Challenges in predicting PROTAC-mediated protein-protein interfaces with AlphaFold reveal a general limitation on small interfaces.

Motivation: Proteolysis Targeting Chimeras (PROTACs) are heterobifunctional molecules composed by ligands binding to a target protein and a E3-ligase complex, connected by a linker, that induce proximity-based target protein degradation. PROTACs are promising alternatives to conventional drugs against cancer. Predicting PROTAC-mediated complexes is often the first step for in silico PROTAC design pipelines. We previously noted that AlphaFold2 (AF2) fails to predict PROTAC-mediated complexes.

Results: Here, we investigate the potential causes of this limitation. We consider a set of 326 protein heterodimers orthogonal to the AF2 training set, and evaluate AF2 models focusing on the interface size and presence of interface ligand. Our results show that AF2-multimer predictions are sensitive to the size of the interface to predict even in the absence of ligands, with the majority of models being incorrect for the smallest interfaces. We also benchmark both AF2 and AF3 on a set of 28 PROTAC-mediated dimers and show that AF3 does not significantly improve upon the accuracy of AF2. The low accuracy of AF2 on complexes with small interfaces has strong implications for computational pipelines for PROTAC design, as these stabilize typically small interfaces, and more generally on any prediction task that involves small interfaces.

Availability and implementation: All the models analyzed in this article are available in the Zenodo archive https://zenodo.org/records/14810843.