Validating pharmacological disruption of protein-protein interactions by acceptor photobleaching FRET imaging

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Abstract

Proteins are the major targets of drug discovery and many of the new drugs are designed to exert their effect by disrupting protein-protein interactions. Validation of the inhibition of molecular interactions is generally done by biochemical methods, however, these are often not feasible when the interaction is not stable enough. Fluorescence resonance energy transfer (FRET) is an excellent tool for determining direct molecular interactions between two molecules in the cell membrane or inside cells in their natural state. Although originally established as a flow cytometric approach, FRET has been adapted for microscopy, allowing for analysis of sub-cellular co-localization at the single cell level. In this chapter, we provide theoretical introduction to the phenomenon of FRET, and a protocol - including labeling techniques, measurement, and evaluation of microscopy images - of the simplest microscopic FRET approach, acceptor photobleaching FRET. This technique is generally usable for studying protein interactions and requires only a standard confocal laser scanning microscope. To demonstrate the value of image based FRET for testing pharmacological disruption of protein-protein interactions, we show how inhibition of the heterodimerization of ErbB2 and ErbB1 by the humanized monoclonal antibody pertuzumab can be validated using this technique.

Original languageEnglish
Title of host publicationTarget Identification and Validation in Drug Discovery
Subtitle of host publicationMethods and Protocols
PublisherHumana Press Inc.
Pages165-178
Number of pages14
ISBN (Print)9781627033107
DOIs
Publication statusPublished - 2013

Publication series

NameMethods in Molecular Biology
Volume986
ISSN (Print)1064-3745

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Keywords

  • ErbB1
  • ErbB2
  • Förster resonance energy transfer
  • Image cytometry
  • Pertuzumab
  • Protein interactions

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Roszik, J., Tóth, G., Szöllosi, J., & Vereb, G. (2013). Validating pharmacological disruption of protein-protein interactions by acceptor photobleaching FRET imaging. In Target Identification and Validation in Drug Discovery: Methods and Protocols (pp. 165-178). (Methods in Molecular Biology; Vol. 986). Humana Press Inc.. https://doi.org/10.1007/978-1-62703-311-4_11