Molecular modelling and experimental validation identified a new therapeutic inhibitor of toxoplasmosis.

Toxoplasmosis is a widespread parasitic disease, caused by Toxoplasma gondii, that affects nearly one-third of the human population. The lack of effective treatments drives the demand for novel anti-toxoplasmosis therapeutic options. In the present study, we used computational approaches and experimental validation to identify therapeutic inhibitors of toxoplasmosis. Initially, using the structure of the co-crystallized ligand of T. gondii calcium-dependent protein kinase 1 (TgCDPK1), we retrieved 3000 compounds from the database of COCONUT (COlleCtion of Open Natural ProdUcTs). These compounds were docked against the crystal structure of TgCDPK1 on the Glide Ligand Docking panel of Maestro 12.5 (Schrödinger Suite 2020-3). Based on the docking scores, we assessed promising molecules for toxicity potential on the ProTox-II online server, while the ADME profiling was done on the SwissADME server. Following the computational studies, we selected nine promising compounds for experimental validation against T. gondii in vitro. Of the compounds, C4, C5, C6, and C8 exhibited dose-dependent anti-T. gondii action with EC values ranging from 3.3 to 120.2 μg/mL. Host toxicity profiling revealed differential cytotoxic action with a selectivity index (SI) of <1 for the compounds except C5, which had an SI of 1.8. To validate our screening assay, we used sulfadiazine, a standard drug for toxoplasmosis and showed that it inhibited parasite growth. Further experiments showed that C5, an imidazole-based natural compound, has strong but reversible anti-parasitic action that peaks within the first 8 h. In addition, C5 exhibited similar toxic tendencies towards T. gondii within (intracellular) and outside (extracellular) the host, suggesting it likely has a parasite target(s). C5 showed no effect on host invasion but strongly impeded parasite replication and growth, thereby affecting the T. gondii lytic cycle. Furthermore, C5 treatment raised the reactive oxygen species level, but this may be a secondary effect because augmentation with Trolox antioxidant failed to block C5 anti-T. gondii action. In addition, molecular dynamics simulations of C5 and TgCDPK1 complex revealed relative stability within 100 ns run time. Collectively, our findings support the potential of imidazole-based compounds as novel, alternative anti-parasitic agents.