Development of cell penetrating peptide-based ubiquitin ligase ligand for the development of PROTAC

PROTAC (PROteolysis TArgeting Chimeric molecule) [1], which induces ubiquitination and degradation of protein of interest by recruiting ubiquitin ligase (E3) to target proteins, is expected to be a novel therapeutic modality.PROTAC is a chimeric molecule that combines the E3 ligand with the target protein ligand via an appropriate linker, and inhibits protein function through its degradation.We have developed peptide-based PROTAC by incorporating cell penetrating peptides and modifications like stapling structures to enhance cell membrane permeability [2, 3].This molecule was expected to expand targets to include proteins considered undruggable by conventional small molecule-based drugs.However, low cell membrane permeability remains a problem for the development of PROTAC.Therefore, in this study, we enhanced the cell membrane permeability to the E3 ligand, a commonly used moiety in PROTACs.We designed a peptidebased E3 ligand moiety by conjugating it with cell penetrating peptides, including oligoarginine, endosomal escape domains (EEDs), and oligo-sarcosines, and attached a small molecule-based fluorophore as a model ligand for target proteins to the designated E3 ligand.The designed peptides were synthesized through solid-phase synthesis, and their cell penetrating ability was evaluated by detecting the fluorescence from the fluorophore conjugated to the peptide.We found that controlling the introduction and sequence of oligoarginine, the EED and oligo-sarcosine could improve the efficiency of intracellular delivery of peptide-based E3 ligands.Therefore, the obtained E3 ligands were applied to PROTAC, in which peptide ligands called peptidomimetic estrogen receptor modulators (PERMs) for estrogen receptor were introduced into the obtained E3 ligand peptides.Their degradation activities were evaluated by western blotting.As a result, the PROTAC with oligoarginine and the EED showed higher degradation activity than that of the parent peptide, suggesting that the developed E3 ligand peptide would be useful for the development of PROTAC.