The first line treatment for Chagas disease, a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi, involves administration of benznidazole (Bzn). Bzn is a 2-nitroimidazole pro-drug which requires nitroreduction to become active, although its mode of action is not fully understood. In the present work we used a non-targeted MS-based metabolomics approach to study the metabolic response of T. cruzi to Bzn. Parasites treated with Bzn were minimally altered compared to untreated trypanosomes, although the redox active thiols trypanothione, homotrypanothione and cysteine were significantly diminished in abundance post-treatment. In addition, multiple Bzn-derived metabolites were detected after treatment. These metabolites included reduction products, fragments and covalent adducts of reduced Bzn linked to each of the major low molecular weight thiols: trypanothione, glutathione, γ-glutamylcysteine, glutathionylspermidine, cysteine and ovothiol A. Bzn products known to be generated in vitro by the unusual trypanosomal nitroreductase, TcNTRI, were found within the parasites, but low molecular weight adducts of glyoxal, a proposed toxic end-product of NTRI Bzn metabolism, were not detected. Our data is indicative of a major role of the thiol binding capacity of Bzn reduction products in the mechanism of Bzn toxicity against T. cruzi.
Ten million people worldwide are infected with Trypanosoma cruzi, the causative agent of Chagas disease, and 40 million are at risk of infection [1], [2]. In spite of a substantial reduction in prevalence over the last few decades, the disease is considered among the world' s 17 most neglected tropical diseases and is responsible for 13,000 annual deaths according to the World Health Organization (WHO). T. cruzi is naturally transmitted to humans and other mammals by reduviid insects of the subfamily Triatominae, and may also be transmitted by blood transfusions, organ transplants, orally through contaminated food, and vertically from mother to child. The disease progresses with an initial acute phase, usually asymptomatic, that can subsequently develop into a chronic form with cardiac and digestive pathologies that can lead to death [3]. Benznidazole (Bzn), formerly commercialized as Rochagan and Radanil (Roche), and nifurtimox (Nfx), marketed as Lampit (Bayer), are the only drugs proven effective against Chagas disease. Both contain a nitro group linked, respectively, to an imidazole or furan ring, and unwanted side effects are common, leading to treatment discontinuation in some cases. Bzn has the best safety and efficacy profile, and is therefore used as first line treatment. A major limitation is the low potency of these drugs against parasites in the established chronic disease, which is the form most commonly encountered clinically [4], [5]. The therapeutic benefit of Bzn in established mild to moderate Chagas disease is currently under scrutiny in the Benznidazole Evaluation for Interrupting Trypanosomiasis (BENEFIT) trial [6]. In spite of the limitations of Bzn and Nfx in treatment, only a few compounds are undergoing clinical trials against chronic Chagas disease, and there are no immediate prospects of a vaccine. Interest in nitro-heterocyclic compounds has recently been reinvigorated given the advancement of several members of the class into clinical trials [7], [8], [9]. Bzn was discovered as an anti-trypanosomal agent through screening against parasites without understanding its mechanism of action. Other nitroimidazoles, including the 5-nitroimidazole metronidazole, are well-established in the treatment of anaerobic protozoal and bacterial infections [10]. The mode of action of nitroheterocyclic compounds appears to involve metabolic activation of the compounds initiated through reduction of the compounds' nitro group. Subsequent metabolism of the compounds can be divergent. The anti-mycobacterial agent PA-824, for example, is reduced by a deazaflavin (F420) -dependent nitroreductase (Ddn) in M. tuberculosis and eventually decomposes to various reactive nitrogen species including nitric oxide [11]. Bzn activity has been proposed to be mediated via reduced intermediates that covalently modify macromolecules in vivo, including lipids, DNA and proteins, rather than by formation of radical intermediates producing reactive oxygen species [12], [13]. An unusual prokaryotic type I nitroreductase (NTRI) was identified in trypanosomatid protozoa which is primarily responsible for the reductive activation of some trypanocidal nitroheterocycles including Bzn, Nfx and also fexinidazole [14], [15], [16]. In vitro TcNTR can catalyse the consecutive two electron reduction of Bzn, leading to formation of a dihydroxy-dihydroimidazole derivative which may decompose to give glyoxal, a well-known toxic metabolite. Glyoxal was postulated to contribute to the pleiotropic effects of Bzn on trypanosomes [17], although roles for each of the metabolic products of Bzn have not been investigated in situ. Here we report, for the first time, an untargeted metabolomics analysis of T. cruzi to investigate changes in the parasite associated with exposure to the drug. Using a platform involving HILIC chromatography to separate low molecular weight metabolites coupled to high resolution mass spectrometry [18] we detected in the order of a thousand T. cruzi metabolites and were able to identify metabolic perturbations associated with Bzn exposure, as well as the production of Bzn metabolites and their in situ reaction products.
