TR-FRET and FP Assay Reagents

TR-FRET is a highly sensitive assay technology used in the field of drug discovery and biological research to study protein-protein interactions, enzyme kinetics, and receptor-ligand binding. This technology works by measuring the energy transfer between two fluorescent molecules: a donor fluorophore, such as CoraFluor, and an acceptor fluorophore. The approach involves excitation of the donor molecule, typically with a 340 nm light source; if the donor molecule and acceptor molecule are in proximity (5 – 10 nm), energy is transferred from the donor to the acceptor through a phenomenon called Förster resonance energy transfer (FRET) (FIGURE 4). Lanthanides, such as terbium or europium, are generally uses as donors in TR-FRET as they have a large Stokes shift and millisecond long lifetimes. This enables time-delayed detection and eliminates background noise derived from scattered excitation light and autofluorescence from media components and proteins.

TR-FRET has been extensively used to identify novel drug targets, screen compound libraries, and optimize small molecules leads in various therapeutic areas, including oncology, cardiovascular disease, infectious disease, and neurodegenerative disorders. TR-FRET assays are particularly useful in measuring the binding affinity of small molecules, such as inhibitors and Degraders, to protein targets in a multi-well plate format, allowing for efficient high-throughput screening (HTS) of target engagement (FIGURE 5).

FP assays involve the excitation of a fluorescent molecule with polarized light and detection of emission using parallel and perpendicular polarizing filters. The technique is based on the observation that the effect of polarized light applied to a fluorescent probe is dependent on multiple factors, such as molecular conformation, orientation, and size. FP assays measure the change in orientation of a molecule in time, between absorption and emission events, and indicate the level of binding of the fluorescent molecule. 

Like TR-FRET, fluorescence polarization (FP) assays are used in biology to study molecular interactions, such as protein-protein, protein-DNA, and protein-small molecule binding, as well as for monitoring the progress of enzyme reactions. The approach provides a rapid, quantitative, and versatile analysis of molecular interactions, and can be used to measure pharmacological parameters for small molecules, such as affinity constants (K_i, K_d) and IC₅₀ values. FP assays are therefore frequently used in HTS for drug discovery.