Speaker
Description
Targeted protein degradation of undruggable proteins is transformative in drug discovery. Molecular glues (MGs) enhance weak interactions between targets and E3 ligase. Native mass spectrometry (nMS) identifies E3-MG-target complexes directly, but manual sample preparation limits throughput.
This study demonstrates high-throughput MG screening using nMS for WEE1 binding to CRBN-DDB1, enabling multiplex screening of over 2,500 compounds per day. Gas-phase ligand release and fragmentation help identify unknown binders, and cryo-electron microscopy (EM) analysis characterizes ligand-bound complexes.
We compressed 96 compounds into 24 mixtures and used a SEC column for rapid online buffer exchange. LC-MS screening of all 96 compounds took under an hour, achieving throughput of over 2,500 compounds per day. Strong ternary complex formation between CRBN-DDB1 and WEE1 was observed in 4 of 24 mixtures, with 2 additional samples showing moderate binding. Identifying individual binders within mixtures was challenged by compound multiplexing, native adduct interference, and non-specific interactions.
To resolve this, ternary complexes were isolated in the quadrupole and subjected to collision-induced dissociation. Released binders, potentially uncharged in the gas phase, were detected via polarity switching. MS2 analysis of low m/z ions enabled accurate mass determination and comparison with the compound library. Ligands with unmatched masses were classified as "Unknown" and further analyzed by MS3 fragmentation for structural elucidation.
This workflow uses MS1 to detect intact complexes, MS2 to identify bound ligands, and MS3 to characterize unknowns, increasing throughput and specificity in MG screening. This approach confirmed 16 of 96 compounds as potential MGs, with varying binding strengths.
Selected hits were further characterized using cryo-EM, yielding high-resolution structures of WEE1-MG-CRBN-DDB1 ternary complexes. These structures reveal how MGs mediate and stabilize protein-protein interactions, offering critical insights to guide drug design and optimization.
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