T. cruzi epimastigotes of the DM28c strain [19] were grown in LIT medium supplemented with yeast extract and 10% foetal bovine serum at 28°C [20]. For metabolite extraction a protocol was adapted from that used for other trypanosomatid protozoa [18], [21]. Cultures were initiated by inoculating exponentially growing epimastigotes to a final concentration of 2. 5×107 parasites per mL. Two days after inoculation, parasites were counted in a Neubauer chamber and 1×108 parasites per sample were taken for treatment (approximately 1 mL). After adding 20 or 50 µM Bzn, parasites were incubated at 28°C for 6 h. Tubes containing parasites in suspension were quenched on ice for 3 min, after which Bzn was added to control cells when necessary (Table S1). Cells were collected immediately by centrifugation (2000× g, 4°C, 3 min). Supernatants were carefully removed from cell pellets. Samples from each supernatant were separated for analysis as medium samples (5 µL). Cell disruption and metabolite extraction was performed using 200 µl chloroform/methanol/water 20/60/20 (v/v/v) during 1 hour in a Thermomixer (1000 rpm, 4°C –Eppendorf AG, Hamburg, Germany). Metabolite extracts were separated from cell debris by centrifugation (13,000× g, 4°C, 3 min). Extracts were stored at −70°C under nitrogen gas until analysis. Biological replicates were grown, incubated and extracted on different days. A short drug incubation period was chosen to impede changes in the number of parasites during treatment. For viability determinations, treated parasites were washed, incubated in fresh medium for 72 h and counted in Neubauer chamber. Samples were analysed on an Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific) in both positive and negative modes (rapid switching), coupled to a HPLC separation with a ZIC-HILIC column (Sequant) as has previously been described [18]. All samples from each experiment were analysed in the same analytical batch in randomised order and the quality of chromatography and signal reproducibility were checked by analysis of quality control samples, internal standards and total ion chromatograms. A standard mix containing approximately 160 authentic metabolite standards was run at the start of each analysis batch to aid metabolite identification. MSMS analysis was performed on an LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific) in positive mode with ZIC-HILIC chromatography as described above. High resolution (15,000) MSMS spectra were obtained with HCD induced fragmentation at normalised collision energy of 35 eV. MSMS spectra for the follow-up 50 µM Bzn experiment were collected at unit resolution. Untargeted metabolite analysis was conducted with the freely available software packages mzMatch [22] and Ideom (http: //mzmatch. sourceforge. net/ideom. php) [23]. Raw LC-MS data was converted to mzXML format and peak detection was performed with XCMS [24] and saved in peakML format. Mzmatch. R was used for sample alignment, peak filtering (based on reproducibility, peak shape and an intensity threshold of 3000), gap filling and annotation of related peaks. Ideom was used to remove contaminants and LC-MS artefact peaks and to perform metabolite identification. Metabolite identities were confirmed by exact mass (within 3 ppm after correction for loss or gain of a proton in negative mode or positive mode ESI respectively) and retention time where authentic standards were available for analysis. Putative identification of all other metabolites was made on the basis of exact mass and predicted retention time from all metabolites from the KEGG, MetaCyc and Lipidmaps databases [18]. The m/z values corrected for proton gain or loss are referred as m/zc in the text. In cases where identification was putative, the most likely metabolite was chosen based on available chemical and biological knowledge [23]. However, LC-MS data alone is often insufficient for accurate isomer identification and lists of alternative identifications with meta-data for each identified formula are accessible in the macro-enabled Ideom files (Files S1 and S2; help documentation available at mzmatch. sourceforge. net/ideom. php). Quantification is based on raw peak heights, and expressed relative to the average peak height observed in untreated cells from the same experiment. Unidentified peaks in the LC-MS data were also investigated for drug-induced changes. After removal of LC-MS artefacts and known contaminants, measured exact masses were compared with theoretical exact masses of Bzn derived metabolites contained in Bznmet database (“targeted sheet”, Files S1 and S2). This database included reported and putative Bzn reduction products and covalent adducts of Bzn with small cellular metabolites, all retrieved from existing