存在GTP的情况下进行测定。在这些条件下，该测定对直接与激酶结构域相互作用的抑制剂和通过直接与包括GT3结构域在内的其他结构域相互作用来调节激酶活性的抑制剂敏感。该测定法进行了优化，并用于在384孔格式中稳健地评估我们的化合物文库。通过筛选鉴定的抑制剂被进一步表征为与ATP和PLK-肽两者的非竞争性抑制剂，并且对LRRK 2 WT和突变体G2019 S显示出类似的抑制。
LRRK2 is a large and complex protein that possesses kinase and GTPase activities and has emerged as the most relevant player in PD pathogenesis possibly through a toxic gain-of-function mechanism. Kinase activity is a critical component of LRRK2 function and represents a viable target for drug discovery. We now report the development of a mechanism-based TR-FRET assay for the LRRK2 kinase activity using full-length LRRK2. In this assay, PLK-peptide was chosen as the phosphoryl acceptor. A combination of steady-state kinetic studies and computer simulations was used to calculate the initial concentrations of ATP and PLK-peptide to generate a steady-state situation that favors the identification of ATP noncompetitive inhibitors. The assay was also run in the absence of GTP. Under these conditions, the assay was sensitive to inhibitors that directly interact with the kinase domain and those that modulate the kinase activity by directly interacting with other domains including the GTPase domain. The assay was optimized and used to robustly evaluate our compound library in a 384-well format. An inhibitor identified through the screen was further characterized as a noncompetitive inhibitor with both ATP and PLK-peptide and showed similar inhibition against LRRK2 WT and the mutant G2019S.