Discovery of PROTACs recruiting the E3 ligase CHIP.

Proteolysis-targeting chimeras (PROTACs) are promising degraders in targeted protein degradation (TPD) systems. Although more than 600 E3 ubiquitin (Ub) ligases exist in the human genome, only a few E3 ligases have been engaged in PROTACs. It is essential to expand the range of available E3 ligase resources to combat drug resistance caused by their mutations and to find suitable candidates for the degradation of certain target proteins. In this study, we aimed to develop novel PROTACs utilizing the carboxyl terminus of the Hsc70-interacting protein (CHIP) E3 ligase, which was previously unutilized. We designed and synthesized PROTACs conjugated with CHIP ligands and target protein ligands via linkers. These PROTACs form a ternary complex with CHIP and a target protein and ubiquitylate the target protein in vitro. Furthermore, taking advantage of the fact that CHIP is not an intricate multi-subunit E3 ligase, unlike other E3 ligases utilized for PROTACs, an in vitro ubiquitylation assay revealed that CHIP induced not only the lysine (K) 48-linked polyubiquitin (poly-Ub) chain, which is a typical proteasome degradation signal, but also the K63 linkage. Furthermore, it induces the proteasome-dependent degradation of HaloTag protein and endogenous bromodomain-containing protein 4 (BRD4). Thus, we successfully expanded the E3 ligase toolbox for PROTACs. These new PROTACs using CHIP, which induce non-canonical proteasome degradation signals containing a mixed linkage of poly-Ub chains on the target protein, can be applied to the degradation of other proteins, especially disease-related proteins.