Structural characterization of CYP51 from Trypanosoma cruzi and Trypanosoma brucei bound to the antifungal drugs posaconazole and fluconazole

Abstract

Background: Chagas Disease is the leading cause of heart failure in Latin America. Current drug therapy is limited by issues of both efficacy and severe side effects. Trypansoma cruzi, the protozoan agent of Chagas Disease, is closely related to two other major global pathogens, Leishmania spp., responsible for leishmaniasis, and Trypansoma brucei, the causative agent of African Sleeping Sickness. Both T. cruzi and Leishmania parasites have an essential requirement for ergosterol, and are thus vulnerable to inhibitors of sterol 14alpha-demethylase (CYP51), which catalyzes the conversion of lanosterol to ergosterol. Clinically employed anti-fungal azoles inhibit ergosterol biosynthesis in fungi, and specific azoles are also effective against both Trypanosoma and Leishmania parasites. However, modification of azoles to enhance efficacy and circumvent potential drug resistance has been problematic for both parasitic and fungal infections due to the lack of structural insights into drug binding.

Methodology/principal findings: We have determined the crystal structures for CYP51 from T. cruzi (resolutions of 2.35 A and 2.27 A), and from the related pathogen T. brucei (resolutions of 2.7 A and 2.6 A), co-crystallized with the antifungal drugs fluconazole and posaconazole. Remarkably, both drugs adopt multiple conformations when binding the target. The fluconazole 2,4-difluorophenyl ring flips 180 degrees depending on the H-bonding interactions with the BC-loop. The terminus of the long functional tail group of posaconazole is bound loosely in the mouth of the hydrophobic substrate binding tunnel, suggesting that the major contribution of the tail to drug efficacy is for pharmacokinetics rather than in interactions with the target.

Conclusions/significance: The structures provide new insights into binding of azoles to CYP51 and mechanisms of potential drug resistance. Our studies define in structural detail the CYP51 therapeutic target in T. cruzi, and offer a starting point for rationally designed anti-Chagasic drugs with improved efficacy and reduced toxicity.

Figure 1. Chemical structures of posaconazole and fluconazole.

Figure 2. Spectral characterization of CYP51_Tc and CYP51_Tb.

Soret and visible regions of the CYP51_Tc (A) and CYP51_Tb (B) spectra are shown. The ferric protein (dashed trace) was reduced with sodium dithionite to a ferrous form (solid trace) in the presence of CO. The spectra were recorded at room temperature in a 1 ml quartz cuvette containing 1 µM CYP51 in 10 mM Tris-HCl, pH 7.5, and 10% glycerol using a Cary UV-visible scanning spectrophotometer (Varian). CYP51_Tc has a Soret maximum at 420 nm which upon reduction with sodium dithionite and CO binding shifts to 449 nm (A). CYP51_Tb has a Soret maximum at 417 nm which upon reduction and CO binding shifts to 446 nm (B).

Figure 3. Overall structures of CYP51.

A, B. Fluconazole-bound CYP51_Tc with selected α-helices labeled (PDB ID Code 2WUZ). A. Distal protein surface with respect to heme. B. Image is rotated ∼90° toward viewer. Protein backbone is depicted by cyan ribbon with the I-helix highlighted in magenta, BC-region in green, FG-region in blue. Heme (orange) and fluconazole are depicted by spheres. Fluconazole color scheme: carbon yellow, oxygen red, nitrogen blue, fluorine cyan. Images were prepared using PYMOL unless indicated otherwise. C, D. CYP51_Tb in surface representation with the hydrophobic residues colored in golden yellow. In C, posaconazole is omitted for clarity, heme prosthetic group (pink) shows through the hydrophobic tunnel entrance. In D, two overlaid posaconazole conformers are shown protruding out of the tunnel opening. Bent conformer (chain B) is in red, extended conformer (chain D) is in blue. Images were generated using VMD program .

Figure 4. Comparison between CYP51 from different phyla.

A. CYP51_Tc (cyan in A, B and C, PDB ID Code 2WUZ) and CYP51_Tb (wheat, PDB ID Code 2WV2) superimposed with r.m.s.d. of 0.89 Å. Helices are represented by labeled cylinders. Fluconazole is omitted for clarity. B. CYP51_Tc and CYP51_Mt (golden yellow, PDB ID Code 2VKU) superimposed with r.m.s.d of 1.83 Å. C. CYP51_Tc and CYP51_h (lemon green, PDB ID Code: 3I3K) superimposed with r.m.s.d of 1.45 Å. In each panel, distal surface is shown on the right. Image on the left is rotated ∼90° toward viewer.

Figure 5. Fluconazole binding in CYP51_Tc.

