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Updated: Jul 27, 2025
High-Throughput Cellular Profiling of Targeted Protein Degradation Compounds Using HiBiT CRISPR Cell Lines
Published on: November 9, 2020
Comparative analysis of biophysical methods for monitoring protein proximity induction in the development of small
Kamil Przytulski1, Przemyslaw Glaza2, Katarzyna Brach2
1Captor Therapeutics Inc, Duńska St 11, PL54427 Wroclaw, Poland; Max Planck Research Group at Malopolska Centre of Biotechnology, Jagiellonian University, Gronostajowa St 7a, PL30387 Cracow, Poland; Doctoral School of Exact and Natural Sciences, Jagiellonian University, Prof. St. Łojasiewicza St 11, PL30348 Cracow, Poland.
Background:
Targeted protein degradation relies on inducing proximity between an E3 ubiquitin ligase and a target protein, and subsequent proteasomal degradation of the latter. Biophysical methods allow the measurement of the ternary complex formation by recombinant target and E3 ligase proteins in the presence of molecular glues and bifunctional degraders. The development of new chemotypes of degraders mediating ternary complex formation of unknown dimensions and geometries requires the use of different biophysical approaches.
Methods:
The TR-FRET and AlphaLISA platforms have been applied to study molecular glues and bifunctional degraders. The performance of the label-based proximity assays was compared with the BLI method, which is a label-free, sensor-based approach.
Results:
We present and compare two commonly used assays to monitor proximity induction, AlphaLISA and TR-FRET. The LinkScape system consisting of the CaptorBait peptide and the CaptorPrey protein is a novel method of protein labeling compatible with TR-FRET assay.
Conclusions:
The TR-FRET and AlphaLISA proximity assays enable detection of ternary complexes formed between an E3 Ligase, a target protein and a small molecule degrader. Experiments with different chemotypes of GSPT1 degraders showed that ALphaLISA was more susceptible to chemotype-dependent interference than TR-FRET assay.
General Significance:
The discovery and optimization of small-molecule inducers of ternary complexes is greatly accelerated by using biophysical assays. The LinkScape-based TR-FRET assay is an alternative to antibody-based proximity assays due to the CaptorPrey's subnanomolar affinity to the CaptorBait-tagged protein target, and the 10-fold lower molecular weight of the CaptorPrey protein compared to the antibody.
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