In Silico Identification of Antifungal Peptide Candidates from Theobroma cacao By-Products: Multi-Target Molecular Docking against CYP51, Chitin Synthase 2, and Myosin-I of Fusarium oxysporum

Fusarium oxysporum is among the most devastating fungal pathogens in global agriculture, causing significant crop losses in tomato, banana, and legumes. The emergence of azole-resistant strains highlights the urgent need for novel antifungal agents with alternative mechanisms of action. Here, we present the first systematic computational evaluation of peptide candidates derived from Theobroma cacao agro-industrial by-products against three validated molecular targets of F. oxysporum: CYP51 lanosterol 14α-demethylase (PDB: 4LXJ ), Chitin Synthase 2 (PDB: 7STO ), and Myosin-I motor domain (PDB: 1LKX ). Two tetrapeptide fragments (KTTF, AFLR) from T. cacao vicilin proteins and three cyclic dipeptides (DKPs: cyclo(Phe-Pro), cyclo(Trp-Pro), cyclo(Leu-Pro)) from cacao endophytic fungi were evaluated by molecular docking (smina, Vina scoring, exhaustiveness=16). All five candidates showed multi-target predicted binding profiles (mean ΔG ≤ −5.0 kcal/mol for ≥2 targets; n=3 independent docking runs per complex), with predicted affinities surpassing fluconazole (ΔG = −5.9 kcal/mol) against CYP51. KTTF showed the highest predicted binding affinity: ΔG = −8.2 ± 0.2 kcal/mol (CYP51) and −8.5 ± 0.1 kcal/mol (Myosin-I). Direct pairwise sequence alignment confirmed the validity of all three docking templates: CYP51 47.4% (4LXJ vs Q9P4A3), CaChs2 68.4% (7STO vs Q9P4C8), and Myosin-I 50.6% (1LKX vs XP_018243624.1) — all substantially above the 30% threshold for valid homologous docking. Molecular dynamics simulation of the KTTF–CYP51 complex (47.98 ns) confirmed binding stability: KTTF maintained hydrogen bonds with active site residues in 100% of all 4,799 analyzed frames (mean 5.7 H-bonds/frame; ≥3 H-bonds in 98.0% of frames). Pose stability analysis showed consistent binding geometry across docking modes (RMSD = 1.45 Å for KTTF in CaChs2). ADMET profiling confirmed drug-like profiles for DKPs (QED: 0.60–0.71) with no toxicity alerts. These results provide a computational foundation for valorizing T. cacao by-products as sources of multi-target antifungal candidates, warranting experimental validation through enzyme inhibition assays and in planta biocontrol trials.