Materials Chemistry and Chemical Analysis, University of Turku, Vatselankatu 2, 20500 Turku, Finland.
Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, 8560 Progress Dr., Frederick, Maryland 21702, United States.
Anal Chem. 2020 Apr 7;92(7):4971-4979. doi: 10.1021/acs.analchem.9b05126. Epub 2020 Mar 9.
We have developed a rapid and sensitive single-well dual-parametric method introduced in linked RAS nucleotide exchange and RAS/RAF-RBD interaction assays. RAS mutations are frequent drivers of multiple different human cancers, but the development of therapeutic strategies has been challenging. Traditionally, efforts to disrupt the RAS function have focused on nucleotide exchange inhibitors, GTP-RAS interaction inhibitors, and activators increasing GTPase activity of mutant RAS proteins. As the amount of biological knowledge grows, targeted biochemical assays enabling high-throughput screening have become increasingly interesting. We have previously introduced a homogeneous quenching resonance energy transfer (QRET) assay for nucleotide binding studies with RAS and heterotrimeric G proteins. Here, we introduce a novel homogeneous signaling technique called QTR-FRET, which combine QRET technology and time-resolved Förster resonance energy transfer (TR-FRET). The dual-parametric QTR-FRET technique enables the linking of guanine nucleotide exchange factor-induced Eu-GTP association to RAS, monitored at 615 nm, and subsequent Eu-GTP-loaded RAS interaction with RAF-RBD-Alexa680 monitored at 730 nm. Both reactions were monitored in a single-well assay applicable for inhibitor screening and real-time reaction monitoring. This homogeneous assay enables separable detection of both nucleotide exchange and RAS/RAF interaction inhibitors using low nanomolar protein concentrations. To demonstrate a wider applicability as a screening and real-time reaction monitoring method, the QTR-FRET technique was also applied for G(i)α GTP-loading and pertussis toxin-catalyzed ADP-ribosylation of G(i)α, for which we synthesized a novel γ-GTP-Eu molecule. The study indicates that the QTR-FRET detection technique presented here can be readily applied to dual-parametric assays for various targets.
我们开发了一种快速灵敏的单孔双参数方法，该方法用于连接的 RAS 核苷酸交换和 RAS/RAF-RBD 相互作用测定。RAS 突变是多种人类癌症的常见驱动因素，但治疗策略的发展具有挑战性。传统上，破坏 RAS 功能的努力集中在核苷酸交换抑制剂、GTP-RAS 相互作用抑制剂和增加突变 RAS 蛋白 GTPase 活性的激活剂上。随着生物知识量的增加，能够进行高通量筛选的靶向生化测定变得越来越有趣。我们之前介绍了一种用于 RAS 和异三聚体 G 蛋白核苷酸结合研究的均相猝灭共振能量转移 (QRET) 测定法。在这里，我们引入了一种称为 QTR-FRET 的新型均相信号技术，它结合了 QRET 技术和时间分辨Förster 共振能量转移 (TR-FRET)。双参数 QTR-FRET 技术能够将 G 核苷酸交换因子诱导的 Eu-GTP 与 RAS 的结合联系起来，在 615nm 处监测，随后 Eu-GTP 负载的 RAS 与 RAF-RBD-Alexa680 的相互作用在 730nm 处监测。这两种反应都在单个孔测定中进行，适用于抑制剂筛选和实时反应监测。这种均相测定法能够使用低纳摩尔蛋白浓度分离检测核苷酸交换和 RAS/RAF 相互作用抑制剂。为了证明作为筛选和实时反应监测方法的更广泛适用性，还将 QTR-FRET 技术应用于 G(i)α GTP 加载和百日咳毒素催化的 G(i)α ADP-核糖基化，为此我们合成了一种新型的γ-GTP-Eu 分子。该研究表明，这里呈现的 QTR-FRET 检测技术可以很容易地应用于各种靶标的双参数测定。
