Fluorescence anisotropy imaging in drug discovery

Abstract

Non-invasive measurement of drug-target engagement can provide critical insights in the molecular pharmacology of small molecule drugs. Fluorescence polarization/fluorescence anisotropy measurements are commonly employed in protein/cell screening assays. However, the expansion of such measurements to the in vivo setting has proven difficult until recently. With the advent of high-resolution fluorescence anisotropy microscopy it is now possible to perform kinetic measurements of intracellular drug distribution and target engagement in commonly used mouse models. In this review we discuss the background, current advances and future perspectives in intravital fluorescence anisotropy measurements to derive pharmacokinetic and pharmacodynamic measurements in single cells and whole organs.

Near‐Infrared‐II Molecular Dyes for Cancer Imaging and Surgery

“…Observing deep tissue information in vivo at a (sub)cellular resolution to explore the molecular signals and cell behaviors is a critically important direction for both oncology and other biological processes. Intravital imaging, affording quantitative and dynamic visualization/mapping into tumor biology and immunology, has realized this goal, with the help of the long‐term imaging windows (cranial window, dorsal skinfold chamber, the mammary window, as well as abdominal window) . There are several reports regarding the intravital imaging of the tumor microenvironment .…”

Creating and screening natural product libraries

“…As with many uorescent homogenous phase assays, this technique was rapidly adopted into MWP format, and has be employed for a wide range of drug discovery campaigns. 157 Since the FP measurement itself can be affected by the chemical characteristics of the uorophore, the nature and location of the uorophore itself on the target molecule is crucial. 156,157 For example, FP measurements are dependent on the excited state lifetime of the uorophore (s), and in general large target molecules will require uorophores with large (s); however, for most FP dependent screening campaigns it will be necessary to empirically determine which uorophores (and linker lengths) are best suited to the assay.…”

“…157 Since the FP measurement itself can be affected by the chemical characteristics of the uorophore, the nature and location of the uorophore itself on the target molecule is crucial. 156,157 For example, FP measurements are dependent on the excited state lifetime of the uorophore (s), and in general large target molecules will require uorophores with large (s); however, for most FP dependent screening campaigns it will be necessary to empirically determine which uorophores (and linker lengths) are best suited to the assay. Interestingly because FP depends on a relational ratio between uorescence at the same wavelength, it suffers less from uorescence interference (since quenching affects both angles of measurement equally).…”

“…Despite these limitations, well-developed FP assays can yield clinically relevant drug candidates. 156,157 Another biophysical assay that has been adopted for HTS assays is the thermal shi assay (TSA). 153,155 This assay measures the thermal stability of a target biomolecule (proteins initially but more recently nucleic acids) across a temperature gradient by monitoring the unfolding dependent binding of a uorogenic dye.…”

Imaging mitochondrial matrix viscosity in live cells via fluorescence lifetime imaging (FLIM) of fluorescent molecular rotors

“…The most established way to visually observe microviscosity -viscosity on the microscopic level -is by using time-resolved fluorescence anisotropy imaging (TR-FAIM) 7,4 . In TR-FAIM, the rotational correlation time, which describes the tumbling of a dye in its microenvironment, is extracted for each pixel.…”