Homogeneous Dual-Parametric-Coupled Assay for Simultaneous Nucleotide Exchange and KRAS/RAF-RBD Interaction Monitoring
Arto T. Pulliainen; Emmiliisa Vuorinen; Harri Härmä; Kari Kopra; Maria Abreu-Blanco; Matthew Holderfield; Qi Wang; Ville Eskonen; William Gillette
Homogeneous Dual-Parametric-Coupled Assay for Simultaneous Nucleotide Exchange and KRAS/RAF-RBD Interaction Monitoring
Arto T. Pulliainen
Emmiliisa Vuorinen
Harri Härmä
Kari Kopra
Maria Abreu-Blanco
Matthew Holderfield
Qi Wang
Ville Eskonen
William Gillette
American Chemical Society
Julkaisun pysyvä osoite on:
https://urn.fi/URN:NBN:fi-fe2021042821311
https://urn.fi/URN:NBN:fi-fe2021042821311
Tiivistelmä
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 Eu3+-GTP association to RAS, monitored at 615 nm, and subsequent Eu3+-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-Eu3+ molecule. The study indicates that the QTR-FRET detection technique presented here can be readily applied to dual-parametric assays for various targets.
Kokoelmat
- Rinnakkaistallenteet [19207]