Trypanosoma cruzi CYP51 Inhibitor Derived from a Mycobacterium tuberculosis Screen Hit

Enzyme sterol 14α-demethylase (CYP51) is a well-established target for anti-fungal therapy and is a prospective target for Chagas' disease therapy. We previously identified a chemical scaffold capable of delivering a variety of chemical structures into the CYP51 active site. In this work the binding modes of several second generation compounds carrying this scaffold were determined in high-resolution co-crystal structures with CYP51 of Mycobacterium tuberculosis. Subsequent assays against CYP51 in Trypanosoma cruzi, the agent of Chagas' disease, demonstrated that two of the compounds bound tightly to the enzyme. Both were tested for inhibitory effects against T. cruzi and the related protozoan parasite Trypanosoma brucei. One of the compounds had potent, selective anti–T. cruzi activity in infected mouse macrophages. This compound is currently being evaluated in animal models of Chagas' disease. Discrimination between T. cruzi and T. brucei CYP51 by the inhibitor was largely based on the variability of a single amino acid residue at a critical position in the active site. Our work is aimed at rational design of potent and highly selective CYP51 inhibitors with potential to become therapeutic drugs. Drug selectivity to prevent host–pathogen cross-reactivity is pharmacologically important, because CYP51 is present in human host

Estriol Bound and Ligand-free Structures of Sterol 14α-Demethylase

AbstractSterol 14α-demethylases (CYP51) are essential enzymes in sterol biosynthesis in eukaryotes and drug targets in antifungal therapy. Here, we report CYP51 structures in ligand-free and estriol bound forms. Using estriol as a probe, we determined orientation of the substrate in the active site, elucidated protein contacts with the invariant 3β-hydroxy group of a sterol, and identified F78 as a key discriminator between 4α-methylated and 4α,β-dimethylated substrates. Analysis of CYP51 dynamics revealed that the C helix undergoes helix-coil transition upon binding and dissociation of a ligand. Loss of helical structure of the C helix in the ligand-free form results in an unprecedented opening of the substrate binding site. Upon binding of estriol, the BC loop loses contacts with molecular surface and tends to adopt a closed conformation. A mechanism for azole resistance in the yeast pathogen Candida albicans associated with mutations in the ERG11 gene encoding CYP51 is suggested based on CYP51 protein dynamics

Crystallographic data and statistics.

a<p>Numbers in parentheses correspond to the highest resolution shell.</p>b<p>R_sym = Σ|<i>I_i</i>−〈<i>I</i>〉|/Σ<i>I_i</i>, where <i>I_i</i> is the intensity of the <i>i</i>^th observation, and 〈<i>I</i>〉 is the mean intensity of reflection.</p>c<p>R_cryst = Σ∥Fo|−|Fc∥/Σ|Fo|, calculated with the working reflection set. R_free is the same as R_cryst but calculated with the reserved reflection set.</p>d<p>Program PROCHECK <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000372#pntd.0000372-Laskowski1" target="_blank">[28]</a>, portions of the protein residues in most favored/additional allowed/generously allowed regions.</p

Overall view of compound binding in the active site.

<p>Compounds 11 (A), 9 (B), and 8 (C) (highlighted in <i>pink</i>) bound in the active site of CYP51_Mt are shown looking in from the active site opening. For clarity only one conformation of each compound is shown. Protein is represented by the semitransparent accessible surface (<i>gray</i>). The ordered BC-loop obstructs the view in the CYP51_Mt-8 complex in (C). The invariable elements of the CYP51 active site, Y76, H259 (<i>yellow</i>), and heme (<i>green</i>), are in a stick mode. Water molecules are shown as <i>red spheres</i>. Oxygen atoms are <i>red</i>, nitrogen <i>blue</i>, sulfur <i>yellow</i>. Images were generated using the VMD program <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000372#pntd.0000372-Humphrey1" target="_blank">[35]</a>.</p

Inhibition of <i>T. cruzi</i> intracellular amastigotes by compound 10.

<p><i>T. cruzi</i> intracellular multiplication was evaluated at 52 hr of incubation at several concentrations of the inhibitor by determining the number of parasites/cell. Intracellular parasites were counted per one hundred cells to estimate a mean number of parasites per cell. Approximation of concentration dependence of mean P/cell±SD data with a smooth curve highlights the 50% drop in parasite count at ∼1 nM compound 10. SD did not exceed 14% of the mean.</p

Spectroscopic binding of compounds.

<p>(A) Type II spectral responses of CYP51_Tc to increasing concentrations of compound 10. The concentration dependence of compound 10, fluconazole (B), and compound 8 (C) binding were deduced from the difference absorption changes obtained from the titration of CYP51_Tc with increasing concentrations of the inhibitor. The concentration dependence of fluconazole (D) was deduced from the difference absorption changes obtained from the titration of CYP51_Tb.</p

Toxicity in mammalian cells.

<p>Toxicity in mammalian cells.</p

Stereo view of compounds in the active site.

<p>Compounds 11 (A), 9 (B), and 8 (C) are shown surrounded by the CYP51 active site residues. The fragments of the electron density 2Fo-Fc map (<i>gray mesh</i>) are cut at 1.2 σ. Different conformers in (A) and (B) are highlighted in <i>pink</i> and <i>cyan</i>. Images were generated using PYMOL <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000372#pntd.0000372-DeLano1" target="_blank">[36]</a>.</p

Spectral characteristics of CYP51_Tc (A) and CYP51_Tb (B).

<p>Main panel shows the absolute protein spectrum, while insert shows CO-bound reduced difference spectrum.</p