Nitroimidazole Drugs Research Articles

The drug ornidazole inhibits photosynthesis in a different mechanism described for protozoa and anaerobic bacteria.

Ornidazole of the 5-nitroimidazole drug family is used to treat protozoan and anaerobic bacterial infections via a mechanism that involves preactivation by reduction of the nitro group, and production of toxic derivatives and radicals. Metronidazole, another drug family member, has been suggested to affect photosynthesis by draining electrons from the electron carrier ferredoxin, thus inhibiting NADP+ reduction and stimulating radical and peroxide production. Here we show, however, that ornidazole inhibits photosynthesis via a different mechanism. While having a minute effect on the photosynthetic electron transport and oxygen photoreduction, ornidazole hinders the activity of two Calvin cycle enzymes, triose-phosphate isomerase (TPI) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Modeling of ornidazole's interaction with ferredoxin of the protozoan Trichomonas suggests efficient electron tunneling from the iron-sulfur cluster to the nitro group of the drug. A similar docking site of ornidazole at the plant-type ferredoxin does not exist, and the best simulated alternative does not support such efficient tunneling. Notably, TPI was inhibited by ornidazole in the dark or when electron transport was blocked by dichloromethyl diphenylurea, indicating that this inhibition was unrelated to the electron transport machinery. Although TPI and GAPDH isoenzymes are involved in glycolysis and gluconeogenesis, ornidazole's effect on respiration of photoautotrophs is moderate, thus raising its value as an efficient inhibitor of photosynthesis. The scarcity of Calvin cycle inhibitors capable of penetrating cell membranes emphasizes on the value of ornidazole for studying the regulation of this cycle.

Crystal structure of di-μ-chlorido-bis-[chlorido-bis-(1,2-dimethyl-5-nitro-1H-imidazole-κN3)copper(II)] acetonitrile disolvate.

1,2-Dimethyl-5-nitro-imidazole (dimetridazole, dimet) is a compound that belongs to a class of nitro-imidazole drugs that are effective at inhibiting the activity of certain parasites and bacteria. However, there are few reports that describe structures of compounds that feature metals complexed by dimet. Therefore, we report here that dimet reacts with CuCl2·H2O to yield a chloride-bridged copper(II) dimer, [Cu2Cl4(C5H7N3O2)4] or [Cu(μ-Cl)Cl(dimet)2]2. In this mol-ecule, the CuII ions are coordinated in an approximately trigonal-bipyramidal manner, and the mol-ecule lies across an inversion center. The dihedral angle between the imidazole rings in the asymmetric unit is 4.28 (7)°. Compared to metronidazole, dimetridazole lacks the hy-droxy-ethyl group, and thus cannot form inter-molecular O⋯H hydrogen-bonding inter-actions. Instead, [Cu(μ-Cl)Cl(dimet)2]2 exhibits weak inter-molecular inter-actions between the hydrogen atoms of C-H groups and (i) oxygen in the nitro groups, and (ii) the terminal and bridging chloride ligands. The unit cell contains four disordered aceto-nitrile mol-ecules. These were modeled as providing a diffuse contribution to the overall scattering by SQUEEZE [Spek (2015 ▸). Acta Cryst. C71, 9-18], which identified two voids, each with a volume of 163 Å3 and a count of 46 electrons, indicative of a total of four aceto-nitrile mol-ecules. These aceto-nitrile mol-ecules are included in the chemical formula to give the expected calculated density and F(000).

The Potential of Lactobacillus casei and Entercoccus faecium Combination as a Preventive Probiotic Against Entamoeba.

