ac9b05126_si_001.pdf (1.08 MB)
Homogeneous Dual-Parametric-Coupled Assay for Simultaneous Nucleotide Exchange and KRAS/RAF-RBD Interaction Monitoring
journal contribution
posted on 2020-03-06, 21:44 authored by Kari Kopra, Emmiliisa Vuorinen, Maria Abreu-Blanco, Qi Wang, Ville Eskonen, William Gillette, Arto T. Pulliainen, Matthew Holderfield, Harri HärmäWe 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.
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Keywords
Simultaneous Nucleotide ExchangeGTP associationhigh-throughput screeningQTR-FRET detection techniqueGTP-loaded RAS interactionheterotrimeric G proteinsKRASRAS functiondual-parametric QTR-FRET techniquesingle-well dual-parametric methodQRET technologysingle-well assaynucleotide binding studiesRAS nucleotide exchangedual-parametric assayspertussis toxin-catalyzed ADP-ribosylationnucleotide exchange inhibitorsGTP-RAS interaction inhibitorsreaction monitoring615 nmRAS mutationsnucleotide exchangenanomolar protein concentrationsRAF-RBD-Alexa 680730 nmquenching resonance energy transferQTR-FRET techniquereaction monitoring methodinhibitor screeningRAS proteinsHomogeneous Dual-Parametric-Coupled AssayGTPase activity
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