reports on in vivo and in vitro modification processes for Bzn and similar nitroimidazoles, covering enzymatic and non-enzymatic conversions [17], [25], [26], [27]. Accurate mass and relative isotope abundance was used to determine the chemical formulae for the remaining unidentified metabolites detected specifically in the 50 µM Bzn treated samples. In most cases these formulae contain the subset C12H14N4O, suggesting that they are adducts of reduced Bzn with a broad range of unexpected metabolites. In addition, a targeted analysis of potential metabolites in the Bznmet database was performed on the raw data using accurate mass within a 3 ppm mass range, to allow detection of metabolites that may have been excluded by the automated data processing due to peak shape, intensity or reproducibility filters. Manually retrieved intensity and RT values from all samples are included in File S3. TcNTRI: GenBank AHD24669. 1 TbNTRI: GenBank AAX69576. 1 Dnd Mycobacterium tuberculosis: UniProtKB/Swiss-Prot P71854. 1 TcCPR-B: GenBank ABI15738. 1 TcOYE: GenBank AAX54861. 1 TcAKR: GenBank ACD93222. 1
Metabolites were extracted from T. cruzi epimastigote cell pellets with a monophasic solvent mixture of chloroform, methanol and water (1∶3∶1); separated and analysed by ZIC-HILIC chromatography coupled to high accuracy MS using an Orbitrap mass spectrometer. Five biological replicates were analysed for each condition. Signal extraction and initial filtering of the LC-MS data yielded 3,117 peaks for negative mode and 5,528 peaks for positive ESI mode. Additional artefact filtering and polarity merging in Ideom reduced the list of features to 1,477 candidate molecules, of which over 70% (1,069) matched compounds in the metabolite databases based on accurate mass and retention time information [18]. A complete list of putatively identified metabolites, with the detected peak heights for each sample and confidence values for their identifications is supplied in File S1 (see “Comparison” sheet). Only putatively identified metabolites were considered for intensity comparisons among groups of samples. Based on the Ideom software' s automated metabolite calling we can divide metabolites into several classes, although additional confirmation of the identity of each individual metabolite would be required to impose certainty on these classifications. The largest class of metabolites was peptides, representing 42. 1% of the total metabolites (135 dipeptides, 183 tripeptides and 132 tetrapeptides) (Figure 1 and File S1). The next largest class of metabolites was amino acids and compounds associated with amino acid metabolism (amino acids, thiol compounds and polyamines), which represented 14. 6% of the total putatively identified metabolites. In addition to peptides and amino acids, metabolites from a diverse range of metabolic pathways were detected (Figure 2), including lipid (110), carbohydrate (44), nucleotide (33) and cofactor metabolism (26). Putatively identified metabolites that lack KEGG [28] or Lipidmaps [29] pathway annotations were classified as unmapped (210), among these there are N-acetylated amino acids and polyamines, acyl-carnitines and acyl-glycines. A hallmark metabolite in kinetoplastid parasites, including trypanosomes, is trypanothione. This di-thiol is composed of two molecules of glutathione linked by one molecule of spermidine, and it is usually detected as a multi charged ion by ESI-MS [30]. Under the conditions used in this study, the tri-charged form of trypanothione disulphide was detected in all parasite samples with very high S/N ratios. Furthermore, a mass consistent with tri-charged homotrypanothione, a metabolite unique to T. cruzi [31], was also detected although with eighty times lower signal intensity than trypanothione. It is important to note that the protocol used here leads to extensive oxidation of thiols, thus the relative quantification of the intracellular redox state of these molecules is not possible. Other common small thiols detected were glutathione, cysteine/cystine and homocysteine. Detected polyamines included cadaverine, spermidine, putrescine and some modified forms such as N-acetylspermidine, N-acetylputrescine and gamma-glutamylputrescine. In an attempt to analyse the metabolic changes induced by Bzn treatment, T. cruzi epimastigotes were exposed to 20 µM Bzn over six hours (cBt samples) after which metabolites were extracted. Approximately 80% of the parasites remained viable after this treatment (not shown). Parasites that were not exposed to Bzn (cTc) and parasites to which Bzn was added just prior to the extraction of metabolites (cBc) (to control for any mass spectrometry related effects due to drug) were included as controls. Medium samples were analysed in parallel including fresh medium (Med) and spent mediums collected from cBc (mBc) and cBt samples (mBt). Principal Component Analysis (PCA) of the automatically filtered data indicated that no obvious differences were found among the different groups of cells samples (cBc, cBt and cTc), although medium samples could be readily separated from cell samples (Figure S1). This indicated that the global structure of the T. cruzi metabolome was little changed by this treatment with Bzn. Univariate analysis of individual metabolites using p<0. 