KRAS4b:RAF-1 Homogenous Time-Resolved Fluorescence Resonance Energy Transfer Assay for Drug Discovery

Erik K Larsen; Maria Abreu-Blanco; Dana Rabara; Andrew G Stephen

doi:10.1007/978-1-0716-3822-4_12

KRAS4b:RAF-1 Homogenous Time-Resolved Fluorescence Resonance Energy Transfer Assay for Drug Discovery

Methods Mol Biol. 2024:2797:159-175. doi: 10.1007/978-1-0716-3822-4_12.

Authors

Erik K Larsen¹, Maria Abreu-Blanco², Dana Rabara², Andrew G Stephen²

Affiliations

¹ NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, MD, USA. erik.larsen@nih.gov.
² NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, MD, USA.

PMID: 38570459
DOI: 10.1007/978-1-0716-3822-4_12

Abstract

Homogenous time-resolved FRET (HTRF) assays have become one of the most popular tools for pharmaceutical drug screening efforts over the last two decades. Large Stokes shifts and long fluorescent lifetimes of lanthanide chelates lead to robust signal to noise, as well as decreased false positive rates compared to traditional assay techniques. In this chapter, we describe an HTRF protein-protein interaction (PPI) assay for the KRAS4b G-domain in the GppNHp-bound state and the RAF-1-RBD currently used for drug screens. Application of this assay contributes to the identification of lead compounds targeting the GTP-bound active state of K-RAS.

Keywords: Assay development; Drug screen; Homogenous time-resolved FRET; MRTX1133; Protein-protein interaction; RAF; RAS.

MeSH terms

Chelating Agents
Drug Discovery*
Fluorescence Resonance Energy Transfer* / methods

Substances

Chelating Agents