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. 2012;6(7):e1736.
doi: 10.1371/journal.pntd.0001736. Epub 2012 Jul 31.

Diverse Inhibitor Chemotypes Targeting Trypanosoma Cruzi CYP51

Free PMC article

Diverse Inhibitor Chemotypes Targeting Trypanosoma Cruzi CYP51

Shamila S Gunatilleke et al. PLoS Negl Trop Dis. .
Free PMC article


Background: Chagas Disease, a WHO- and NIH-designated neglected tropical disease, is endemic in Latin America and an emerging infection in North America and Europe as a result of population moves. Although a major cause of morbidity and mortality due to heart failure, as well as inflicting a heavy economic burden in affected regions, Chagas Disease elicits scant notice from the pharmaceutical industry because of adverse economic incentives. The discovery and development of new routes to chemotherapy for Chagas Disease is a clear priority.

Methodology/principal findings: The similarity between the membrane sterol requirements of pathogenic fungi and those of the parasitic protozoon Trypanosoma cruzi, the causative agent of Chagas human cardiopathy, has led to repurposing anti-fungal azole inhibitors of sterol 14α-demethylase (CYP51) for the treatment of Chagas Disease. To diversify the therapeutic pipeline of anti-Chagasic drug candidates we exploited an approach that included directly probing the T. cruzi CYP51 active site with a library of synthetic small molecules. Target-based high-throughput screening reduced the library of ∼104,000 small molecules to 185 hits with estimated nanomolar K(D) values, while cross-validation against T. cruzi-infected skeletal myoblast cells yielded 57 active hits with EC(50) <10 µM. Two pools of hits partially overlapped. The top hit inhibited T. cruzi with EC(50) of 17 nM and was trypanocidal at 40 nM.

Conclusions/significance: The hits are structurally diverse, demonstrating that CYP51 is a rather permissive enzyme target for small molecules. Cheminformatic analysis of the hits suggests that CYP51 pharmacology is similar to that of other cytochromes P450 therapeutic targets, including thromboxane synthase (CYP5), fatty acid ω-hydroxylases (CYP4), 17α-hydroxylase/17,20-lyase (CYP17) and aromatase (CYP19). Surprisingly, strong similarity is suggested to glutaminyl-peptide cyclotransferase, which is unrelated to CYP51 by sequence or structure. Lead compounds developed by pharmaceutical companies against these targets could also be explored for efficacy against T. cruzi.

Conflict of interest statement

The authors have declared that no competing interests exist.


Figure 1
Figure 1. High throughput assay overview.
T. cruzi CYP51 target is shown clipped by plane through the substrate binding tunnel (top, center). Type II difference spectrum is recorded for 1 µM CYP51 at saturated concentration of reference compound (top, right). Screen plates were prepared in duplicate, A and B, with target protein loaded only in A set (left). HTS scatter plot shows absorbance differences of test compounds (ΔA = A425−A390) measured using the two-wavelength detection mode; each point represents a single test compound (bottom, center). Overlapped spectra are shown for a sub-micromolar hit re-evaluated in spectral mode at four serial dilutions (bottom, right).
Figure 2
Figure 2. Scoring positive hits in spectral mode.
Absorbance was monitored at four serial dilutions for each tested compound. (A) Lack of characteristic spectra-score 0; (B) concentration dependence with no spectra overlap – score 1; (C) any two spectra overlap-score 2; (D) any three spectra overlap-score 3; (E) three highest concentrations overlap-score 4; (F) all four spectra overlap-score 5.
Figure 3
Figure 3. Cross-validation of hit compounds in intracellular T. cruzi amastigotes.
Segmented images of DAPI-stained T.cruzi-infected myoblasts processed with image processing software are shown for cells cultured after 72 h treatment with Compounds 1 (C, E, G) or 13 (D, F, H) at indicated concentrations. Nuclei of host cells are highlighted in blue and nuclei and kinetoplasts of parasites are highlighted in red. The inserts show original DAPI staining in blue. Note the elimination of intracellular amastigotes at 0.123 µM and 10 µM of Compound 1 (E, G) and 10 µM of Compound 13 (H). In the negative control, DMSO-treated myoblasts show abundant parasitemia (A). Posaconazole was used as a positive control (B). Bars = 20 µM.
Figure 4
Figure 4. Nitrogen-containing aromatic heterocyclic pharmacophores.
Distribution of nitrogen-containing aromatic heterocyclic pharmacophores among 185 positive hits with binding score 4 or 5 (A) echoes their frequency in the library (B). R represents diverse chemical structures as shown in Table S3.
Figure 5
Figure 5. Sub-micromolar T. cruzi inhibitors.
aEC50 values obtained against T. cruzi parasites in HTS assay used to rank order the hits. bIn parenthesis are EC50 averaged from two independent cross-validation assays for individually repurchased hits.
Figure 6
Figure 6. Comparative analysis of spectral (blue) vs. T. cruzi-active (red) hits.
Distribution of the (A) logP and (B) molecular weight (MW) values.
Figure 7
Figure 7. Hit validation in binding assay.
(A) Hit compound structures. Compound 13 shows structural similarity to miconazole. Stoichiometric binding of 1 (B) and 13 (C) to CYP51 at 0.5 µM by UV-vis titration suggests low nanomolar binding affinity as approximated by quadratic tight-binding equation (1).
Figure 8
Figure 8. Highest rated hit compound cluster.
aEC50 values obtained against T. cruzi parasites in HTS assay used to rank order the hits. bIn parenthesis are EC50 averaged from two independent cross-validation assays for individually repurchased hits. N/A – not applicable; N/D – not determined.
Figure 9
Figure 9. Second highest rated hit compound cluster.
aEC50 values obtained against T.cruzi parasites in HTS assay used to rank order the hits. bIn parenthesis is the EC50 obtained in validation assays for individually repurchased hits. N/A - not applicable; N/D - not determined.
Figure 10
Figure 10. GC-MS analysis of the T. cruzi lipid extracts treated with the CYP51 inhibitors.
DMSO and K777 were used as negative controls; posaconazole served as a positive control. Uninfected host cell panel (top) demonstrates that chromatographic peaks labeled with small-case letters from a to i are of T. cruzi origin, corresponding to a - cholesta-7,24-dien-3β-ol, [M]•+ = m/z 454; b - cholesta-8,24-dien-3β-ol (zymosterol), [M]•+ = m/z 470; c - 24-methyl-7-en-cholesta-en-3β-ol, [M]•+ = m/z 472; d - ergosta-7,24-diene-3β-ol (episterol), [M]•+ = m/z 470; e - ergosta-8,24-diene-3β-ol (fecosterol), [M]•+ = m/z 470; f - lanosterol, [M]•+ = m/z 498; g - 4-methylepisterol, [M]•+ = m/z 484; h - eburicol, [M]•+ = m/z 512; i - 24-ethyl-7,24(24′)-en-cholesta-dien-3β-ol, [M]•+ = m/z 484. Cholesterol is the only peak originating from host cells. Increase in the CYP51 substrates lanosterol (f) and eburicol (h) is due to inhibition of T. cruzi CYP51, as is the decline in the downstream major products episterol (d), fecosterol (e), cholesta-7,24-dien-3β-ol (a) and the minor products 4-methyl-episterol (g) and 24-ethyl-7,24(24′)en-cholestadien-3β-ol (i).
Figure 11
Figure 11. Flowchart diagram of screening and validation steps.

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