Chapter 10
Ligand Discovery: High-Throughput Binding: Fluorescence
Polarization (Anisotropy)
Geoffrey A. Holdgate and Paul E. Hemsley
Abstract
High-throughput assays based on fluorescence polarization (or fluorescence anisotropy) technology have
often been employed for primary hit-finding in drug discovery. These binding assays provide a homogeneous format and consistent performance and offer advantages over some other optical methods. Developments in assay design and improvements in fluorescent probes have enabled the application of the
technique to even complex biological systems. Here we describe the practical considerations for development of FP assays applied in high-throughput screening, including fluorophore selection, assay design, data
analysis, and approaches for detecting compound interference.
Key words Fluorescence Polarization, Fluorescence Anisotropy, High-throughput Screening, Drug
Discovery, Dye, Ratiometric
1 Introduction
Screening large compound libraries to identify small molecule
modulators of biological targets is a well-established and commonly
used procedure in drug discovery [1–3]. The choice and configuration of the primary screen technology is influenced by many
factors, including the biological target, the range of desired
modes of action of hit molecules, access to tools and reagents, the
follow-up cascade of assays available, and any previous experience
with screening technologies applied to the actual or similar targets.
Typically, primary screening methods are then followed up with
orthogonal approaches designed to remove false hits arising from
primary assay interference or to demonstrate functional activity of
compounds identified as binding to the target [4]. Thus, drug
discovery often involves the use of both binding assays and activity
assays, and these can often be used in combination to provide
confidence in the primary screen output. One technology for identifying compounds that bind to the biological target is fluorescence
polarization (or fluorescence anisotropy) [5, 6]. This method relies
Tina Daviter et al. (eds.), Protein-Ligand Interactions: Methods and Applications, Methods in Molecular Biology, vol. 2263,
https://doi.org/10.1007/978-1-0716-1197-5_10, © Springer Science+Business Media, LLC, part of Springer Nature 2021
231
Ligand Discovery: High-Throughput Binding: Fluorescence
Polarization (Anisotropy)
Geoffrey A. Holdgate and Paul E. Hemsley
Abstract
High-throughput assays based on fluorescence polarization (or fluorescence anisotropy) technology have
often been employed for primary hit-finding in drug discovery. These binding assays provide a homogeneous format and consistent performance and offer advantages over some other optical methods. Developments in assay design and improvements in fluorescent probes have enabled the application of the
technique to even complex biological systems. Here we describe the practical considerations for development of FP assays applied in high-throughput screening, including fluorophore selection, assay design, data
analysis, and approaches for detecting compound interference.
Key words Fluorescence Polarization, Fluorescence Anisotropy, High-throughput Screening, Drug
Discovery, Dye, Ratiometric
1 Introduction
Screening large compound libraries to identify small molecule
modulators of biological targets is a well-established and commonly
used procedure in drug discovery [1–3]. The choice and configuration of the primary screen technology is influenced by many
factors, including the biological target, the range of desired
modes of action of hit molecules, access to tools and reagents, the
follow-up cascade of assays available, and any previous experience
with screening technologies applied to the actual or similar targets.
Typically, primary screening methods are then followed up with
orthogonal approaches designed to remove false hits arising from
primary assay interference or to demonstrate functional activity of
compounds identified as binding to the target [4]. Thus, drug
discovery often involves the use of both binding assays and activity
assays, and these can often be used in combination to provide
confidence in the primary screen output. One technology for identifying compounds that bind to the biological target is fluorescence
polarization (or fluorescence anisotropy) [5, 6]. This method relies
Tina Daviter et al. (eds.), Protein-Ligand Interactions: Methods and Applications, Methods in Molecular Biology, vol. 2263,
https://doi.org/10.1007/978-1-0716-1197-5_10, © Springer Science+Business Media, LLC, part of Springer Nature 2021
231