Small-molecule mimicry intercepts PEX5 recognition by trypanosomal PEX14 as revealed by Molecular Dynamics

Human trypanosomiases, including Chagas disease and human African trypanosomiasis, remain major neglected tropical diseases with limited therapeutic options and substantial treatment-associated toxicity. In trypanoso- matids, glycolytic enzymes are compartmentalised within glycosomes, peroxisome-like organelles whose biogenesis depends on the essential interaction between the peroxins PEX5 and PEX14. Interference with this protein– protein interface therefore represents a promising strategy for developing new trypanocidal agents. Here, molecular docking, MD simulations, and MM–PBSA calculations were used to examine the PEX5–PEX14 interaction and small-molecule binding to the PEX14 recognition groove. The PEX5–PEX14 complex was stabilised by contacts centred on the canonical Wxxx(F/Y) motif of PEX5, particularly Trp60 and Tyr64, with additional contributions from a secondary non-canonical PEX5 region. Hydrogen bonds, salt bridges, π–cation contacts, and T-shaped aromatic interactions involving PEX14 residues Phe12, Phe29, Lys33, and Thr21 were key contributors to com- plex stability. The reported inhibitor ET7 reproduced central features of the native PEX5 recognition motif and remained associated with the PEX14 groove throughout the simulations. SuFEx-functionalised naphthoquinones from a previously reported series were then assessed as potential PEX14 ligands. While the initial sulfonyl fluoride derivative associated only transiently with the groove, SuFEx derivatisation at the sulfonyl centre to introduce an additional aromatic substituent increased residence time and gave more favourable MM–PBSA binding free ener- gies. These results identify aromatic and electrostatic contacts as major determinants of ligand stabilisation within the PEX14 groove and support SuFEx-derived naphthoquinones as promising scaffolds for inhibitors targeting glycosomal protein import in Trypanosoma species.