Leveraging high-throughput proteomics and AI-based protein folding to accelerate VAV1 molecular glue discovery.

This study reports a series of CRBN molecular glues that selectively induce proteasomal degradation of VAV1, a hematopoietic-specific signaling protein and key target in hematological malignancies and autoimmune diseases. Using unbiased global proteomics, we identify phenyl-glutarimide derivatives (e.g., NGT-201-12) as effective VAV1 degraders that engage its C-terminal SH3 domain (SH3-2). We elucidate a non-canonical RT-loop degron (RDxS motif, residues 796-799) in SH3-2, distinct from previously characterized G-loop degrons, supported by the physics- and AI-driven GluePlex workflow and site-directed mutagenesis. Applying Free Energy Perturbation (FEP) to predicted ternary structures yields cooperativity metrics that correlate with degradation potency, overcoming limitations of standard docking and enabling prospective ranking of analogs-even from weak initial binders. We further show that conformational restriction via halogen substitution (e.g., NGT-201-18) potentiates degradation, a principle rationalized by DFT calculations, while comprehensive SAR studies chart a roadmap for next-generation degraders. Dose-response proteomics confirms VAV1 as the primary target and reveals LIMD1, bearing a canonical G-loop, as an off-target, showing that one glue can engage disparate degrons. These findings expand the landscape of CRBN neosubstrate recognition and offer a therapeutic strategy for targeting VAV1.