High-Throughput Dual Screening Method for Ras Activities and Inhibitors

A1 Journal article (refereed)


Internal Authors/Editors


Publication Details

List of Authors: Kari Kopra, Arjan J. van Adrichem, Outi M. H. Salo-Ahen, Juha Peltonen, Krister Wennerberg, Harri Härmä
Publisher: American Chemical Society
Publication year: 2017
Journal: Analytical Chemistry
Journal acronym: Anal. Chem.
Volume number: 89
Issue number: 8
Start page: 4508
End page: 4516
eISSN: 1520-6882


Abstract

Ras GTPases act as "molecular switches", alternating between inactive GDP-bound and active GTP-bound conformation. Ras-oncogenes were discovered over three decades ago, but there are still no effective therapies for Ras-driven cancers. So far, drug discovery strategies have been unsuccessful, because of a lack of suitable screening methodologies and well-defined binding pockets on the Ras proteins. Here, we addressed the former by introducing a homogeneous quenching resonance energy transfer (QRET) technique-based screening strategy for Ras interfacial and competitive inhibitors. We demonstrate that using a unique GTP-specific antibody fragment to monitor GTPase cycling in the presence of a guanine nucleotide exchange factor (GEF) and a GTPase activating protein (GAP) is an efficient method for Ras inhibitor high-throughput screening. When compared to a conventional GEF-stimulated nucleotide exchange assay in a proof-of-concept screen, we identified an overlapping set of potential inhibitor compounds but also compounds found exclusively with the new GTP hydrolysis monitoring-based GTPase cycling assay.


Keywords

Drug screening, Inhibitor, Molecular docking, Quenching resonance energy transfer, Ras

Last updated on 2019-08-12 at 03:31