Discovery of small molecules that reprogram the substrate specificity of the CRL4^CRBN E3 ubiquitin ligase offers powerful opportunities for targeted protein degradation (TPD). Here we report an unbiased proteomics workflow to evaluate a series of novel immunomodulatory imide-derived compounds for cereblon-dependent degradation activity. Eight cereblon-binding analogues were synthesized (>95% purity) and six analogues demonstrating adequate solubility and stability for cell-based assays were profiled in Jurkat T-cells alongside lenalidomide as references. Cells were treated (DMSO vehicle; reference: 10 µM; test compounds: 100 µM; 18 h), proteins were extracted, trypsin-digested and analyzed by nanoLC–MS/MS on a Q-Exactive Plus. Data were processed with FragPipe (1% FDR) and differential abundance assessed using limma (|log₂FC| > 1, adjusted p < 0.05). Several compounds induced reproducible depletion of known CRBN neo-substrates (e.g., IKZF1, GSPT1, ZFP91). While SNT-7443, SNT-7467, and SNT-7541 showed the strongest concordance with lenalidomide-like degradation signatures, others displayed distinct substrate selectivity patterns, suggesting both lenalidomide-like and novel modes of cereblon modulation. Pathway enrichment of downregulated proteins highlighted impacts on transcriptional regulation, RNA processing, and DNA-damage response, consistent with pleiotropic modulation of proteostasis. These findings identify multiple candidate molecular glues and nominate SNT-7443 and SNT-7467 for follow-up biochemical validation. Collectively, this study highlights the power of unbiased quantitative proteomics for the discovery of next-generation molecular glues, offering new opportunities for CRBN-targeted therapeutic design.
This study, conducted with Syntara Pharmaceuticals, has the potential to advances the translational development of novel imide molecular glues with differentiated profiles.