Evaluation of targeted protein degraders using high-throughput ubiquitination assays for mechanistic and SAR analysis

Targeted Protein Degraders (TPDs) represent a paradigm shift from traditional occupancy-driven inhibition to event-driven pharmacology by harnessing the ubiquitin-proteasome system for selective protein elimination. Despite their therapeutic potential, the rational design of effective degraders, particularly Molecular Glues, remains a significant challenge due to the complex interplay between binding cooperativity, ubiquitination efficiency, and degradation kinetics. To better understand these mechanisms, we established a suite of preclinical assays to systematically evaluate TPD activity across key mechanistic steps, including ternary complex formation, ubiquitination induction, cellular uptake, and target protein degradation in cellular and in vivo models. During the early screening of degrader libraries, medicinal chemists require efficient methods to assess structure-activity relationships (SARs). Comparative studies revealed that the ubiquitination assay provides a more predictive measure of cellular degradation efficiency than ternary complex formation assays. While ternary complex assays characterize binding affinity, cooperativity, and complex stability among the target, degrader, and E3 ligase, they fail to capture conformational changes necessary for productive ubiquitination. Our data demonstrate that ubiquitination assays offer deeper mechanistic insights with high throughput and scalability, enabling rapid generation of robust datasets for data-driven SAR development. This approach effectively bridges the gap between structural optimization and functional outcomes, accelerating the rational design of next-generation degraders with improved potency and selectivity for therapeutic applications.