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 hetero-dimerization of ErbB2 and ErbB1 by the humanized monoclonal antibody pertuzumab can be validated using this technique.
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Acknowledgments
The authors were supported by the following grants: Hungarian National Research Fund K75752 and NK101337; Hungarian National Development Agency TAMOP-4.2.1/B-09/1/KONV-2010-0007 and 4.2.2/A-11/1/KONV-2012-0025; Baross Gabor programme (REG-EA-09-1-2009-0010) and ETT 362-01/2009 grants.
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Roszik, J., Tóth, G., Szöllősi, J., Vereb, G. (2013). Validating Pharmacological Disruption of Protein–Protein Interactions by Acceptor Photobleaching FRET Imaging. In: Moll, J., Colombo, R. (eds) Target Identification and Validation in Drug Discovery. Methods in Molecular Biology, vol 986. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-311-4_11
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DOI: https://doi.org/10.1007/978-1-62703-311-4_11
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