title: Two-photon Fluorescence Anisotropy Microscopy for Imaging and Direct Measurement of Intracellular Drug Target Engagement.
abstract: Small molecule therapeutic drugs must reach their intended cellular targets (pharmacokinetics) and engage them to modulate therapeutic effects (pharmacodynamics). These processes are often difficult to measure in vivo due to their complexities and occurrence within single cells. It has been particularly difficult to directly measure cellular drug target binding. Fluorescence polarization is commonly used in pharmacological screening assays to measure drug-protein or protein-protein interactions. We hypothesized that fluorescence polarization imaging could be adapted and used with fluorescently labeled drugs to measure drug target engagement in vivo. Here we summarize recent results using two photon fluorescence anisotropy microscopy. Our imaging technique offers quantitative pharmacological binding information of diverse molecular interactions at the microscopic level, differentiating between bound and unbound states. Used in combination with other recent advances in the development of novel fluorescently labeled drugs, we expect that the described imaging modality will provide a window into the distribution and efficacy of drugs in real time and in vivo at the cellular and subcellular level.
authors.affiliation: Center for System Biology, Massachusetts General Hospital and Harvard Medical School, Richard B. Simches Research Center, 185 Cambridge Street, Boston 02114, USA.
authors.forename: Claudio
authors.initials: C
authors.lastname: Vinegoni
authors.affiliation: Center for System Biology, Massachusetts General Hospital and Harvard Medical School, Richard B. Simches Research Center, 185 Cambridge Street, Boston 02114, USA.
authors.forename: John M
authors.initials: JM
authors.lastname: Dubach
authors.affiliation: Center for System Biology, Massachusetts General Hospital and Harvard Medical School, Richard B. Simches Research Center, 185 Cambridge Street, Boston 02114, USA and with the Department of Neurological and Movement Sciences, University of Verona, Strada Le Grazie 8, 37134 Verona, Italy.
authors.forename: Paolo Fumene
authors.initials: PF
authors.lastname: Feruglio
authors.affiliation: Center for System Biology, Massachusetts General Hospital and Harvard Medical School, Richard B. Simches Research Center, 185 Cambridge Street, Boston 02114, USA.
authors.forename: Ralph
authors.initials: R
authors.lastname: Weissleder
keywords: Intravital microscopy; drug imaging; fluorescence anisotropy; fluorescence polarization; in vivo imaging; pharmacology