Biosensor Technologies for Accelerated TPD Drug Discovery and Development

Proteolysis-targeting chimeras (PROTACs) and molecular glues promise selective protein degradation, but their characterization is anything but straightforward.

Binary affinities alone don’t explain activity. What matters is how target, ligase, and degrader behave together.

Failure to account for ternary complex behavior is a frequent reason why promising PROTAC candidates underperform.

Biophysical methods can resolve this complexity—if they capture the full system:

• switchSENSE^® quantifies both binary and ternary interaction kinetics, revealing cooperativity, avidity, and ternary complex stability 
• With switchSENSE^®, PROTAC-induced conformational changes can be directly detected, providing additional insight into functional mechanisms
• High-throughput SPR enables rapid binary screening to assess selectivity early

Explore how these approaches help disentangle PROTAC behavior and make more confident decisions in your development process.

A prototypical workflow for PROTAC design and characterization is presented here, employing BRD4 as a model target and combining comprehensive biophysical and cellular analyses. Binders selective for bromodomain 1 of BRD4 were initially identified via DNA-encoded library (DEL) screening, and PROTAC molecules were subsequently designed to recruit the E3 ligase Cereblon (CRBN).

Binary binding kinetics across various BRD4 constructs and PROTAC variants were determined by surface plasmon resonance (Triceratops SPR #64), providing insights into PROTAC specificity, while ternary complex formation was evaluated and validated using switchSENSE^® technology and the recently developed Y-structure proximity assay, yielding important information on the complex binding mechanism.

Importantly, the results of these biophysical analyses were supported by cellular studies, which demonstrated clear evidence of BRD4 ubiquitination and subsequent degradation, highlighting the functional relevance of the PROTAC-induced mechanism in a cellular context. By integrating high-resolution biophysical profiling with cellular degradation assays, detailed mechanistic insights into PROTAC mode of action were obtained, laying the foundation for data-driven optimization of degrader candidates.

The structural and biophysical characterization of PROTACs is often hampered by the complexity of full-length E3 ligases, particularly cereblon (CRBN), one of the most extensively studied and clinically relevant ligases. CRBN^midi, a truncated CRBN construct, overcomes these limitations and enables detailed analysis of degrader-induced ternary complex formation and stability.

Here, we compare PROTAC-mediated ternary complexes formed with CRBN^midi or the full-length DDB1:CRBN complex in the presence of Brd4 using the switchSENSE^® Y-structure binding assay. Our results demonstrate that CRBN^midi forms more stable ternary complexes with slower dissociation kinetics than the full-length E3 ligase.

This effect likely arises from the absence of DDB1, which leaves the sensor loop unbound, destabilizes the open state, and favors the closed, ligand-bound conformation. Together, these findings establish CRBN^midi as a valuable tool for mechanistic studies and highlight the utility of the DNA Y-structure assay for the characterization and optimization of protein degraders.

Ternary complexes, consisting of two proteins connected by small molecules like PROTACs or molecular glues pose new challenges for the analysis of molecular interactions, because they depend not only on binary affinities, but are orchestrated by cooperativity and avidity effects.

Here, we introduce a proximity binding assay for the simultaneous measurement of binary and ternary interaction kinetics on a biosensor surface. Target proteins and ubiquitin E3 ligase substrate receptors are tethered to mobile swivel arms of a Y-shaped DNA scaffold, which presents them in close proximity to PROTAC analytes flown across the sensor. PROTAC-induced ternary complex formation is measured by fluorescence energy transfer (FRET), while binary interactions are detected by fluorescence quenching.

The assay is applied to cereblon (CRBN) and von Hippel-Lindau (VHL) as E3 ligase substrate receptors, a range of compounds including AT1, MZ1, dBETs, and ARV-825 as PROTACs, and the two bromodomains of Brd2, Brd3, Brd4, and BrdT proteins as targets. Automated workflows enable the measurement of 384 real-time sensorgrams in a single run using picomolar sample quantities. Ternary and binary binding kinetics and proximity-mediated binding enhancements are analyzed.

Ternary complex stability is shown to arise from a dynamic interplay of associations and dissociations, suggesting that proximity assays can be utilized to identify weak interactions. The insights into proximity-mediated binding kinetics can enable the development of PROTACs and molecular glues with improved properties for targeted protein Degradation.

Better data leads to better decisions.
Let’s explore how you can de-risk your PROTAC development with the right biophysical insights.