Advanced Drug Delivery Reviews 151–152 (2019) 262–288 Contents lists available at ScienceDirect Advanced Drug Delivery Reviews journal homepage: www.elsevier.com/locate/addr Fluorescence anisotropy imaging in drug discovery Claudio Vinegoni a,⁎, Paolo Fumene Feruglio a,b, Ignacy Gryczynski c, Ralph Mazitschek a, Ralph Weissleder a a Center for System Biology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA b Department of Neurological, Biomedical and Movement Sciences, University of Verona, Verona, Italy c University of North Texas Health Science Center, Institute for Molecular Medicine, Fort Worth, TX, United States article info abstract Article history: Non-invasive measurement of drug-target engagement can provide critical insights in the molecular pharmacol- Received 17 July 2017 ogy of small molecule drugs. Fluorescence polarization/fluorescence anisotropy measurements are commonly Received in revised form 29 January 2018 employed in protein/cell screening assays. However, the expansion of such measurements to the in vivo setting Accepted 30 January 2018 has proven difficult until recently. With the advent of high-resolution fluorescence anisotropy microscopy it is Available online 2 February 2018 now possible to perform kinetic measurements of intracellular drug distribution and target engagement in com- monly used mouse models. In this review we discuss the background, current advances and future perspectives Keywords: fl Two photon microscopy in intravital uorescence anisotropy measurements to derive pharmacokinetic and pharmacodynamic measure- Fluorescence polarization, fluorescence anisot- ments in single cells and whole organs. ropy © 2018 Elsevier B.V. All rights reserved. Fluorescence anisotropy imaging Fluorescently labeled drugs Drug-target engagement Single-cell pharmacodynamics Intravital imaging Contents 1. Introduction.............................................................. 263 2. Fluorescenceanisotropybasicprinciples.................................................. 264 2.1. History............................................................. 264 2.2. Definitions............................................................ 264 2.3. One-photonsteadystateandtime-resolvedanisotropy........................................ 265 2.3.1. Limiting and fundamental fluorescenceanisotropies..................................... 265 2.3.2. Perrinequation..................................................... 265 2.3.3. Additive property of fluorescenceanisotropy........................................ 266 2.3.4. Fluorescenceanisotropywithexcitationbyunpolarized(natural)light............................ 266 2.3.5. Anisotropydecays.................................................... 266 2.3.6. Associatedanisotropydecays............................................... 266 2.4. Two-photon fluorescenceanisotropy................................................ 266 2.4.1. Simultaneousmulti-photonexcitation........................................... 266 2.4.2. Two-colortwo-photonexcitation............................................. 267 2.5. Depolarizationfactors....................................................... 267 3. Fluorescence polarization/fluorescenceanisotropyassay.......................................... 267 3.1. Design of fluorescentlylabeledcompounds............................................. 267 3.2. High-throughputscreening.................................................... 268 4. Imagingmicroscopy........................................................... 268 4.1. Wide fieldmicroscopy...................................................... 268 4.2. Confocalmicroscopy....................................................... 269 4.3. Spinningdiskmicroscopy..................................................... 270 4.4. Homo-FRETimaging....................................................... 270 ⁎ Corresponding author. E-mail address: [email protected] (C. Vinegoni). https://doi.org/10.1016/j.addr.2018.01.019 0169-409X/© 2018 Elsevier B.V. All rights reserved. C. Vinegoni et al. / Advanced Drug Delivery Reviews 151–152 (2019) 262–288 263 4.5. Time resolved fluorescenceimagingmicroscopy.......................................... 270 4.6. Two-photonmicroscopy..................................................... 270 4.7. Super-resolutionmicroscopy................................................... 271 5. Methods in fluorescenceanisotropymicroscopy.............................................. 271 5.1. Experimentalsetup....................................................... 271 5.2. Fluorescencemicroscopyimageacquisition............................................. 272 5.3. Noise in fluorescencemicroscopy................................................. 272 5.4. Effectsofnoiseinconfocalmicroscopy............................................... 