High-Throughput Dual Screening Method for Ras Activities and Inhibitors.
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.
Institute of Biomedicine, University of Turku , Kiinamyllynkatu 10 C, FI-20520 Turku, Finland.
Kari
0000-0001-7585-6020
K
Kopra
Institute for Molecular Medicine Finland, University of Helsinki , Tukholmankatu 8, FI-00290 Helsinki, Finland.
Arjan J
AJ
van Adrichem
Structural Bioinformatics Laboratory and Pharmaceutical Sciences Laboratory, Faculty of Science and Engineering, Biochemistry and Pharmacy, Åbo Akademi University , Tykistökatu 6A, FI-20520 Turku, Finland.
Outi M H
OMH
Salo-Ahen
Institute of Biomedicine, University of Turku , Kiinamyllynkatu 10 C, FI-20520 Turku, Finland.
Juha
J
Peltonen
Institute for Molecular Medicine Finland, University of Helsinki , Tukholmankatu 8, FI-00290 Helsinki, Finland.
Krister
K
Wennerberg
Institute of Biomedicine, University of Turku , Kiinamyllynkatu 10 C, FI-20520 Turku, Finland.
Harri
H
Härmä
D001665:Binding Sites; D045744:Cell Line, Tumor; D056831:Coordination Complexes / Q000737:chemistry; D005063:Europium / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D057166:High-Throughput Screening Assays; D006801:Humans; D062105:Molecular Docking Simulation; D011485:Protein Binding; D018631:ras Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*
D056831:Coordination Complexes; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D005063:Europium; D006160:Guanosine Triphosphate; D018631:ras Proteins