AlphaFold3 for Non-canonical Cyclic Peptide Modeling: Hierarchical Benchmarking Reveals Accuracy and Practical Guidelines

Despite the revolutionary impact of AlphaFold3 on structural biology, this models capability in predicting non-canonical cyclic peptides remains unexplored. Given the clinical significance of cyclic peptides containing unnatural residues as a therapeutic modality, we present the first systematic evaluation of AlphaFold3 for this class of molecules. To facilitate benchmarking, we developed an automated input pipeline to streamline AlphaFold3 predictions for cyclic peptides. Our study aims to: (1) quantify the hierarchical accuracy (all-atoms, C atoms, and atoms of unnatural residue levels) of AlphaFold3 in predicting both non-canonical cyclic peptide monomers and complexes; (2) assess the reliability of AlphaFold3s confidence metrics; (3) evaluate the influence of multiple sequence alignment (MSA) and structural templates; and (4) identify systematic biases in AlphaFold3s predictions. Based on these analyses, we provide practical guidelines for applying AlphaFold3 in cyclic peptide structure prediction for facilitating the related research of bioactive cyclic peptides.