Synthesis and biological evaluation of Retro-2-based PROTACs reveal PEG-linker length and warhead impact on GSPT1 degradation

PROteolysis TArgeting Chimeras are attracting growing interest in pharmaceutical research thanks to their catalytic and irreversible mechanism, which is capable of targeting proteins previously considered "undruggable". These bifunctional molecules hijack the cellular ubiquitin-proteasome system by recruiting E3 ligases (such as CRBN or VHL) to induce the selective degradation of a target protein. Their efficacy has been demonstrated against various disorders and several compounds have reached phase I-III clinical trials, reinforcing their appeal. In the fight against toxins, derivatives of the Retro-2 molecule (active against Shigatoxins, ricin, and various pathogens) have been developed. These molecules act on host cells by disrupting the intracellular trafficking of pathogens, targeting in particular the Sec16A and/or ASNA1 proteins. Our study presents the synthesis and characterization of new chemical probes based on PROTAC technology, combining a CRBN ligand derived from thalidomide, a Retro-2 derivative, and variable-length PEG chain linkers to better understand the mechanism of action of Retro-2-derived molecules. Contrary to initial assumptions, our results do not show proteasome-dependent degradation of the protein targets, but demonstrate that PEG-2 molecules can degrade the translation termination factor GSPT1 despite the normally propitious anchoring of the PEG linker at position 4 of the phthalimide ring. Furthermore, this study shows for the first time that GSPT1 degradation depends on the length of the flexible PEG chain linker.