05 as a significance threshold in t-tests and a fold abundance change above 1. 4 revealed relatively few differences between treated and untreated parasites (see “Comparison” sheet, File S1). Among these, trypanothione disulphide, homotrypanothione disulphide and cystine (cysteine disulphide) were significantly diminished in abundance after treatment. Three glutamate containing di-peptides were elevated in Bzn treated samples (Figure 3), and although they were not structurally characterised, they likely represent the gamma-glutamyl dipeptides involved in glutathione recycling. In addition to the relatively small changes in the metabolites discussed above, two metabolites which were substantially elevated in treated cells over controls were identified through the automated screening of the datasets. One metabolite (m/zc 278. 14, RT 11. 1) was assigned as N-Benzoyl-D-arginine and another (m/zc 292. 15, RT 10. 2) as 4-coumaroyl-3-hydroxyagmatine, based on mass similarity. However, as discussed below, these were mis-identifications of Bzn metabolites that have masses (i. e. molecular formulae) identical to these representatives from the IDEOM metabolite database. Considering that Bzn acts as a pro-drug which undergoes reductive metabolism to generate toxic intermediates [4], [5], [13], the presence of unidentified peaks at significantly higher concentrations in treated than in untreated cells prompted us to re-scan the filtered data seeking putative molecules derived from in situ Bzn metabolism. We applied a correlation analysis on the intensities of all the LC-MS peaks, searching for molecules that were highly correlated with the metabolites detected in treated samples (and thus assumed to be derived from Bzn) and with low or no abundance in all the other samples (control parasites, media and solvent samples) (“all Data” sheet, File S1). Six additional ions corresponding to putative Bzn metabolites were found after carefully removing additional MS artefacts derived from these molecules (Table 1). To further investigate the identity of the Bzn metabolites and to search for additional Bzn metabolites, an in-house database of reported and putative Bzn metabolites was built: Bznmet database (“Targeted” sheet, File S1). The theoretical m/zc values contained in the database (109 ions belonging to 56 different molecules) were compared with the accurate measured m/zc values from the collected filtered data and also the raw data, resulting in identification of a number of Bzn metabolites (Table 1 and File S3). Finally, to gain additional information to support the proposed metabolite structures, MSMS fragmentation spectra were collected for the ions of interest (File S4). Once the LC-MS data were examined, a number of reduced derivatives of Bzn were found to be highly abundant in treated samples. The signal at m/zc 264. 12 (RT 13 min) was attributed to a dihydroxy-dihydro derivative of Bzn: 2- (2-amino-4,5-dihydroxy-4,5-dihydroimidazol-1-yl) -N-benzylacetamide (3). Additionally, the two metabolites initially detected in Bzn-treated samples and mis-identified, as N-Benzoyl-D-arginine (m/zc 278. 13, RT 11. 1 min) and 4-coumaroyl-3-hydroxyagmatine (m/zc 292. 15, RT 10. 2 min), could be assigned as methoxy derivatives of the Bzn dihydroxy-dihydro derivative (2,6). Also, a molecule with an m/z value expected for the hydroxylamine or some hydroxy derivatives of Bzn was found (m/zc 246. 11, RT 11 min) (11). N-benzyl-2-guanidinoacetamide (m/zc 206. 11, RT 11. 7 min) (5) was also detected. This molecule was reported to arise after the Bzn dihydroxy-dihydro derivative decomposes, or reacts with other molecules, to release glyoxal [17], [32]. Nevertheless, glyoxal adducts with nucleotides, nitrogenated bases or amino acids could not be detected in our data. Also, 2- (2-amino-1H-imidazol-1-yl) -N-benzylacetamide (m/zc 230. 11, RT 10. 85 min) (1), a six electron reduction product of Bzn, was highly abundant in treated samples. Multiple detected signals were assigned to covalent adducts of Bzn reduction products with low molecular weight thiols. Three signals were assigned to different Bzn-trypanothione adducts, all detected as tri-charged ions (m/zc 322. 47, RT 29. 1; m/zc 316. 46, RT 28 and m/zc 356. 