Repurposing of Milciclib as a Potential Inhibitor of Toxoplasma gondii in Human Trophoblasts and Placental Explants

Abstract Congenital toxoplasmosis persists as a significant public health challenge due to the limited efficacy and safety of current treatments during pregnancy. In this study, we assessed the antiparasitic activity and mechanism of action of milciclib (MCB), a cyclin-dependent kinase inhibitor from the Medicines for Malaria Venture Pathogen Box, against Toxoplasma gondii using in vitro and ex vivo models of the maternal–fetal interface. MCB exhibited no cytotoxic effects in human trophoblastic BeWo cells or placental villous explants at concentrations up to 1.62 μM. The compound significantly reduced intracellular parasite proliferation in a dose-dependent manner, achieving approximately 39.72, 73.22, and 91.52% of inhibition at 0.40, 0.81, and 1.62 μM, respectively, and maintained a persistent antiproliferative effect following drug withdrawal. Importantly, MCB also reduced parasite burden in human placental explants (approximately 47.69% of inhibition) while preserving tissue viability and architecture. Mechanistically, MCB induced G1 phase arrest in T. gondii without affecting parasite adhesion or invasion, suggesting a specific action on intracellular replication. Ultrastructural analyses revealed marked morphological alterations in treated parasites, supporting a direct antiparasitic effect. In addition, MCB modulated host cell responses by reducing macrophage migration inhibitory factor (MIF) and reactive oxygen species (ROS) production accompanied by decrease in parasite burden, without triggering a pro-inflammatory profile. In silico analyses indicated potential interactions with parasite cyclin-dependent kinase-related proteins, particularly CRK2. Together, these findings identify MCB as a promising candidate for drug repositioning in congenital toxoplasmosis and support further investigation of its efficacy and safety in vivo.