Abstract
Raltitrexed is a classical antifolate drug with antimetabolite and anticancer properties. In this research, we provide its detailed antitrypanosomal inhibition against six Trypanosoma species and investigate its potential mode of action. Molecular dynamics (MD) simulations and in silico analyses were used to track the binding strength and stability. Raltitrexed showed broad-spectrum trypanocidal actions against Trypanosoma brucei brucei GUTat3.1, T. b. rhodesiense IL1501, T. b. gambiense IL1922, T. evansi Tansui, T. equiperdum IVM-t1 and T. congolense IL3000. The estimated IC50 was found to be in the range of 5.18-24.13 mu g/mL, indicating inhibition of Trypanosoma in the low micromolar range. Although the co-crystallized ligand had robust hydrogen bonding and lipophilic characteristics, its docking score was only -4.6 compared to raltitrexed's -7.78, indicating strong binding with T. brucei dihydrofolate reductase-thymidylate synthase (TbDHFR-TS). MD simulations support the strong binding of raltitrexed with TbDHFR-TS evidenced by low root mean square deviation (RMSD), low residues fluctuations, a tight radius of gyration (ROG) and an average of 3.38 +/- 1.3 hydrogen bonds during 50 ns MD simulation. The prospective extended spectrum of raltitrexed against Trypanosoma species grants further research for the synthesis of raltitrexed derivatives and repurposing against other protozoa.