8, RT 29. 9 min) (10,13,16). Likewise, a glutathionylspermidine adduct was detected as a tri-charged ion (m/zc 220. 78, RT 34. 5 min) (15). The mono-charged ion with m/zc 349. 12 (RT 11 min) was assigned to a cysteine-containing adduct (9). In addition, we found a glutathione adduct with reduced Bzn, both as a mono and a di-charged ion, with m/zc 535. 18 and 267. 59 respectively. Each of these ions were detected at two different retention times (RT 17. 6 and 18. 4 min), most probably representing two isomers (7,8). Ovothiol A covalent adducts were assigned to signals present at two retention times on HILIC chromatography (m/zc 429. 16 and m/zc 214. 58; both at RT 19. 5 and 21. 5 min) (14,12). High quality MSMS spectra were obtained for a number of putative Bzn-derived molecules, even though some metabolites were detected with very low intensity signals. The analysis of the fragmentation patterns is summarized in File S4. Although additional structural data would be necessary to unequivocally identify all the metabolites, the fragmentation data, together with the high resolution accurate mass MS and RT data, are supportive for the proposed structures. In this sense, all MSMS spectra contained a fragment of m/zc 90. 047 assigned to the benzene ring moiety of Bzn and also present in the Bzn fragmentation pattern. In addition, molecules proposed as related structures displayed fragmentation patterns with shared peaks. Metabolites with m/zc 264. 12 (3) and 278. 13 (2) (hydroxy and methoxy derivatives) included 14 shared signals, while cysteine (9) and glutathione adducts (7,8) (Bzn-thiol conjugates) displayed five shared peaks. Furthermore, shared peaks were obtained when glutathione and trypanothione MSMS fragmentation spectra were compared with the corresponding spectra of the proposed Bzn-thiol adducts. Among the common peaks we found the typical fragment encountered in glutathione containing molecules of mass 129 Da [33] and also fragment masses that correspond to the neutral loss of pyroglutamic acid. Also, 34S isotopic peaks were observed for a number of metabolites, confirming the presence of thiol moieties in these covalent adducts (Table 1). Finally, a group of cell-free control samples were analysed separately: a control group in which the drug was incubated with the growth medium was compared with the medium alone (not-shown). In this analysis we observed that no Bzn metabolites arise after the drug incubation with the medium components after 6 hours at 28°C. Thus, all of the observed Bzn derived metabolites are produced by T. cruzi-mediated metabolism of the drug. Since a number of the Bzn-related metabolites were found only with very low abundance when T. cruzi epimastigotes were incubated with 20 µM Bzn over six hours, we collected additional metabolomics data using a higher concentration of Bzn, to allow the detection of additional signals arising from Bzn derived molecules and to observe effects on endogenous metabolites. For this purpose, parasites were treated with 50 µM Bzn over 6 h in the same general conditions (cBzt samples). Parasites that were not exposed to Bzn (cBec) and parasites to which Bzn was added just prior to the extraction of metabolites (cBzc) were used as controls. Fifty percent (50%) of the parasites remained viable after this treatment compared to untreated controls (not shown). After data filtering, and analogous to the 20 µM Bzn treatment, we observed several endogenous metabolites that showed significant differences between treated and control samples (File S2). Consistent with the lower Bzn dose, trypanothione and homotrypanothione showed diminished levels after drug exposure, probably a consequence of the formation of the drug-thiol conjugates. Three lipids putatively identified as vitamin D-related sterols showed diminished levels after treatment, whereas two long-chain acyl-carnitines showed augmented levels, along with the dipeptide γ-glutamylcysteine (T-test P values<0. 05 and FC>2). Treatment with 50 µM Bzn induced the appearance of a high number of Bzn related signals, with 124 ions included in the filtered data. After careful removal of LC-MS artefact ions, 36 of these Bzn-specific peaks were listed as putative Bzn metabolites (Table 2), although some of the analysed signals may still represent in-source MS-derived fragments or adduct ions from true Bzn metabolites. The raw data was scanned for putative Bzn metabolites contained in the Bznmet database, and 14 additional ions were retrieved (Table 2). The Bzn metabolites identified using 20 µM Bzn were also found in the 50 µM assay, with the exception of a metabolite with m/zc 356. 80 (16). An additional signal at m/zc 164. 09 (RT 13. 