For several years, the authors of this study have been developing chemical compounds capable of inhibiting the entry of plant and bacterial toxins into eukaryotic cells through the endocytic pathway. Retro-2, identified through a high-throughput screening campaign, disrupts this intracellular transport route without affecting the trafficking of endogenous proteins. Subsequent work by the team demonstrated that Retro-2 provides both in vitro and in vivo protection against a range of pathogens, including viruses and bacterial toxins that exploit this intracellular transport pathway, making it a broad-spectrum inhibitor. The therapeutic development of optimized Retro-2 derivatives (Retro-2.x) led to the identification of several compounds with improved in vitro activity, although with reduced efficacy in vivo. Because Retro-2 compounds act on host-cell trafficking pathways rather than directly on pathogens, and despite the identification of two molecular targets (Sec16A and ASNA1), the precise mechanism of action of Retro-2.x remains unresolved.
In this context, and to validate and/or identify new molecular targets of Retro-2 compounds, the researchers synthesized and characterized a series of chemical probes based on PROTAC® technology, an emerging pharmaceutical strategy that enables the irreversible degradation of target proteins (see Box). The PROTAC molecules designed for this study consisted of an E3 ubiquitin ligase recruiter (CRBN ligand), a Retro-2 derivative capable of binding the protein of interest, and a polyethylene glycol (PEG) linker of variable length connecting the two moieties. The results did not demonstrate that any of the six synthesized PROTAC analogs could selectively degrade the putative protein targets of Retro-2, despite evidence of interaction with one or more targets, highlighting the structural sensitivity of these PROTACs. However, contrary to expectations based on the literature, the authors found that these molecules promoted the degradation of the protein GSPT1, which therefore appears to act as a neosubstrate of the CRBN ligase. In this case, the PROTACs behaved more like molecular glues, with their efficacy strongly dependent on PEG linker length, shorter PEG chains producing the most pronounced effect.
The results of this study, which aimed to identify one or more molecular targets of Retro-2 compounds using PROTAC® technology, indicate that the structural features responsible for the degradation of a neosubstrate remain poorly understood. This knowledge gap currently limits the rational design of PROTACs capable of avoiding GSPT1 degradation. With regard to Retro-2-based PROTACs, further empirical investigations will be required and may involve the use of alternative linker chemistries or ligands recruiting different E3 ubiquitin ligases, thereby circumventing GSPT1 degradation through distinct substrate-recognition mechanisms.
Legend : Illustration of the structure of a chimeric molecule (PROTAC) for target degradation of a protein. Molecules that induce targeted proteolysis are small compounds composed of two ligands (yellow and cyan) linked by a spacer (purple). One ligand recruits the target protein, whilst the other binds a ligase ; the spatial proximity between the proteins leads to ubiquitination and subsequently the degradation of the target protein. © Thom Leach / Science Photo Library via Getty Images
Joliot contact : Julien Barbier (julien.barbier@cea.fr)
- Endocytosis is a mechanism for transporting molecules, or even particles (viral, bacterial, etc.), into the cell.
- PROTAC®s (PROteolysis-TArgeting Chimeras) are small bifunctional molecules designed to induce the targeted degradation of unwanted or disease-associated proteins. They consist of a ligand that binds the protein of interest, linked through a chemical spacer to a ligand that recruits an E3 ubiquitin ligase, such as CRBN. One end of the PROTAC binds the target protein, while the other recruits the E3 ligase complex, bringing the two proteins into close proximity and promoting ubiquitination of the target. The ubiquitinated protein is then recognized and degraded by the ubiquitin–proteasome system (UPS). By hijacking the cell's natural protein degradation machinery through E3 ligase recruitment, PROTACs enable the selective elimination of specific target proteins.
- GSPT1 is a protein involved in translation termination, and its dysregulation has been implicated in oncogenesis. Although GSPT1 is not a natural substrate of the E3 ubiquitin ligase CRBN, it can become a neosubstrate in the presence of certain molecular glues (small molecules that promote the interaction between CRBN and specific target proteins, leading to their ubiquitination and subsequent degradation by the proteasome).