Molecular Interaction and Biological Activity of Fatty Acids and Sterols: An In Silico and In Vitro Approach Against Haemonchus contortus

<b>Background:</b><i>Haemonchus contortus</i> is a gastrointestinal nematode that affects small ruminants and exhibits widespread resistance to commercial anthelmintics. This has driven interest in natural compounds such as fatty acids and sterols; however, their biological relevance against resistant parasite strains remains insufficiently understood. <b>Methods:</b> The nematicidal potential of four fatty acids (palmitic, linoleic, pentadecanoic, and stearic acids) and two sterols (β-sitosterol and ergosterol), all of them commercially available in Mexico, was evaluated against infective L3 larvae of a benzimidazole-resistant <i>H. contortus</i> strain. <i>In vitro</i> larval mortality and migration inhibition assays were performed, and molecular docking analyses were conducted to explore interactions with the glutamate-gated chloride channel (GluCl) using AutoDock4. Statistical analyses were performed using ANOVA followed by Tukey's post hoc test (<i>p</i> < 0.05). <b>Results:</b> Molecular docking indicated strong binding affinities of ergosterol and β-sitosterol to GluCl, comparable to that of ivermectin. <i>In vitro</i> assays showed that fatty acids, particularly linoleic acid, produced more pronounced effects on larval motility, suggesting predominantly nematostatic activity. No clear dose-response relationship was observed in migration assays, and <i>in vitro</i> mortality remained limited across treatments. <b>Conclusions:</b> The results highlight a disconnect between <i>in silico</i> binding affinity and <i>in vitro</i> biological activity, particularly in a drug-resistant <i>H. contortus</i> strain. Integrating <i>in vitro</i> bioassays with computational approaches provides valuable mechanistic insight but also underscores the limitations of affinity-based predictions for assessing anthelmintic efficacy.