Comparative anthelmintic activity of artemisinin, dihydroartemisinin, and artesunate against sheep gastrointestinal nematodes: in vitro evaluation and exploratory β-tubulin molecular modeling

Gastrointestinal nematode infections are an important constraint on small-ruminant production, and increasing anthelmintic resistance has intensified the search for alternative therapeutic compounds. Artemisinin and its derivatives have demonstrated activity against several helminth species, although comparative information regarding their effects on gastrointestinal nematodes remains limited. This study compared the in vitro anthelmintic activity of artemisinin (ART), dihydroartemisinin (DHA), and artesunate (ARTE) and explored β-tubulin as a candidate molecular target using complementary computational analyses. All three compounds showed concentration-dependent activity against gastrointestinal nematode eggs and third-stage larvae. In the egg hatch assay, ARTE showed the lowest EC₅₀ value (63.45 µg/mL), followed by ART (82.02 µg/mL) and DHA (99.06 µg/mL). A similar activity pattern was observed for the predefined 24-h larval non-viability endpoint, with operational LC₅₀ values of 66.08 µg/mL for ARTE, 102.82 µg/mL for ART, and 185.41 µg/mL for DHA. Exploratory molecular docking against β-tubulin showed broadly comparable predicted binding at the colchicine-binding pocket, whereas ARTE exhibited the most favorable predicted interaction at the GTP-associated region. Molecular dynamics simulation supported the conformational stability of the predicted ARTE–β-tubulin complex over 150 ns, including persistent residue contacts and Mg²⁺ coordination. In silico ADMET profiling indicated distinct permeability and distribution characteristics among the three compounds without major structural toxicity alerts in the applied prediction tools. ARTE showed the highest in vitro anthelmintic activity among the three evaluated artemisinin derivatives, with a consistent activity ranking of ARTE > ART > DHA in both experimental assays. The computational findings provide hypothesis-generating support for a potential interaction of ARTE with the GTP-associated region of β-tubulin but do not establish β-tubulin as its molecular target. Further parasite-specific target studies, in vivo efficacy experiments, pharmacokinetic evaluation, and direct target-engagement studies are required to clarify the biological mechanism and veterinary relevance of these findings.