Evaluating the potential of non-immunosuppressive cyclosporin analogs for targeting Toxoplasma gondii cyclophilin: Insights from structural studies

Abstract

Toxoplasmosis persists as a prevalent disease, facing challenges from parasite resistance and treatment side effects. Consequently, identifying new drugs by exploring novel protein targets is essential for effective intervention. Cyclosporin A (CsA) possesses antiparasitic activity against Toxoplasma gondii, with cyclophilins identified as possible targets. However, CsA immunosuppressive nature hinders its use as an antitoxoplasmosis agent. Here, we evaluate the potential of three CsA derivatives devoid of immunosuppressive activity, namely, NIM811, Alisporivir, and dihydrocyclosporin A to target a previously characterized cyclophilin from Toxoplasma gondii (TgCyp23). We determined the X-ray crystal structures of TgCyp23 in complex with the three analogs and elucidated their binding and inhibitory properties. The high resolution of the structures revealed the precise positioning of ligands within the TgCyp23 binding site and the details of protein-ligand interactions. A comparison with the established ternary structure involving calcineurin indicates that substitutions at position 4 in CsA derivatives prevent calcineurin binding. This finding provides a molecular explanation for why CsA analogs can target Toxoplasma cyclophilins without compromising the human immune response.

Keywords: Toxoplasma gondii; X‐ray crystal structure; cyclophilin inhibitors; cyclophilins; cyclosporin A; peptidyl‐prolyl isomerases.

FIGURE 1

Chemical structure of Cyclosporin A (CsA), NIM811, dihydrocyclosporin A (dhCsA), and Alisporivir (Ali). Modifications are highlighted in blue.

FIGURE 2

Crystallographic structures of TgCyp23‐ligand complexes. (a) Overall structure of TgCyp23 complexes. The TgCyp23 protein (as observed in chain B of the TgCyp23:dhCsA complex) is shown in gray cartoon while NIM811, dhCsA, and Alisporivir are represented in sticks (purple, pink, and cyan, respectively). N indicates the N‐terminus region and C the C‐terminus. Secondary structural elements are labeled. (b) Structural superimposition of the three non‐immunosuppressive compounds bound to TgCyp23. The TgCyp23 protein (as observed in chain A of the TgCyp23:NIM811 complex) is shown as a gray surface, NIM811, dhCsA, and Alisporivir are represented in sticks (purple, pink, and cyan, respectively). (c) 2Fo‐Fc electron density maps observed for the three ligands. Maps are contoured at 1σ. A detailed view of each ligand's modification compared to CsA is shown on the right side following the same color's legend.

FIGURE 3

Comparison of loops L108–140 and L144–156 between TgCyp23:CsA and TgCyp23:Alisporivir complexes. Both structures are aligned and displayed in cartoon, TgCyp23:CsA in orange and TgCyp23:Ali in cyan. CsA and Alisporivir are also displayed in orange and cyan sticks, respectively. Thr117 is labeled with one‐letter code.

FIGURE 4

NMR analysis of the interaction between TgCyp23 and CsA analogs. (a) ¹H‐¹⁵N‐HSQC spectra of TgCyp23 in the absence (black) and in the presence of a two‐fold molar excess of CsA (red), dhCsA (violet), NIM811 (green), and Alisporivir (blue). Selected peaks are indicated. (b) Residue specific chemical shift perturbation (CSP) analysis of the interaction observed upon binding of the three compounds to ¹⁵N‐TgCyp23. Secondary structure elements are shown on the top of the panels (α‐helix in gray, β‐sheets in black). The color code is as in panel (a).

FIGURE 5

Chemical shift perturbation difference between TgCyp23 in complex with CsA or Alisporivir. (a) Superposition of ¹H‐¹⁵N‐HSQC spectra of TgCyp23:CsA (red) and TgCyp23:Alisporivir (blue). (b) Mapping of the chemical shift perturbation analyzed in panel (a) on TgCyp23 structure in complex with Alisporivir. The most affected residues are reported in orange.

FIGURE 6

Effects of CsA analogs on TgCyp23 properties. (a) Pseudo‐first order kinetics of the binding reaction between TgCyp23 versus different concentrations of CsA and CsA analogs. (b) DSC data for denaturation of TgCyp23 in the absence and presence of CsA analogs. (c) CD thermal denaturation profiles of TgCyp23 in the absence and presence of CsA analogs. (d) PPIase activity of TgCyp23 in the presence of increasing amounts of CsA analogs. The color code in all panels is as follows: TgCyp23 alone (black), TgCyp23 in the presence of CsA (red), NIM811 (green), Alisporivir (blue), and dhCsA (violet).

FIGURE 7

Interaction basis of CypA‐CsA‐Calcineurin human complex (PDB code 1M63) and potential impact of CsA analogs. (a) Assembly of the human ternary complex, proteins CypA, CnA, and CnB are displayed as a surface (gray, yellow, and blue respectively). CsA is presented in orange spheres. (b, c) Hydrophobic pocket of Cn (residues from CnA and CnB in yellow and blue sticks/spheres, respectively). MeLeu‐4 (CsA), in orange sticks/spheres, is tightly fitted into the cavity. (d) TgCyp23:NIM811 complex superposition onto the ternary Cn–CypA–CsA human complex (CsA and CypA have been omitted for clarity reasons). (e) Predicted clashes between MeIle‐4 (NIM811), and Trp352 (CnA). NIM811 is depicted in purple sticks. (f) Predicted clashes between NEV‐4 (Alisporivir), Trp352 (CnA), and Phe356 (CnA). Alisporivir is painted in cyan sticks.