273 5.5. Anisotropy imaging and noise filtering............................................... 273 6. Sources of errors in fluorescenceanisotropyimaging............................................ 273 6.1. Scattering............................................................ 274 6.2. Objectivenumericalaperture................................................... 275 6.3. Instrumentcalibration...................................................... 275 6.4. Detectorsacquisitionschemes................................................... 276 7. Measurementsofdrug-targetengagement................................................ 276 7.1. Fluorescenceanisotropyvisualization............................................... 276 7.2. Cellsegmentation........................................................ 277 7.3. Measurement and quantificationofsinglecelldrug-targetengagement................................ 277 7.3.1. Labeling fluorophorechoice................................................ 278 7.3.2. Fluorescence anisotropy two photon imaging of fluorescentlylabeleddrug.......................... 278 7.4. Competitive binding of matched fluorescentlylabeleddrugs..................................... 281 8. Conclusions.............................................................. 281 Acknowledgements............................................................. 282 References................................................................. 282 1. Introduction quantitative measurements without a separation step i.e. without re- moving one of the components from the solution. Thanks to these fea- For a drug to become successful clinically it must produce a desired tures the current FP/FA methods have enjoyed wide distribution to therapeutic effect at no, or only minimal and acceptable, toxicities. To study protein-ligand and protein-protein interactions [13,14] and to de- better understand drug effects (or the lack thereof) in vivo it is highly termine the fraction of bound vs. free ligand for resolving their dissoci- desirable to directly measure drug-target engagement in single cells as ation constants [12,15,16]. FP/FA have also been used in assays to well as in populations of cells making up tissues and organs [1]. Conven- enable high-throughput screening of small molecule libraries for drug tional pharmacology and most pharmacokinetics/pharmacodynamics discoveries [17]. (PK/PD) studies rely on bulk sampling of tissue or plasma where subtle Optical imaging technologies offer high spatial and temporal resolu- nuances can be easily missed or “diluted out”.Conversely,in vitro assays tion, extended penetration depth, and the ability to distinguish multiple against purified targets lack the barriers, pressures and effects that reporter. They are therefore the ideal detection technologies for in vitro drugs face in vivo. Furthermore, even genetically identical cells are and in vivo single-cell phenotypic screening [18]. Their availability con- often heterogenous and these effects are difficult to model in vitro. current with the emergence of a growing list of fluorescent drug deriv- A number of recent technologies have been described to directly atives that maintain comparable target specificity and affinity as the measure drug binding in cells. Among them are the drug affinity respon- unlabeled drug [19], has enabled direct insight into drug delivery and sive target stability (DARTS) assay [2], competitive positron emission drug action in vitro and in vivo, including target-selectivity, kinetics, tomography (PET) [3,4], or mass spectroscopy imaging (MSI) [5,6]. All drug exposure and resistance, and pharmacodynamics effects [20–24]. of these methods have inherent limitations with respect to cellular res- By extending FP/FA to optical microscopy imaging modalities, in olution (PET), subsequent analysis (MSI) or others. combination with measurements of fluorescence intensity, and co- The cellular thermal shift assay (CETSA) [7] has also been used to de- localization with fluorescent reporter proteins, we have demonstrated termine target engagement of unlabeled drugs in specimen as well as in recent works from our group that one can obtain spatially and tempo- in vivo, and to measure off-target binding of thousands of proteins rally resolved cellular mapping both in vivo and in vitro, enabling in- using mass spectrometry [8]. However CETSA yields average measure- sights into the degree of drug accumulation within individual cells, the ments of cell populations and temporal resolution is limited. quantification of drug target expression, and the degree of specific Fluorescence polarization (FP) and fluorescence anisotropy (FA) [9] drug-target binding and unspecific binding to off-target proteins