CoraFluors: A User-friendly TR-FRET Assay Technology for PROTAC Profiling and Beyond.

Abstract: The pace of progress in biomedical research directly depends on techniques that enable the quantitative characterization of interactions between proteins and other biopolymers, or with their small molecule ligands. Here, time-resolved Förster resonance energy transfer (TR-FRET) assay platforms stand out with their high sensitivity and specificity. However, the paucity of accessible and biocompatible luminescent lanthanide complexes, which are essential reagents for TR-FRET-based approaches, and their poor cellular permeability have limited broader adaptation of TR-FRET beyond homogenous and extracellular assay applications.
To address this challenge, we have recently developed CoraFluors, a new class of macrotricyclic terbium complexes. CoraFluors are synthetically readily accessible, stable in biological media, and exhibit photophysical and physicochemical properties desirable for biological studies. Importantly, CoraFluors offer improved sensitivity and stability in biological settings over other published and/or commercial TR-FRET donor complexes. Furthermore, we have identified CoraFluors that are cell-permeable and demonstrated their utility for quantitative target engagement assays in living cells. These strategies have facilitated the development and implementation of novel experimental approaches, including the complex-specific characterization of inhibitor binding and quantification of endogenous target proteins directly in lysates of unmodified cells.
Here, we present the development of a set of complementary assay systems harnessing CoraFluor technology to enable the comprehensive characterization of PROTACs and molecular glue degraders. Our approach facilitates the direct quantification of endogenous target proteins in time and dose-dependent fashion following PROTAC treatment of genetically unmodified cell lines, enables the kinetic and thermodynamic characterization of ligand binding using cell lysates or purified proteins, and allows for the quantitative determination of ternary complex cooperativity in high-throughput.