Synthesis and Application of BRD4‐Targeting ByeTACs (Bypassing E‐Ligase Targeting Chimeras)

Targeted protein degradation (TPD) has revolutionized the way we think of drug discovery and has the potential for substantial therapeutic benefits. Traditional mechanisms rely on taking advantage of the cells endogenous protein degradation pathway known as the ubiquitin proteasome system (UPS). Traditional proteolysis targeting chimeras (PROTACs) rely on this mechanism by developing heterobifunctional molecules, which are compounds that contain two different ligands that bind two different proteins linked together with varying linker lengths. These compounds typically contain a ligand to a protein of interest that is to be degraded and a linker to the other ligand that binds to an E3 ligase. Once these compounds bind both proteins of interest with the proper confirmation, the E-ligase complex can facilitate the ubiquitination of the protein, leading to its recognition by the proteasome for degradation. This approach has been effective at developing degraders for a wide variety of proteins, yet there remain several challenges, such as limited ligands to E3 ligases, selectivity, and degrading proteins that cannot be ubiquitinated. To overcome these limitations, we developed a new targeted protein degradation approach that can bypass the need for E3 ligases and ubiquitination that we have named ByeTACs. This was accomplished by developing a bifunctional molecule that recruits proteins directly to the 26S proteasome, no longer requiring the E ligase cascade. The protocols presented here describe the synthesis and application of a ByeTAC targeting bromodomain-containing protein 4 (BRD4), that can be generalized to other POIs to assess their "ByeTACability." © 2025 Wiley Periodicals LLC. Basic Protocol 1: Synthesis and characterization of a ByeTAC library targeting BRD4 Basic Protocol 2: Assessing degradation of BRD4 ByeTACs in cells Basic Protocol 3: Validating BRD4 ByeTACs mechanism of action.