Travellers' diarrhoea caused by enteric protozoa like Entamoeba histolytica is among the most common protozoan diseases in developing countries. In developing countries, amoebiasis is the second most prevalent protozoan disease. This protozoan parasite is often known to coexist as a part of the normal gut microbiota. It is estimated that around 50-60% of population in developing countries might be harbouring Entamoeba in an asymptomatic manner. Due to physiological perturbation or upon immuno-compromise, it can become virulent and then cause diarrhoea, bloody stools and may invade other organs if left untreated. Nitroimidazole drugs, namely metronidazole and tinidazole, are widely used to treat protozoan infections. These drugs often show dose-dependent side effects. With emerging antibiotic resistance, novel therapeutics to prevent parasitic infections is required. This study aims to study effect of probiotics on prevention of Amoebiasis. In this study, we have investigated the effect of selected probiotics on the growth of Entamoeba. From the list of probiotics being currently used, five bacterial strains were selected for testing. These probiotic strains were co-cultured with Entamoeba, and their effect on Entamoeba proliferation was checked. Of the five probiotics chosen, individual treatments of Lactobacillus casei and Enterococcus faecium showed a significant reduction of up to 71% in parasite survival only at higher CFUs. When the two probiotics were used in combination, the percentage of survival reduced gradually further to 80% at a total CFU of 109cells/ml of bacteria. The study lays the foundation for providing cost-effective prophylactic treatment for amoebiasis without the overuse of antibiotics.

Imaging latent tuberculosis infection with radiolabeled nitroimidazoles.

Imaging latent tuberculosis infection with radiolabeled nitroimidazoles.

Allergy to Benznidazole: Cross-Reactivity with Other Nitroimidazoles

Benznidazole (Bzn) is a nitroimidazole drug used as first-line treatment for Chagas disease, a potentially life-threatening illness caused by Trypanosoma cruzi. Many adverse effects had been reported with it, including allergy reactions such as exanthemas, dermatitis and AGEP. Alternative therapeutic nitroimidazole drugs for this group of patients are important for treatment of other parasitic infections. We aim to study hypersensitivity reactions to Bzn and cross-reactivity with metronidazole (Mtn), another nitroimidazole drug. Five patients with non-immediate skin reactions due to Bzn, all presented with maculo-papular exanthema, were referred to our Allergy Department. Patch tests (PT) with Bzn 10% and Mtn 10% in DMSO, were made with readings at day 2 (D2) and D4. Single blind oral challenge test (SBOCT) was performed in patients with allergy confirmed to Bzn and negative PT with Mtn. PT with Bzn were positive in 5/5 patients (100%) at D2 and D4. PT with Mtn were negative in 4/5. Only one patient had both positive PT. SBOCT with Mtn was negative in all of them (4/4). PT with Bzn was diagnostic in 100% of patients so was very useful for the diagnosis, suggesting type IV-hypersensitivity mechanism. Cross-reactivity between Bzn and Mtn was low, only found in 1 patient that was sensitizated to both nitroimidazoles. 100% of patients with negative PT to Mtn had negative SBOCT (negative predictive value of 100%). We think that is important to test both drugs in these patients in order to give them alternative therapeutic treatment for other parasitosis disease.

Metabolomics reveals novel biomarkers of illegal 5-nitromimidazole treatment in pigs. Further evidence of drug toxicity uncovered

The aim of the study was to investigate the potential of a metabolomics platform to distinguish between pigs treated with ronidazole, dimetridazole and metronidazole and non-medicated animals (controls), at two withdrawal periods (day 0 and 5). Livers from each animal were biochemically profiled using UHPLC–QTof-MS in ESI+ mode of acquisition. Several Orthogonal Partial Least Squares-Discriminant Analysis models were generated from the acquired mass spectrometry data. The models classified the two groups control and treated animals. A total of 42 ions of interest explained the variation in ESI+. It was possible to find the identity of 3 of the ions and to positively classify 4 of the ionic features, which can be used as potential biomarkers of illicit 5-nitroimidazole abuse. Further evidence of the toxic mechanisms of 5-nitroimidazole drugs has been revealed, which may be of substantial importance as metronidazole is widely used in human medicine.

Adenosine/guanosine preferring nucleoside ribohydrolase is a distinct, druggable antitrichomonal target.

