High-Throughput Dual Screening Method for Ras Activities and Inhibitors

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

Research output: Contribution to journalArticleScientificpeer-review

8 Citations (Scopus)

Abstract

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 languageUndefined/Unknown
Pages (from-to)4508–4516
JournalAnalytical Chemistry
Volume89
Issue number8
DOIs
Publication statusPublished - 2017
MoE publication typeA1 Journal article-refereed

Keywords

  • Ras
  • Drug screening
  • Quenching resonance energy transfer
  • Inhibitor
  • Molecular docking

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