Table 1.
EC50 concentrations calculated by alamar Blue for several trypanosomatids.
Fig 1.
Metabolic changes associated with AN5568 treatment.
A) A volcano plot showing the global metabolic changes associated with AN5568 treatment. Numbers at red dots correspond to the following metabolites that were significantly increased 1) S-adenosyl-L-methionine (m/z: 398.1374, RT: 18.02 min, 7.67-fold), 2) 1,2-Dihydroxy-5-(methylthio)pent-1-en-3-one (m/z: 162.0350, RT: 17.24 min, 46.33-fold), 3) 5’-methylthioadenosine (m/z: 297.0896, RT: 7.75 min, 6.17-fold), 4) N6-acetyl-L-lysine (m/z: 188.1162, RT: 14.70 min, 2.83-fold), 5) adenine (m/z: 135.0546, RT: 7.71 min, 12.55-fold), 6) 4-hydroxy-4-methylglutamate (m/z: 177.0637, RT: 15.3 min, 3.46-fold), 7) N6,N6,N6-trimethyl-L-lysine (m/z: 188.1524, RT: 23.54 min, 7.40-fold), 8) cyclic ADP-ribose (m/z: 541.0608, RT: 16.90 min, 43.78-fold), 9) aminoacetone (m/z: 73.0528, RT: 7.67 min, 12.06-fold), 10) 8-amino-7-oxononanoate (m/z: 187.1208, RT: 13.75 min, 8.43-fold). Numbers at blue dots correspond to the following metabolites that were significantly decreased: 1) [PC(14:0)] 1-tetradecanoyl-sn-glycero-3-phosphocholine (m/z: 467.3014, RT: 4.75 min, 0.35-fold), 2) sn-glycerol 3-phosphate (m/z: 172.0136, RT: 16.44 min, 0.40-fold), 3) D-glucosamine 6-phosphate (m/z: 259.0457, RT: 17.68 min, 0.47-fold), 4) 2-deoxy-D-ribose 5-phosphate (m/z: 214.0242, RT: 16.45 min, 0.39-fold), 5) Asp-Asp-Cys-Pro (peptide) (m/z: 448.1256, RT: 17.64 min, 0.22-fold). These metabolites all have p-values of <0.05. B) Plots of individual metabolites perturbed after AN5568 treatment. There was an enrichment in metabolites involved in L-methionine metabolism. WT; wild type untreated, AN5568; wildtype treated for six hours at 10x EC50. C) A schematic to show the metabolites involved in methyltransferase reactions. Red ‘x’ indicates the typical S-adenosyl-L-methionine-dependent methyltransferase reaction potentially affected by the benzoxaborole.
Fig 2.
Metabolic changes in PCF T. b. brucei after AN5568 treatment.
Plots of individual metabolites perturbed after AN5568 treatment. WT; wild-type untreated, AN5568; wildtype treated for eight hours at 15 μM (10x EC50).
Fig 3.
Comparative analyses of T. b. brucei treated with AN5568 and sinefungin, a non-specific methyltransferase inhibitor.
A) A fixed-ratio isobologram analysis was conducted using sinefungin and AN5568. B) Comparative metabolomics analysis was carried out to assess any common metabolic alterations between sinefungin- and AN5568-treated cells. A principal component analysis (PCA) plot of the 3 sample group was generated. C) Individual metabolites were compared in terms of their fold change over a control group. D) AN5568 treatment leads to increased abundance in components of the trypanothione biosynthesis pathway, whilst sinefungin treatment does not.
Fig 4.
Tracing 13C distribution in AN5568-treated cells incubated with 13C-U-L-methionine.
Cells were incubated with 200 μM [U-13C]-L-methionine for 6 hours. In addition, one sample group was incubated with 1.9 μM (10× EC50) AN5568. Metabolites involved in L-methionine metabolism detected in this experiment were overlaid onto metabolic maps. In cases where 13C isotopes were detected, graphs incorporating the percentage labelling are shown. The methionine salvage pathway (Yang cycle) is shown.
Fig 5.
VSG biosynthesis in AN5568-treated cells.
A) Three metabolites, all known to be involved in glycoprotein biosynthesis, were found to be at higher levels in AN5568-treated cells. B) Western blots carried out on protein lysates taken from AN5568-treated cells at a 6-hour time-point showed no significant changes in VSG expression at this time-point. When the contrast of the resulting blot was increased, an extra band was seen in the 2× EC50-treated sample. C) In addition to VSG expression, blots were probed with a VSG cross-reacting determinant (VSG XR) antibody. For this experiment, protein lysates were fractionated to isolate the membrane fraction and separate it from the soluble fraction. Most of the cellular GPI is found in the membrane, and no changes in expression could be seen in cells treated with 10× EC50 for 6 hours. WT; untreated wild-type cells, AN5568; wild-type cells treated with AN5568 for 6 hours at 10× EC50.
Fig 6.
Comparing metabolic profiles of DTT and AN5568 treated cells.
A heatmap was generated to highlight similarities and differences in metabolism after treatment with the ER stress inducer dithiothreitol (DTT) and AN5568. A) AdoMet metabolism was unaffected by DTT-treatment. B) Components of the oxidative stress response pathway were altered after both treatments. In particular, DTT treatment led to a significant decrease in trypanothione disulphide. C) Both DTT and AN5568 treatment result in increased levels of modified lysines and arginines, suggesting these are part of a conserved trypanosomatid stress response. WT; wild-type untreated, AN5568; wild-type cells treated with AN5568 for 6 hours at 10× EC50, DTT; wild-type cells treated with DTT for 6 hours at 10× EC50.
Fig 7.
The T. brucei “methyltransferome”.
A) A stylised tree depicting the entire methyltransferase complement of T. brucei, clustered based on domain class as designated by Schubert and colleagues [52]. The arrangements of genes and branch lengths carry no significance as methyltransferases are highly diverse, making generation of a phylogenetic tree challenging. Gene IDs in bold italics have been previously characterised. Abbreviations: TM: Tetrapyrrole methylase. B) Number of members in each class of methyltransferase. Enzymes containing Rossmann-like folds and SET domains are numerous compared to the remaining classes. C) Methyltransferases grouped by the substrate they methylate. The majority of MTases studied include spliced leader RNA MTases and protein arginine MTases. SET domain-containing proteins are likely lysine MTases.
Fig 8.
Effect of AN5568 treatment on T. b. brucei cell division and morphology.
T. b. brucei cells were incubated with 2× EC50 concentration of AN5568 and cells were isolated for analysis by microscopy at specific time points. A) After 6 hours of drug exposure, cells appeared normal under both direct light and DAPI, whilst mitochondria were swollen. This phenotype was exaggerated by 12-hours post-treatment. After 24 hours, DAPI staining indicated a build-up of DNA-containing compartments in a significant number of cells, suggesting a cytokinetic defect. Scale bar represents 5 μm. B) Cell cycle analysis was carried out by counting the numbers of nuclei and kinetoplasts in AN5568-treated cells. Drug-treated cells were compared to a DMSO control at 6, 12 and 24 hour time-points, as indicated by the horizontal lines.