Protein Age Bias in Target Degradation by PROTACs

Targeted protein degradation (TPD) by PROteolysis TArgeting Chimeras (PROTACs) has emerged as a powerful chemical biology and therapeutic modality, yet many degraders exhibit incomplete target clearance and characteristic rebound kinetics despite continuous exposure. The mechanistic basis for this behavior remains poorly understood. Here, we uncover protein age as a previously unrecognized determinant of PROTAC efficacy. Using CG-SLENP, a chemical genetics strategy that selectively labels newly synthesized and pre-existing proteins within the same living cell, we directly resolve PROTAC-induced degradation of distinct intracellular protein populations. Applying this approach to the bromodomain protein BRD4, we show that two mechanistically and structurally distinct PROTACs, dBET6 and MZ-1, preferentially degrade pre-existing BRD4, while newly synthesized BRD4 is degraded substantially more slowly and incompletely. This age-dependent degradation bias is observed in live-cell imaging, across compound concentrations and time scales, and for both reporter and endogenous BRD4. BRD9-targeting PROTAC VZ185 exhibited similar degradation preference. These findings reveal that PROTAC-mediated degradation is governed not only by target engagement and ternary complex formation but also by the dynamic balance between protein synthesis and degradation. By identifying temporal proteostasis as a critical parameter in TPD, this work provides a mechanistic framework for incomplete degradation and rebound kinetics and therefore establishes protein maturation state as an important consideration for degrader design and evaluation.