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

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KRAS

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2797))

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.

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Acknowledgments

We would like to acknowledge Bridge Bio for supplying the library of compounds to assay. Additionally, we would like to acknowledge Dominic Esposito, Bill Gillette, Gulcin Gulten, Jennifer Mehalko, Simon Messing, Nitya Ramakrishnan, Troy Taylor, and Vanessa Wall for cloning, protein expression, protein purification, and electrospray ionization mass spectroscopy. We would also like to acknowledge Anna Maciag for facilitating lead compound acquisition. This project was funded in part with federal funds from the National Cancer Institute, National Institutes of Health Contract 75N91019D00024. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, and the mention of trade names, commercial products, or organizations does not imply endorsement by the US government.

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Authors and Affiliations

  1. NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, MD, USA

    Erik K. Larsen, Maria Abreu-Blanco, Dana Rabara & Andrew G. Stephen

Authors
  1. Erik K. Larsen
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  2. Maria Abreu-Blanco
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  3. Dana Rabara
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  4. Andrew G. Stephen
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Corresponding author

Correspondence to Erik K. Larsen .

Editor information

Editors and Affiliations

  1. Frederick National Laboratory for Cancer Research, NCI RAS Initiative, Frederick, MD, USA

    Andrew G. Stephen

  2. Frederick National Laboratory for Cancer Research, NCI RAS Initiative, Frederick, MD, USA

    Dominic Esposito

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Larsen, E.K., Abreu-Blanco, M., Rabara, D., Stephen, A.G. (2024). KRAS4b:RAF-1 Homogenous Time-Resolved Fluorescence Resonance Energy Transfer Assay for Drug Discovery. In: Stephen, A.G., Esposito, D. (eds) KRAS. Methods in Molecular Biology, vol 2797. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3822-4_12

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  • DOI: https://doi.org/10.1007/978-1-0716-3822-4_12

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3821-7

  • Online ISBN: 978-1-0716-3822-4

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