Fluorescence polarization (FP), also known as Fluorescence anisotropy, is commonly used in early-phase drug discovery. When a fluorescent molecule is excited by polarized light, the polarization degree of the emitted wavelength is determined by the rate of molecular rotation. A fluorescently labeled small molecule rotates rapidly in solution, therefore the light emitted is depolarized. On the other hand, when a fluorescent probe is binding to a target protein, its rotation is slower, and the emission wavelength is polarized.

This method can be used to study molecular interactions (binding, kinetics, etc.) and enzyme activities in solution. The main advantage of fluorescence polarization is the possibility of performing anhomogenous assay, making it especially suitable for High Throughput Screening approaches.

We have validated several of our fluorescent ligands as optimal tools for FP, both on membrane preparations from cell cultures and on membrane extracts from baculovirus.

Fluorescence Polarization-a

CELT-228 hA3 Adenosine receptor fluorescent antagonist is suitable to measure the affinity of compounds for the hA3 receptor in Fluorescence Polarization binding assays. A) Concentration−percent of specific binding of CELT-228 curves obtained with compounds 7d (red), 9a (black), 9c (green), and 10a (blue); B) Comparison of hA3 binding affinities of the series obtained by radioligand binding assays using [3H]NECA and fluorescence polarization binding assay using CELT-228.

FP validation performed in Innopharma Laboratory (EU-OPENSCREEN network).

Figure from Miranda-Pastoriza, D. et al. “Exploring Non-orthosteric Interactions with a Series of Potent and Selective A3 Antagonists”ACS Med. Chem. Lett. 2022, 1 3, 2, 243–249 https://doi.org/10.1021/acsmedchemlett.1c00598

Celtarys Ligands validated for Fluorescence Polarization assays

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