Complexes of Trypanosoma cruzi sterol 14α-demethylase (CYP51) with two pyridine-based drug candidates for Chagas disease: structural basis for pathogen selectivity

Abstract

Chagas disease, caused by the eukaryotic (protozoan) parasite Trypanosoma cruzi, is an alarming emerging global health problem with no clinical drugs available to treat the chronic stage. Azole inhibitors of sterol 14α-demethylase (CYP51) were proven effective against Chagas, and antifungal drugs posaconazole and ravuconazole have entered clinical trials in Spain, Bolivia, and Argentina. Here we present the x-ray structures of T. cruzi CYP51 in complexes with two alternative drug candidates, pyridine derivatives (S)-(4-chlorophenyl)-1-(4-(4-(trifluoromethyl)phenyl)-piperazin-1-yl)-2-(pyridin-3-yl)ethanone (UDO; Protein Data Bank code 3ZG2) and N-[4-(trifluoromethyl)phenyl]-N-[1-[5-(trifluoromethyl)-2-pyridyl]-4-piperi-dyl]pyridin-3-amine (UDD; Protein Data Bank code 3ZG3). These compounds have been developed by the Drugs for Neglected Diseases initiative (DNDi) and are highly promising antichagasic agents in both cellular and in vivo experiments. The binding parameters and inhibitory effects on sterol 14α-demethylase activity in reconstituted enzyme reactions confirmed UDO and UDD as potent and selective T. cruzi CYP51 inhibitors. Comparative analysis of the pyridine- and azole-bound CYP51 structures uncovered the features that make UDO and UDD T. cruzi CYP51-specific. The structures suggest that although a precise fit between the shape of the inhibitor molecules and T. cruzi CYP51 active site topology underlies their high inhibitory potency, a longer coordination bond between the catalytic heme iron and the pyridine nitrogen implies a weaker influence of pyridines on the iron reduction potential, which may be the basis for the observed selectivity of these compounds toward the target enzyme versus other cytochrome P450s, including human drug-metabolizing P450s. These findings may pave the way for the development of novel CYP51-targeted drugs with optimized metabolic properties that are very much needed for the treatment of human infections caused by eukaryotic microbial pathogens.

Keywords: Cytochrome P450; Drug Discovery; Enzyme Inhibitors; Infectious Diseases; Protein Complexes; Protein Structure; Sterol 14α-Demethylase; Trypanosoma cruzi; X-ray Crystallography.

FIGURE 1.

Structural formulas and antiparasitic effects of pyridine derivatives UDO (EPL-BS1246) and UDD (EPL-BS0967) in comparison with the antifungal drug posaconazole and an experimental T. cruzi CYP51 inhibitor, VNI. EC₅₀, drug concentration that gives half-maximal response in cellular growth reduction; ED₅₀, drug regimen that is required to reach parasitological cure in 50% animals; IC₅₀, drug concentration required to inhibit the activity of the enzyme by 50%. *, data from Villalta et al. (22); **, data from Keenan et al. (38).

FIGURE 2.

2 Fo − Fc electron density map of T. cruzi CYP51 in complex with UDO and UDD weighted at 1.5 σ. The distance between Fe³⁺ and the pyridine ring nitrogen (Å) is marked with a dashed purple line.

FIGURE 3.

Analysis of CYP51 interaction with different iron-coordinating ligands. Shown are the absolute (upper) and difference (lower) spectra and binding curves (insets, right side). Spectral responses of: T. cruzi CYP51 to pyridine derivatives UDO (A) and UDD (B), triazole posaconazole (C), and imidazole VNI (D); L. infantum CYP51 to UDO (E) and UDD (F); and human CYP51 to UDO (G) and UDD (H). The titration experiments were performed in 5-cm optical path cuvettes at a P450 concentration 0.4 μm; titration range, 0.1–2 μm; titration steps, 0.1 μm. The binding curves show absorbance changes per 1-cm optical path/1 nmol P450.

FIGURE 4.

