From DNA-encoded library to cellular degradation
Developing effective PROTACs is challenging because productive degradation depends not just on binding affinity, but also on forming stable and functional ternary complexes. Having both quantitative biophysical data and robust cellular readouts play a pivotal role in driving degrader optimization.
This application note showing joint forces with Bruker Biosensors presents an integrated workflow that addresses this challenge by transforming an initial DEL hit into PROTAC molecules capable of inducing ternary complex formation. The workflow combines SPR kinetic profiling using the Triceratops SPR#64 with advanced switchSENSE® proximity assays to directly measure PROTAC‑induced ternary complex formation. These high‑quality biophysical data provide unprecedented clarity on specificity, binding kinetics, and complex stability.
Importantly, the biophysical insights are validated in cells by cellular degradation assays, demonstrating BRD4 ubiquitination and degradation. By aligning molecular interaction data with functional cellular readouts, this approach enables more confident, data‑driven PROTAC design and faster prioritization of the most promising degrader candidates.
Abstract:
Targeted protein degradation (TPD) has rapidly advanced as a new therapeutic modality, with PROTACs standing out as the leading and most broadly applied approach. To fully harness the therapeutic potential of PROTACs, a systematic workflow that integrates biophysical characterization is essential for understanding E3 ligase recruitment and ternary complex formation, key determinants of degradation efficiency and selectivity. To this end, 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.
For more details on Bruker Biosensors technologies for accelerated TPD drug discovery and development, visit this page.