Fluorescence anisotropy (polarization): from drug screening to precision medicine

Abstract

Introduction Fluorescence anisotropy (FA) is one of the major established methods accepted by industry and regulatory agencies for understanding the mechanisms of drug action and selecting drug candidates utilizing a high-throughput format. Areas covered This review covers the basics of FA and complementary methods, such as fluorescence lifetime anisotropy and their roles in the drug discovery process. The authors highlight the factors affecting FA readouts, fluorophore selection, and instrumentation. Furthermore, the authors describe the recent development of a successful, commercially valuable FA assay for Long QT syndrome drug toxicity to illustrate the role that FA can play in the early stages of drug discovery. Expert opinion Despite the success in drug discovery, the FA-based technique experiences competitive pressure from other homogeneous assays. That being said, FA is an established yet rapidly developing technique, recognized by academic institutions, the pharmaceutical industry, and regulatory agencies across the globe. The technical problems encountered in working with small molecules in homogeneous assays are largely solved, and new challenges come from more complex biological molecules and nanoparticles. With that, FA will remain one of the major work-horse techniques leading to precision (personalized) medicine.

Quantification of Membrane Protein-Detergent Complex Interactions

“…This rather large separation between excitation and emission (~ 60 nm) ensures that scattering is minimal in our data; (ii) the large wavelength of emission was strategically used to avoid Raman and Rayleigh scattering effects. This is because the light intensities of both scattering contributions are proportional to λ −4 , where λ is the wavelength; 58 (iii) in our very preliminary stage of these studies, we have increased the concentration of labeled proteins up to a level, in which the signal was independent on protein concentration; 59 (iv) we conducted control experiments with proteins of closely similar molecular mass, but that exhibit a broad range of detergent solubilization properties under identical micellization conditions. The basal fluorescence anisotropy of the unfolded FhuA variants under excess of Gdm-HCl was ~0.16.…”

Nanothermometry: From Microscopy to Thermal Treatments

“…FA is commonly used in diverse life science applications,a nywhere from analytical chemistry [45] and drug design, [46] to probing the cellular microenvironment [47] andi maging. [48] FA directly relates to the rotational correlation time (q)o ft he molecules and is inversely proportionalt ot emperature accordingt ot he Stokes-Einstein-Debye equation [Eq.…”

“…Addressing the availability of fluorescent,s tably transfected cell lines, Donner and colleagues [53] proposed FA form apping temperature with green fluorescent protein (GFP). [46] This criterion is an important prerequisite fora ccurate measurements of FA.F urthermore, the methodh as been tested on GFP transfected cancerc ell lines and at emperature sensitivity of about 0.6 K À1 has been achieved. [46] This criterion is an important prerequisite fora ccurate measurements of FA.F urthermore, the methodh as been tested on GFP transfected cancerc ell lines and at emperature sensitivity of about 0.6 K À1 has been achieved.…”

Binding Assays for Bromodomain Proteins: Their Utility in Drug Discovery in Oncology and Inflammatory Disease

“…Their high sensitivity is particularly important, because the interactions of bromodomains and their cognate peptides are often weak and therefore difficult to detect (Filippakopoulos et al., ). Fluorescence polarization assays are suitable when higher‐affinity ligands are available and provide excellent reproducibility (Zhang et al., ). Both of these techniques are used in competitive binding assays, where the test compound displaces a probe molecule.…”

“…Hence, the affinity of ligands or peptides that bind to this site is determined using a competitive binding assay. Fluorescence polarization measurements, also referred to as fluorescence anisotropy measurements, have been used for years as biochemical, biophysical and drug discovery tools, including as a means for studying a variety of bromodomains (Hu et al, 2014;Zhang, Wu, & Berezin, 2015). where I par and I perp are the intensity measured parallel and perpendicular to the incident light, respectively.…”