Annunziato, S.; Quan, C.; Donckele, E. J.; Lamberto, I.; Bunker, R. D.; Zlotosch, M.; Schwander, L.; Murthy, A.; Wiedmer, L.; Staehly, C.; Matysik, M.; Gilberto, S.; Kapsitidou, D.; Wible, D.; De Donatis, G. M.; Trenh, P.; SriRamaratnam, R.; Strande, V.; Almeida, R.; Dolgikh, L.; DeMarco, B.; Tsai, J.; Sadok, A.; Zarayskiy, V.; Walter, M.; Tiedt, R.; Lumb, K. J.; Bonenfant, D.; Fasching, B.; Castle, J. C.; Townson, S. A.; Gainza, P.; Petzold, G.

Molecular glue degraders (MGDs) are small molecule compounds that repurpose the ubiquitin-proteasome system to induce degradation of challenging therapeutic targets. Thalidomide-analogs are clinically effective MGDs that bind cereblon (CRBN), a substrate receptor of the Cullin-4/RING E3 ubiquitin ligase (CRL4CRBN), and impose a gain-of-function activity to recruit, ubiquitinate and degrade so-called neosubstrate proteins. Known neosubstrates bind the CRBN/MGD neosurface on the CRBN CULT domain through a structural G-loop recognition motif that mimics contacts of a natural CRBN degron. Here we report the binding mode of G3BP2, a CRBN neosubstrate that bypasses the G-loop requirement by engaging an unconventional binding site on the CRBN LON domain. The MGD-induced ternary complex interface does not resemble known protein-protein interactions (PPI) with CRBN. Instead, CRBN mimics an endogenous binding partner of G3BP2 and repurposes a preexisting PPI hotspot on the target protein. Our findings provide a novel generalizable concept for the rationalization of unconventional neosubstrate binding modes on CRBN, demonstrate unprecedented potential for the reprogrammability of this substrate receptor by MGDs, and offer opportunities for rational expansion of the target repertoire accessible to this modality.