High-Throughput Dual Screening Method for Ras Activities and Inhibitors
Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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.
Originalsprog | Engelsk |
---|---|
Tidsskrift | Analytical Chemistry |
Vol/bind | 89 |
Udgave nummer | 8 |
Sider (fra-til) | 4508-4516 |
Antal sider | 9 |
ISSN | 0003-2700 |
DOI | |
Status | Udgivet - 18 apr. 2017 |
Eksternt udgivet | Ja |
ID: 199423550