A, B. Stereoscopic view of CYP51_Tc with fluconazole bound in active site. Side chains of the residues within 4 Å of fluconazole are in green. For clarity, A287, A291 and T295 are omitted. Main chain atoms are shown for M360. Fluconazole color scheme as in Fig. 3 . Fragments of 2F_o-F_c electron density map calculated with the fluconazole coordinates omitted from the input file are shown as grey wire mesh. Chain A has been used in both structures to generate the images. A. Fluconazole orientation 1 in 2WX2 structure; OH-group of Y103 H-bonds to the amide nitrogen of M360. B. Fluconazole orientation 2 in the 2WUZ structure; peak in the positive F_o-F_c map (pink mesh) calculated with 2,4-difluorophenyl ring in flipped orientation superimposes with the 2-fluorine H-bonding to Y103.

Figure 6. Hydrophobic tunnel.

A. Fluconazole in orientation 2 bound to CYP51_Tc. Fluorinated edge of the 2,4-difluorophenyl ring faces heme macrocycle. Fluconazole and heme are shown as van der Waals spheres; residues within 7 Å of fluconazole as sticks. Color scheme for heme and fluconazole as in Fig. 3 . F105 superimposed from CYP51_Tb is shown as semitransparent pink spheres. B. Front view of CYP51_Tc clipped by plane (cyan) through substrate binding tunnel. Hydrophobic areas are orange, hydrophilic areas blue. Heme at end of tunnel: with van der Waals spheres in red. Fluconazole is removed for clarity. Image was prepared using CHIMERA .

Figure 7. Sequence alignments between host and pathogen CYP51.

Sequence alignments between CYP51 from Trypanosoma cruzi, Trypanosoma brucei, Aspergillus fumigatus, Candida albicans and human. Accession numbers of the proteins in the Swiss-Prot/TrEMBL (http://us.expasy.org/sprot) and NCBI (http://www.ncbi.nlm.nih.gov/) databases are given next to the name of the protein. Alignments were performed using CLUSTALW program online . The figure was generated using ESPript . The secondary structure annotation and residue numbering at the top correspond to CYP51_Tc, residue numbering at the bottom corresponds to human CYP51. The α-helices are labeled with capital letters according to generally accepted P450 nomenclature. The β-strands of large β-sheets are labeled with dashed numbers. Sequential numbers are used to label short two-residue β-strands. Residues within 7 Å of fluconazole are labeled with blue triangles. Additional residues constituting the hydrophobic tunnel are labeled with green triangles. Human H236 and H489 and the corresponding residues in the pathogenic species are highlighted in yellow. Residues corresponding to CYP51_Tc I105 are highlighted in cyan. Mutation hot spots at the tunnel opening are marked with black stars. Gray stars highlight residues in alternate conformations.

Figure 8. Posaconazole conformation.

A. Fragments of the F_o-F_c electron density map (grey mesh) in chains A, B and D calculated with the posaconazole coordinates omitted from the input file. B, C. Posaconazole protruding from the tunnel entrance is shown in the chain B (B) and chain D (C). Protein backbone is depicted by yellow ribbon, protein surface is represented by black mesh, residues surrounding tunnel entrance (cyan) are in stick mode. Arrows point at the residues corresponding mutation hot spots in posaconazole resistant isolates of A. fumigatus and C. albicans. Posaconazole is shown by spheres with the carbon atoms white, oxygen red, nitrogen blue, fluorine light green. Heme in the background is orange. K50 side chain is not defined in the electron density and therefore modeled as alanine. Images were prepared using MAESTRO .

Figure 9. Posaconazole binding.

Stereoscopic view of posaconazole in chain B (A) and in chain D (B) surrounded by the side chains within 4 Å. Image in B is rotated ∼90° to the right along the vertical axis in the plane of drawing. To avoid image cluttering, four residues from this range, Y103, Y116, A211 and M360, were excluded from the view in A. Posaconazole carbons are highlighted in yellow, amino acid residues are in green. Otherwise color scheme is as in Fig. 8 .

Figure 10. Posaconazole docking.

Stereoscopic view of posaconazole docked in the active site CYP51_Tc in pose 1 (yellow) and pose 2 (pink). Residues within 4 Å of posaconazole (green) are shown. Binding ambiguity of the long posaconazole tail is likely due to the hydrophobic/aromatic stacking nature of the interactions in the substrate binding tunnel. Posaconazole and protein are shown by stick, heme by van der Waals spheres. Posaconazole carbon atoms are yellow or pink, oxygen red, nitrogen blue, sulfur yellow and fluorine pale cyan; heme is orange, For clarity, residues L208 and M360 are excluded from the view.