Targeted Protein Degradation by Chimeric Compounds using Hydrophobic E3 Ligands and Adamantane Moiety.

Takuji Shoda,Genichiro Tsuji,Mikihiko Naito,Hideshi Inoue,Takuma Fujisato,Yosuke Demizu,Nobumichi Ohoka,Masaaki Kurihara

doi:10.3390/ph13030034

Abstract

Targeted protein degradation using small chimeric molecules, such as proteolysis-targeting chimeras (PROTACs) and specific and nongenetic inhibitors of apoptosis protein [IAP]-dependent protein erasers (SNIPERs), is a promising technology in drug discovery. We recently developed a novel class of chimeric compounds that recruit the aryl hydrocarbon receptor (AhR) E3 ligase complex and induce the AhR-dependent degradation of target proteins. However, these chimeras contain a hydrophobic AhR E3 ligand, and thus, degrade target proteins even in cells that do not express AhR. In this study, we synthesized new compounds in which the AhR ligands were replaced with a hydrophobic adamantane moiety to investigate the mechanisms of AhR-independent degradation. Our results showed that the compounds, 2, 3, and 16 induced significant degradation of some target proteins in cells that do not express AhR, similar to the chimeras containing AhR ligands. However, in cells expressing AhR, 2, 3, and 16 did not induce the degradation of other target proteins, in contrast with their response to chimeras containing AhR ligands. Overall, it was suggested that target proteins susceptible to the hydrophobic tagging system are degraded by chimeras containing hydrophobic AhR ligands even without AhR.

Highlights

A technology for selectively degrading a target protein is expected to elucidate the physiological function of proteins and to develop a therapeutic drug for a disease caused by the aberrant protein
Previous studies reported a series of chimeric compounds that recruit E3 ubiquitin ligases to degrade target proteins via the ubiquitin-proteasome system [1,2]
These chimeras, which are called specific and nongenetic inhibitors of apoptosis protein [IAP]-dependent protein erasers (SNIPERs) and proteolysis-targeting chimeras (PROTACs), contain two ligands connected with a linker: one ligand specific to an E3 ubiquitin ligase and another ligand specific to the target protein

Summary

Introduction

A technology for selectively degrading a target protein is expected to elucidate the physiological function of proteins and to develop a therapeutic drug for a disease caused by the aberrant protein. We designed and synthesized compounds with adamantane moiety to investigate whether the AhR-independent degradation of CRABP-2 and BRD3 by the chimeric compounds using AhR ligands are induced by the HyT system. Bn-ATRA 4, which is less hydrophobic than 1, 2, and 3, induced a weaker activity than 1, 2, and 3 These results indicated that CRABP-2 and BRD3 are susceptible to HyT-mediated degradation and that the AhR-independent reduction of these proteins was caused by the HyT system. These results provided useful information for drug development using chimeric compounds with a highly hydrophobic E3 ligand, which may occasionally induce targeted protein degradation by the HyT system. In addition to our findings, it is important to clarify the types of ubiquitin chains on target proteins and the specific E3 ligases involved in the ubiquitination

Materials and Methods

Cell Culture

Western Blotting

Findings

Cell Viability Assay