High-Throughput Dual Screening Method for Ras Activities and Inhibitors
Research output: Contribution to journal › Journal article › Research › peer-review
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.
Original language | English |
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Journal | Analytical Chemistry |
Volume | 89 |
Issue number | 8 |
Pages (from-to) | 4508-4516 |
Number of pages | 9 |
ISSN | 0003-2700 |
DOIs | |
Publication status | Published - 18 Apr 2017 |
Externally published | Yes |
ID: 199423550