Development of Selective Steroid Inhibitors for the Glucose-6-phosphate Dehydrogenase from Trypanosoma cruzi

Abstract

Chagas disease is a parasitic infection affecting millions of people across Latin America, imposing a dramatic socioeconomic burden. Despite the availability of drugs, nifurtimox and benznidazole, lack of efficacy and incidence of side-effects prompt the identification of novel, efficient, and affordable drug candidates. To address this issue, one strategy could be probing the susceptibility of Trypanosoma parasites toward NADP-dependent enzyme inhibitors. Recently, steroids of the androstane group have been described as highly potent but nonselective inhibitors of parasitic glucose-6-phosphate dehydrogenase (G6PDH). In order to promote selectivity, we have synthesized and evaluated 26 steroid derivatives of epiandrosterone in enzymatic assays, whereby 17 compounds were shown to display moderate to high selectivity for T. cruzi over the human G6PDH. In addition, three compounds were effective in killing intracellular T. cruzi forms infecting rat cardiomyocytes. Altogether, this study provides new SAR data around G6PDH and further supports this target for treating Chagas disease.

Figure 1

Endogenous steroid hormone DHEA (1) and synthetic analogues (2–7).

Scheme 1. Synthesis of EA (2) and BrEA (3) Derivatives Comprising: (A) Aliphatic and Heterocycle-Containing Esters; (B) Aliphatic and Heterocycle-Containing Ethers; (C) Nonhydrolyzable Moieties

Target compounds are highlighted in blue rectangles.

Figure 2

SAR analysis of EA (2) and BrEA (3) derivatives against the parasitic and human G6PDHs. Affinities are shown as pIC50 values, where IC50 is expressed in μM (pIC50 = −log(IC50.10^–6). The green dotted triangle represents highly Tc-selective compounds, defined by an IC50_Hs/IC50_Tc ratio >40. Moderate Tc-selectivity (5 < ratio <30) is represented by a dotted orange rectangle. The light green filled rectangle covers BrEA derivatives, whereas the light orange filled rectangle represents EA derivatives. The chart was generated with the DataWarrior software (Osiris).

Figure 3

Anti-T. cruzi intracellular assay at single concentration of 20 μM. Black and gray bars represent mean and standard deviation of normalized total host and infected cells, respectively. DMSO values were used for data normalization.

Figure 4

Dose–response curves for normalized host (black) and infected cells (gray) for BNZ, compounds 32, 15, and 40. Plots prepared with GraphPad Prism.

Figure 5

Image analysis of anti-T. cruzi intracellular assay of compounds 32, 15, and 40 (10 μM). Mean and standard deviation for total host cells (A), infected cells (B), and infection ratio (C) per image (N = 15). (D) Representative images (left) and zoom from blue boxes (right) for DMSO, BNZ, and compounds 32, 15, and 40. Host cell nuclei are colored in yellow, and T. cruzi amastigotes appear as white spots in the cytoplasm of infected cells (indicated by blue arrows).