High-Throughput Dual Screening Method for Ras Activities and Inhibitors
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High-Throughput Dual Screening Method for Ras Activities and Inhibitors. / Kopra, Kari; van Adrichem, Arjan J; Salo-Ahen, Outi M H; Peltonen, Juha; Wennerberg, Krister; Härmä, Harri.
I: Analytical Chemistry, Bind 89, Nr. 8, 18.04.2017, s. 4508-4516.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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TY - JOUR
T1 - High-Throughput Dual Screening Method for Ras Activities and Inhibitors
AU - Kopra, Kari
AU - van Adrichem, Arjan J
AU - Salo-Ahen, Outi M H
AU - Peltonen, Juha
AU - Wennerberg, Krister
AU - Härmä, Harri
PY - 2017/4/18
Y1 - 2017/4/18
N2 - 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.
AB - 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.
U2 - 10.1021/acs.analchem.6b04904
DO - 10.1021/acs.analchem.6b04904
M3 - Journal article
C2 - 28318223
VL - 89
SP - 4508
EP - 4516
JO - Industrial And Engineering Chemistry Analytical Edition
JF - Industrial And Engineering Chemistry Analytical Edition
SN - 0003-2700
IS - 8
ER -
ID: 199423550