High-Throughput Dual Screening Method for Ras Activities and Inhibitors

: Kari Kopra, Arjan J. van Adrichem, Outi M. H. Salo-Ahen, Juha Peltonen, Krister Wennerberg, Harri Härmä

Publisher: AMER CHEMICAL SOC

: 2017

: Analytical Chemistry

: ANALYTICAL CHEMISTRY

: ANAL CHEM

: 89

: 8

: 4508

: 4516

: 9

: 0003-2700

: 1520-6882

DOI: https://doi.org/10.1021/acs.analchem.6b04904

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.