Comparative assessment of co-folding methods for molecular glue ternary structure prediction

Molecular glues (MGs) represent an emerging therapeutic paradigm capable of inducing or stabilizing protein-protein interactions, with broad applications in creating neomorphic interactomes and targeted protein degradation. However, current discovery efforts remain largely confined to experimental screening, while in silico rational design of MGs persists as a formidable challenge. A critical step toward rational design lies in accurate ternary complex modeling. Given the scarcity of such complexes in the Protein Data Bank for training specialized predictive models, we tested the ability of recently developed co-folding models, including AlphaFold 3, Boltz-1, Chai-1, Protenix, and RoseTTAFold All-Atom in building the complex models. We systematically curated a dataset, named MG-PDB, with 221 non-covalent MG-engaged ternary complexes. MGBench were further introduced as a comprehensive benchmark set, which comprises 88 ternary structures excluded from co-folding models’ training data through rigorous time-based partitioning. Our benchmark results demonstrated that AlphaFold3 achieved the best overall performance among co-folding methods, in terms of both protein-protein interaction interface prediction (50.6% success rate) and MG-protein interaction recovery (32.9% success rate). However, our homology study showed that most of their successful predictions actually stemmed from memorization. Further analysis revealed three phenomena of current co-folding methods for MG ternary structure prediction. Firstly, these methods struggle to accurately model large interaction interfaces. Secondly, their predictive accuracy is notably reduced for domain-domain complexes compared to domain-motif interactions. Lastly, they face specific challenges in modeling MG degrader complexes with sufficient accuracy. We showcased they relied on the existing interaction patterns, and highlighted the need for further improved in novel E3 ligase systems. These findings reveal fundamental gaps in existing methods to learn atomic-level interaction rules for MG-engaged ternary complex modeling. MG-PDB and MGBench provide critical resources and mechanistic insights to advance computational MG discovery.