6 min) was found and attributed to 2-amino-N-benzylacetamide (22). This compound was described as one of the products of the in vitro enzymatic conversion of Bzn by NTRI [17]. The metabolite with m/zc 246. 11 was found in both positive and negative ESI modes and with two different retention times (12 and 13. 5 min) (11,19). These could represent both the Bzn hydroxylamine and/or hydroxy metabolites. Also, a conjugate of reduced Bzn with γ-glutamylcysteine was detected in 50 µM Bzn treated samples (m/zc 478. 16 and m/zc 239. 08, RT 17. 5 and 18. 3 min) (20,31). The signal at m/zc 393. 17 (RT 32. 4 min) was assigned to a molecule composed of trypanothione linked to two reduced Bzn molecules (41). A triple and a quadruple charged ion were both attributed to a conjugate of reduced Bzn with a mixed disulphide of trypanothione and glutathione (m/zc 418. 82 and m/zc 314. 12 at RT 30. 3 min) (29). The remaining adducts included mercaptohistidine (35), an intermediate in ovothiol A synthesis, and non-sulphur containing metabolites including valine (36) and pyroglutamic acid (27). Some of the proposed structures were supported by MSMS data, although fragmentation patterns could not be obtained for all metabolites due to low abundance (File S4). Figure 4 depicts proposed structures for some of the Bzn metabolites detected in 20 µM and/or 50 µM Bzn metabolomics analysis. Many of the in situ detected Bzn-thiol conjugates display two different but close retention times, which likely correspond to structures equivalent to the ones described for reduced misonidazole adducts with glutathione [25]. These structures include the thiol moiety bound to the imidazole ring through either carbons at position 4 or position 5 (see Figure 4 for proposed structures). As an example, Bzn-glutathione adduct chromatograms, and MS and MSMS spectra are shown in Figure 5. Similar intensities were observed at the two different retention times where Bzn-glutathione adducts were detected, whereas Bzn-ovothiol A and Bzn-glutathionylspermidine adducts displayed very low intensity peaks at one of the retention times, which possibly results from preferred binding to one of the two imidazole carbons (4-C or 5-C).
Metabolomics technology has allowed us to gain further insight into Bzn mechanism of action. The MS based metabolomics analysis is a powerful tool and can be used to analyse the mode of action of different types of drugs in Trypanosoma cruzi. These in turn should help us understand resistance mechanisms as well as the natural variation in T. cruzi susceptibility to nitroheterocycles and to improve available and future drugs. Through this work we have shown that Bzn is extensively metabolized to a number of molecules once it enters T. cruzi. These molecules include reduction products and covalent adducts with low molecular weight thiols and other small molecules. In addition, the metabolomics analysis of endogenous metabolites identified low molecular weight thiol depletion and turnover as the major metabolic impact of Bzn treatment. We here propose that the covalent binding of Bzn with low molecular weight thiols as well as with protein thiols is a primary cause of the drug' s toxicity against T. cruzi.
The unicellular parasite Trypanosoma cruzi infects humans, leading to Chagas disease, endemic in Central and South America and responsible for 13,000 annual deaths. Only two drugs have proven effective against Chagas, nifurtimox and benznidazole (Bzn). Bzn has the best safety and efficacy profiles and is thus used as first line treatment. Bzn is a pro-drug, and possesses a nitro group which needs to be enzymatically reduced within the parasite to become active. We have investigated for the first time, by means of mass spectrometry based metabolomics, the global changes to small metabolites that occur once Bzn enters the parasite. A decrease in the levels of several thiols, including cysteine and trypanothione, and an increase in gamma-glutamyl containing dipeptides were observed after treatment. Reduced metabolites of Bzn were also detected, together with numerous covalent conjugates of the drug combined with low molecular weight thiols and some non-thiol metabolites. Overall, Bzn treatment primarily affects thiol containing molecules in T. cruzi, and this interference with thiol metabolism contributes to the drug' s mode of action.
biotechnology
medicine and health sciences
medicinal chemistry
small molecules
tropical diseases
chemical biology
parasitic diseases
neglected tropical diseases
pharmacology
infectious diseases
metabolic pathways
chemistry
protozoan infections
drug discovery
biochemistry
chagas disease
drug research and development
biology and life sciences
physical sciences
metabolism
Benznidazole Biotransformation and Multiple Targets in Trypanosoma cruzi Revealed by Metabolomics