Abstract
<title>Abstract</title> <p> The rapid propagation of drug-resistant <italic>Plasmodium falciparum</italic> poses a challenge to the worldwide control of malaria, making the development of antimalarial drugs with new modes of action imperative. The antimalarial potential of metabolites from the medicinal fungus <italic>Cordyceps militaris</italic> has not been thoroughly investigated, despite the fact that natural products are still a promising source of therapeutic leads. In order to assess 42 fungal metabolites against four verified <italic>P. falciparum</italic> targets—PfDHFR-TS, PfLDH, PfMDR1, and PfAMA1—we used an integrated computational pipeline that included drug-likeness screening, molecular docking, docking validation, molecular dynamics (MD) simulations, receptor–ligand interaction analysis, and ADMET prediction. Sterol-derived metabolites showed the largest binding affinities among the evaluated substances; Brassicasterol outperformed the reference medication Artemisinin (−6.2 kcal/mol) with the highest affinity for PfDHFR-TS (−10.5 kcal/mol). While 50 ns MD simulations verified the PfDHFR-TS–Brassicasterol complex's better structural stability through favorable RMSD, RMSF, and radius of gyration profiles, interaction analysis demonstrated stable hydrophobic and π-alkyl interactions inside the active site. Promising pharmacokinetic and safety features were also revealed by ADMET prediction. Brassicasterol is a promising fungal-derived antimalarial lead for further experimental validation and drug development, according to this study, which offers the first thorough multi-target <italic>in silico</italic> evaluation of <italic>C. militaris</italic> metabolites against <italic>P. falciparum</italic> . </p>