Inhibition of L-threonine dehydrogenase from Trypanosoma cruzi reduces glycine and acetate production and interferes with parasite growth and viability

Abstract

Trypanosoma cruzi is a flagellated protozoan and the etiological agent of Chagas disease, a neglected tropical disease described by Carlos Chagas in 1909 that remains without appropriate diagnostics and treatment. Throughout its life cycle, T. cruzi undergoes through many different environments, requiring adaptation of its metabolism to different nutrition sources. Recent studies have confirmed the adaptability of T. cruzi metabolism to different carbon sources and encouraged a deeper investigation of related metabolic pathways. In the present study, we investigated the catabolism of threonine in T. cruzi epimastigotes cultivated in LIT medium and following 24h of starvation in PBS. In LIT medium, threonine, serine, and histidine were rapidly consumed concomitantly with carbohydrates during parasite exponential growth. When threonine was provided as the only carbon source to starved parasites, they excreted acetate and glycine, corroborating the activity of a mitochondrial threonine degradation pathway. Subsequently, we used a recombinant T. cruzi L-threonine dehydrogrenase (TcTDH) to screen the Chagas Box, an open-source collection of phenotypic hits, and identified compound TCMDC-143160 as a low micromolar TcTDH inhibitor (IC50 = 3.5 μM). When TCMDC-143160 was administrated to starved parasites, it inhibited the threonine degradation pathway. Finally, we report the crystal structure of TcTDH and characterize its allosteric activation by potassium. Collectively, these data demonstrate the relevance of threonine catabolism in T. cruzi metabolism and provide a set of tools to further investigate TcTDH as a potential drug target for Chagas disease.

Keywords: Chagas Box; drug discovery; metabolism; potassium binding; substrate-level phosphorylation; target identification.

Figure 1

Exometabolomics of T. cruzi epimastigotes growing in LIT medium for 13 days. A, growth curve of T. cruzi epimastigotes in LIT medium. Exponential and stationary phases are colored blue and red, respectively. B–D, carbohydrates and amino acids consumed during parasite growth. E, amino acids excreted during the exponential growth phase and consumed in the stationary phase. F, main end-products of T. cruzi metabolism. G, other amino acids whose concentration increase over time. Dots represent the means of three technical replicates prepared from the same culture flask at distinc time points.

Figure 2

Metabolites secreted by T. cruzi under nutrient restriction conditions. 1 × 10⁷ parasites growing in LIT medium were submitted to 24h starvation in PBS and then rescued with 5 mM of different nutrients (A–E). After additional 24h, metabolites in the extracellular medium were quantified by ¹H-NMR and the parasite redox state evaluated by resazurin reduction assay (F). Multiple comparisons of normalized resazurin reduction means of distinct nutrients treatment versus PBS were conducted by ordinary one-way ANOVA followed by Dunnett's test for significance (p < 0.05 for a significant difference). Bars and lines indicate the means and SD of three independent experiments.

Figure 3

Effect of Qc1 on TcTDH enzymatic activity and in vitro parasite growth. A, Qc1 inhibition of TcTDH enzymatic activity. B, Comparison of Qc1 and Benznidazole, the reference drug for Chagas disease, on T. cruzi epimastigotes viability assay. Effect of Qc1 (C) and Benznidazole (D) on T. cruzi intracellular image-based assay. Blue dots represent the total number of host cells (H9c2 rat cardiomyocytes) imaged per well, while red dots account for the infection rate, calculated as the ratio of cells containing at least three amastigotes in the cytoplasm area. Representative images of T. cruzi infected host cells, after 72h treatment with Qc1 (E) and Benznidazole (F), both at 20 μM. Intracellular parasites are observed as small dots around the larger host nuclei. All the experiments were performed in biological triplicate.

Figure 4

Screening of ChagasBox for inhibitors of T. cruzi TDH. A, correlation plot between two screening assays of Chagas Box compounds against TcTDH activity. In both runs, compounds were assayed at 10 μM, and the two hits that reduced TcTDH activity to less than 20% (in red) were selected for further characterization. B, Inhibition of TcTDH by different concentrations of TCMDC-143160 (purple) and TCMDC-143463 (green). C, structure of TcTDH inhibitors, IC50 (and 95% confidence interval) values for TcTDH inhibition and EC50 values for T. cruzi amastigotes growth inhibition as previously reported (17). D, inhibition of T. cruzi epimastigotes viability by different concentrations of TCMDC-143160 (EC50 = 31 μM, 95%CI: 29–34 μM). E, concentration of acetate and glycine, end-products of L-threonine catabolism, excreted by starved T. cruzi epimastigotes feed exclusively with 5 mM L-threonine in the presence of 20 μM of TCMDC-143160 or 0,5% DMSO as a control (n = 3, paired t test, significant difference for p < 0.05).

Figure 5

TcTDH activation by potassium and inhibition by TCMDC143160. A, effect of different salts (50 mM) on TcTDH activity. B, effect of different concentrations of KCl on TcTDH activity. (C) and (D) TcTDH Michaelis-Menten plots for L-threonine and NAD⁺ at different concentrations of KCl. (E) and (F) Double-reciprocal plots of TcTDH inhibition at different concentrations of TCMDC-143160 measured for both L-threonine and NAD⁺.

Figure 6

Crystal structure of T. cruzi L-threonine dehydrogenase.A, monomer organization of TcTDH (pdbcode, 8gjb). Cofactor domain is formed by segments N1, N2 and N3, while the catalytic domain comprises segments C1, C2 and C3. B, TcTDH quaternary structure highlighting the different orientations of loop1, which control the access of NAD⁺ and acetate (in balls) to the catalytic site. C, surface representation of chains A and B with ligands (in balls) and loop-1 in red. D, superposition of chain A and B. Protomers were superimposed by the N-terminal domains. The largest Cα – Cα distances are observed for loop-1 residues. E, Potassium coordination and (F) remote switch region around the potassium binding site. Figures were prepared in PYMOL v.2.5 (Schrodinger, LLC, New York, NY, USA).

Figure 7

Mitochondrial pathways associated to threonine, alanine, histidine and glucose catabolism by T. cruzi epimastigotes under nutrient restrict condition. The filled and dashed boxes indicate consumed and excreted metabolites, respectively. L-threonine dehydrogenase (TDH), 2-amino-3-ketobutyrate CoA ligase (KBL), pyruvate dehydrogenase (PDH), acetate:succinate CoA-transferase (ASCT), succinyl-CoA synthetase (SCoAS), α-ketoglutarate dehydrogenase, (KDH) and glutamate dehydrogenase (GDH). (created with Affinity Designer (Version 1.10.6.1665), Serif Ltd).