Inhibitory effects of UDO and UDD on CYP51 activity. Eburicol, obtusifoliol, and lanosterol, final concentration 50 μm) were used as substrates for T. cruzi, L. infantum, and human CYP51s, respectively; molar ratio of enzyme/substrate = 1/50; 1-h reaction (no substrate conversion was observed after a 5-min reaction in the presence of each of the compounds at a 1/1 molar ratio of inhibitor/enzyme). A, HPLC profiles of substrate (S) conversion by T. cruzi and L. infantum CYP51 orthologs in the presence of a 2-fold molar excess of the inhibitors over the enzyme. Posaconazole was used as a control. I1, 14α-carboxyalcohol intermediate, I2, 14α-carboxyaldehyde intermediate; P, 14α-demethylated CYP51 reaction product. Panel B, T. cruzi; C, L. infantum; D, human CYP51 activity at increasing molar excesses of UDO an UDD.

FIGURE 5.

Structural characterization of T. cruzi CYP51 complexes with UDO (3ZG2, salmon) and UDD (3ZG3, gray). A, superimposition of UDO and UDD with posaconazole (3K1O, yellow. B, superimposition of UDO and UDD with VNI (3GW9, yellow). C, overall view of the protein (rainbow color, from blue terminus) with the bound inhibitors. The P450 orientation is the same as in A and B. Surfaces of the inhibitors are shown as a semitransparent mesh. D, distal view of superimposed 3ZG2 (salmon), 3ZG3 (gray), and 3K1O (gold) ribbon diagram. In 3K1O, the structural elements forming the entrance into the substrate access channel are shown in green.

FIGURE 6.

Views of the T. cruzi CYP51 active site illustrating the interactions with UDO (salmon-colored carbon atoms) and UDD (gray-colored carbon atoms). A, binding of UDO. The inhibitor molecule is shown as a spherical model, and the carbon atoms of the side chains of the inhibitor-contacting amino acid residues are depicted as light blue sticks; the corresponding secondary structural elements are shown as semitransparent gray ribbons and numbered; the carbon atoms of the heme are in yellow. UDO-specific T. cruzi CYP51 residues are underlined. B, binding of UDD (spherical model, gray-colored carbon atoms). The carbon atoms of the side chains of the inhibitor-contacting residues and the semitransparent ribbon of the corresponding secondary structural residues are colored pink and numbered. UDD-specific T. cruzi CYP51 residues are underlined. C, comparative location of the inhibitor-contacting residues in the superimposed T. cruzi CYP51 co-structures with UDO and UDD. The P450 orientation is the same as in A and B. This is a stereo view. The side chains of the inhibitor-contacting residues remain in very similar positions, with only the long arms of Met-106 and Met-460 being shifted to the right in the UDO structure to better accommodate the piperazinyl ring of the inhibitor. D, while both of the conserved in the CYP51 family heme supporting hydrogen bonds (between the hydroxyl group of Tyr-103 and the propionate of porphyrin ring A as well as between the hydroxyl group of Tyr-116 and the propionate of porphyrin ring D are preserved in the T. cruzi CYP51 complex with UDO, binding of UDD requires wedging of its aromatic ring between Tyr-116 and the heme plane, which results in disruption of the Tyr-116 OH-porphyrin ring D hydrogen bond.

FIGURE 7.

Structural explanation for UDO/UDD pathogen selectivity. A, Phe-104 in the L. infantum CYP51 structure (PDB code 3L4D) relative to Ile-105 in the superimposed T. cruzi CYP51 (PDB code 3ZG3). At 72% average amino acid sequence identity, L. infantum and T. cruzi have only one amino acid difference in the residues in which side chains line the surface of the substrate binding cavity. B, 12 amino acid differences inside the substrate binding cavity in T. cruzi (PDB code 3ZG2) and human (PDB code 3LD3 (61)) CYP51s. UDO, UDD, the heme, and six iron coordination bonds including the thiolate of Cys-422 (the fifth (proximal) iron ligand) are shown. The color code for T. cruzi CYP51 structures is the same as described in the legend for Fig. 6. L. infantum and human CYP51s are colored in cyan and tan, respectively.