Targeting Trypanosoma cruzi sterol 14α-demethylase (CYP51)

Abstract

There are at least two obvious features that must be considered upon targeting specific metabolic pathways/enzymes for drug development: the pathway must be essential and the enzyme must allow the design of pharmacologically useful inhibitors. Here, we describe Trypanosoma cruzi sterol 14α-demethylase as a promising target for anti-Chagasic chemotherapy. The use of anti-fungal azoles, which block sterol biosynthesis and therefore membrane formation in fungi, against the protozoan parasite has turned out to be highly successful: a broad spectrum anti-fungal drug, the triazole compound posaconazole, is now entering phase II clinical trials for treatment of Chagas disease. This review summarizes comparative information on anti-fungal azoles and novel inhibitory scaffolds selective for Trypanosomatidae sterol 14α-demethylase through the lens of recent structure/functional characterization of the target enzyme. We believe our studies open wide opportunities for rational design of novel, pathogen-specific and therefore more potent and efficient anti-trypanosomal drugs.

FIGURE 4.1

Sterol biosynthetic pathway in T. cruzi. (A) Mevalonate portion. (B) First eukaryote-specific steps. (C) T. cruzi-specific steps.

FIGURE 4.2

Three-step catalytic reaction of T. cruzi CYP51. Each step requires molecular oxygen, two electrons and two catalytic protons. The 14α-methyl group of eburicol is converted into first the alcohol, second the aldehyde and third removed as formic acid. The substrates of CYP51s from other biological kingdoms are all structurally very similar. Eburicol also serves as the substrate for sterol 14α-demethylases in filamentous fungi. Plant and T. brucei orthologs metabolize its C4-monomethylated analog obtusifoliol. Lanosterol and 24,25-dihydrolanosterol, both lacking the methylene group at C24, are the natural substrates for the human (mammalian) enzyme.

FIGURE 4.3

Selected inhibitors and their effects on T. cruzi CYP51 activity. (A) Anti-fungal and experimental azoles; I : E : S, molar ratio inhibitor/enzyme/substrate. (B) Non-azole compounds; I/E2, inhibitor/enzyme ratio that causes a twofold decrease in activity.

FIGURE 4.4

CYP51 crystal structure. (A) Distal view, helices (grey) and β-sheets bundles (black) are shown in ribbon representation and marked. The haem is shown as a stick model. The active site cavity surface is depicted as the grey mesh. (B) Ligand-free T. brucei CYP51 (black) superimposed with VNI-bound T. brucei CYP51, posaconazole-bound T. cruzi (grey) and ketoconazole-bound human (dark grey) orthologs. Only minor alterations are seen in the CYP51 active site cavity area. (C) An example of a xenobioticmetabolizing P450, CYP2B4, in ligand-free form (black) and bound to two different ligands, 4-(4-chlorophenyl)imidazole and bifonazole (grey). Large-scale conformational changes significantly alter shape and volume of its active site pocket.

FIGURE 4.5

Specific details of inhibitor-CYP51 complexes. (A) Surface binding subsite in posaconazole-bound T. cruzi CYP51 (surface representation, I-helix is shown). (B) The carboxamide fragment of VNI forms a hydrogen-bond network with T. brucei CYP51 helices B′ and I. The haem is shown as spheres. (C) VNF binds in the orientation opposite to VNI, its long arm being directed deeper into the active site cavity.

FIGURE 4.6

Active site forming residues in T. cruzi (black) and human (grey) CYP51s. The corresponding secondary structural elements in T. cruzi are depicted as the transparent ribbon and marked, the haem is seen as ball and stick model. The active site cavity is outlined as grey mesh. The cavity residues which are conserved the human and T. cruzi enzymes are shown in line representation. Some of the residues conserved in the whole CYP51 family are marked (T. cruzi CYP51 numbering). The active site residues that differ in the two proteins are displayed as stick models.

FIGURE 4.7

Cellular effects of VNI/VNF in T. cruzi. (A) TLC of sterol standards (1) and unsaponified lipids extracted from T. cruzi amastigotes, untreated (2) and treated with 1 µM VNI (3). Eb, eburicol; Ob, obtusifoliol; Ch, cholesterol (exogenous); Er, ergosterol. (B) Scanning electron microscopy of T. cruzi amastigotes treated with 1 µM VNI; bar = I µm. Membrane disruption is seen as blebs on the surface. (C) Anti-parasitic effect of VNF (1 µM) on T. cruzi amastigotes within cardiomyocytes (GFP-expressing transgenic T. cruzi) is seen as small light dots, cardiomyocytes nuclei are seen as circles. (D) CYP51 gene expression in T. cruzi, immunoblotting. (E) CYP51 gene expression upon treatment of T. cruzi with 1 µM CYP51 inhibitors. C, control. (F) Inhibition of T. cruzi growth with different concentrations of posaconazole and VNF.