A crucial stage in the development of novel drugs is the initial compound screening of biological samples for pharmacological hits to identify potential novel drug targets. Fluorescence polarization is central to the vast advancements in screening technologies crucial to drug discovery, analyzing interactions between a vast array of molecules from DNA to proteins.
Basic Principles of Fluorescence Polarization
Fluorescence polarization - akin to fluorescence anisotropy - assays are based on the concept that fluorescently labeled molecules emit light when excited with plane-polarized light. The degree of polarization of the light emitted during excitation in fluorescence polarization is inversely proportional to its molecular rotation (a product of Brownian molecular rotation theory1), which can reflect the size of the molecule when lifetime, viscosity, and temperature are controlled.
In quantitative terms, fluorescence polarization records the difference in the intensity of the emission of the excitation light plane (normalized by the intensity of fluorescence emitted) in comparison to the intensity of both the parallel and perpendicular light emission. Changes in molecular size during either binding or breakdown between different molecules (such as membrane proteins and their ligands) alter the degree of depolarization of plane-polarized light, which is calculated by the fluorescence polarization value. This polarization value is the basis of identifying new potential druggable targets with fluorescence polarization.2
Fluorescence Polarization Assays For Drug Discovery
Generally, high-throughput screening of compound libraries using ligand binding assays is conducted to identify potential new drug targets. High-throughput fluorescence polarization screening methods can provide information on a variety of biological activities, including membrane receptors, transcription factors, epigenetic regulators, kinases, and proteases.3 This information is gained through polarization screening binding assays between protein receptors and their ligands, proteins and peptides, proteins and proteins, as well as proteins and nucleic acid.
One of the major druggable targets in pharmaceutical research is G-protein coupled receptors. Traditionally radioligand binding assays have been used to identify potential drug targets from target binding. However, this approach has steadily been replaced by fluorescence polarization assays. Fluorescence polarization assays have led to the discovery of numerous new agonists and antagonists of G-protein coupled receptors that are potential drug targets through quantifying binding affinities between them.2
Fluorescence Polarization in Fragment-Based Drug Design
Fluorescence polarization is also used for functional evaluation in fragment-based assays, which detect the binding of extremely small molecules/ “fragments” to specific targets for drug discovery. Fragments are desirable because they have low complexity and are small enough to bind to regions on target cells that ligands could not. Fragment-based assays that utilize fluorescence lifetime and fluorescence probes include functional enzyme assays that evaluate enzyme inhibitors, thermal shift assays that provide essential information about protein stability and microscale thermophoresis that can characterize any biomolecular interaction.4
Benefits and Limitations of Fluorescence Polarization
One main advantage of fluorescence polarization is that fewer, inexpensive reagents are required due to fewer separation and filtration stages. Repeated fluorescence polarization assays can be conducted to increase reliability in discovering potential druggable targets, as the process does not damage samples.
However, a significant limitation of fluorescence polarization assays is that a high amount of the target protein is required to gain fluorescence polarization-based measurements. This is only possible for some proteins, thus limiting the application of fluorescence polarization to a selection of protein families.5 Fluorescence polarization also sometimes lacks its ability to detect ‘weak’ binding interactions, but this is combatted with radiometric assays.2
The Future of Fluorescence Polarization in Drug Discovery
Recent success in designing and fabricating bright and high-affinity tracers with low, non-specific binding affinities, combined with proficient protein expression systems, is resulting in the discovery of novel antagonists and agonists, which will significantly expand the discovery of potential new drug targets. 5 Fluorescence polarization is also opening new doors in examining interactions between proteins and carbohydrates which, although involved in many biological functions, have been much less studied in drug discovery.2
Fluorescence polarization is a well-established method used in both high-throughput and analytical assays for drug discovery. It has been developed for the majority of druggable targets and offers a precise, safer, and cheaper method for the initial stage of developing new therapeutic drugs. The current development and improvement in fluorescence probes and an increase in their accessibility are steering fluorescence polarization assays toward a bright future.
- Jameson, D.M. and Croney, J.C. 2003. Fluorescence polarization: past, present, and future. Combinatorial chemistry & high throughput screening. 6(3), pp.167-176.
- Lea, W.A. and Simeonov, A. 2011. Fluorescence polarization assays in small molecule screening. Expert opinion on drug discovery. 6(1), pp.17-32.
- Hall, M.D., Yasgar, A., Peryea, T., Braisted, J.C., Jadhav, A., Simeonov, A. and Coussens, N.P. 2016. Fluorescence polarization assays in high-throughput screening and drug discovery: a review. Methods and applications in fluorescence. 4(2), p.022001.
- Kirsch, P., Hartman, A.M., Hirsch, A.K. and Empting, M. 2019. Concepts and core principles of fragment-based drug design. Molecules. 24(23), p.4309.
- Uri, A. and Nonga, O.E. 2020. What is the current value of fluorescence polarization assays in small molecule screening? Expert opinion on drug discovery. 15(2), pp.131-133.