Virtual screening and in vitro validation of isoform-specific inhibitors targeting toxoplasma gondii cyclophilins.

Abstract

Cyclophilins (Cyps) regulate several biological and cellular processes and act as essential virulence factors in many parasitic pathogens, making them attractive therapeutic targets. However, the high structural conservation among cyclophilins has hindered the development of isoform-specific inhibitors. Here, we focused on TgCyp23, a cyclophilin from Toxoplasma gondii, the causative agent of toxoplasmosis. We conducted a small-scale virtual screening of small molecules and cyclic peptides, to identify selective inhibitors of TgCyp23, using human CypA (hCypA) as a selectivity control. This strategy identified several candidate compounds with potential inhibitory activity, including ziprasidone, Mant-dADP, ertapenem, bemcentinib, PMX-53 and PMX-205. Biochemical assays revealed that ziprasidone, a clinically approved drug, is a potent and selective TgCyp23 inhibitor in vitro (IC50 = 71 nM), with no detectable activity against hCypA. In contrast, Mant-dADP inhibited both enzymes with similar potency (IC50 ≈ 300 nM), indicating a lack of selectivity. NMR spectroscopy confirmed direct binding of Mant-dADP to TgCyp23, while molecular dynamics simulations supported the stability of ligand-protein complexes. Together, these results validate our computational screening pipeline, highlighting ziprasidone as a selective TgCyp23 inhibitor and establishing Mant-dADP as a valuable scaffold for future structure-based optimization.

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