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Nat Protoc. 2017 Jul;12(7):1472-1497. doi: 10.1038/nprot.2017.043. Epub 2017 Jun 29.

Measurement of drug-target engagement in live cells by two-photon fluorescence anisotropy imaging.

Author information

1
Center for System Biology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.
2
Department of Neurosciences, Biomedicine, and Movement Sciences, University of Verona, Verona, Italy.
3
Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, New York, USA.
4
School of Electrical Engineering, Hanyang University, Ansan, Republic of Korea.
5
Institute for Molecular Medicine, University of North Texas Health Science Center, Fort Worth, Texas, USA.

Abstract

The ability to directly image and quantify drug-target engagement and drug distribution with subcellular resolution in live cells and whole organisms is a prerequisite to establishing accurate models of the kinetics and dynamics of drug action. Such methods would thus have far-reaching applications in drug development and molecular pharmacology. We recently presented one such technique based on fluorescence anisotropy, a spectroscopic method based on polarization light analysis and capable of measuring the binding interaction between molecules. Our technique allows the direct characterization of target engagement of fluorescently labeled drugs, using fluorophores with a fluorescence lifetime larger than the rotational correlation of the bound complex. Here we describe an optimized protocol for simultaneous dual-channel two-photon fluorescence anisotropy microscopy acquisition to perform drug-target measurements. We also provide the necessary software to implement stream processing to visualize images and to calculate quantitative parameters. The assembly and characterization part of the protocol can be implemented in 1 d. Sample preparation, characterization and imaging of drug binding can be completed in 2 d. Although currently adapted to an Olympus FV1000MPE microscope, the protocol can be extended to other commercial or custom-built microscopes.

PMID:
28686582
PMCID:
PMC5928516
DOI:
10.1038/nprot.2017.043
[Indexed for MEDLINE]
Free PMC Article

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