Nucleoside salvage pathway enzymes used by Trichomonas vaginalis are distinct from the pathway involved in activation of existing 5-nitroimidazole drugs. They thus represent excellent targets for developing novel, mechanism-based antitrichomonal agents. The purine-specific adenosine/guanosine preferring ribohydrolase (AGNH) was screened against the NIH Clinical Collection to assess its druggability. Eight compounds, including five flavonoids, were identified with IC50 values ⩽10 μM and confirmed in counter screens run in the presence of detergent. The inhibitors are structurally distinct from inhibitors of the pyrimidine-specific uridine ribohydrolase (UNH) thus indicating that AGNH is a distinct, druggable target from UNH.

Determination of 5-nitroimidazole residues in milk by capillary electrochromatography with packed C18 silica beds

This work presents a novel methodology for analysing 5-nitroimidazole residues in milk samples by capillary electrochromatography using lab-made packed columns, produced by carrying out a high pressure packing procedure using acetone as driving solvent and C18 silica uncapped particles (5µm particle size) as packing material. Column frits resulted from sintering the proper stationary phase by heating the packed material for 20s with a nichrome ribbon (80% Ni–20% Cr, 28cm×2mm×0.2mm, electric resistance 1.3Ω) connected to a 7V AC power supply. Lab-made C18 silica packed capillaries (40cmx50µm i.d.) were employed for the determination of 5-nitroimidazole drugs. Milk samples were treated by a salting-out assisted liquid–liquid extraction followed by a solid phase extraction with Oasis®HLB cartridges prior to their injection. Samples were hydrodynamically injected into the column for 120s at 11.5bar. Afterwards eight 5-nitroimidazole compounds were separated in isocratic mode under an applied voltage of 27kV and a temperature of 30°C. The selected mobile phase consisted of a mixture 60:40 acetonitrile:ammonium acetate (2.5mM, pH=5). Separation was monitored at 320nm and it was performed in less than 15min. The method was characterized in terms of linearity (R2≥0.993) and precision (repeatability, RSD≤12.2% and reproducibility, RSD≤14.5%), obtaining detection limits lower than 29µg/L for all compounds under study.

Anti-Trichomonas vaginalis properties of the oil of Amomum tsao-ko and its major component, geraniol

Context: Trichomonosis, caused by the flagellate protozoan Trichomonas vaginalis, is the most common non-viral sexually transmitted disease (STD) and 5-nitroimidazole drugs are used for the treatment. However, a growing number of T. vaginalis isolates are resistant to these drugs, which make it becomes an urgent issue.Objective: The current study was designed to evaluate the anti-T. vaginalis activity of the essential oil from A. tsao-ko used in traditional Chinese medicine and as a spice and its main component, geraniol.Materials and methods: The anti-T. vaginalis activities of A. tsao-ko essential oil and geraniol were evaluated by the minimum lethal concentration (MLC) and 50% inhibitory concentration (IC50) in vitro. The morphological changes of T. vaginalis were observed by transmission electron microscopy (TEM). Additionally, sub-MLC concentration treatment with sub-MLC A. tsao-ko essential oil and geraniol was also performed.Results: This study shows that MLC/IC50 of A. tsao-ko essential oil was 44.97 µg/ml/22.49 µg/ml for T. vaginalis isolate Tv1, and 89.93 µg/ml/44.97 µg/ml for T. vaginalis isolate Tv2. Those of geraniol were 342.96 µg/ml/171.48 µg/ml, respectively. After A. tsao-ko essential oil or geraniol treatment, obvious similar morphological changes of T. vaginalis were observed by TEM: the nuclear membrane was damaged, nuclei were dissolved, and the chromatin was accumulated; in the cytoplasm, numerous vacuoles appeared, rough endoplasmic reticulum dilated, the number of ribosomes were reduced, organelles disintegrated, the cell membrane was partially damaged, with cytoplasmic leakage, and cell disintegration was observed. The action time did not increase the effect of A. tsao-ko essential oil or geraniol against T. vaginalis, as no significant difference was observed after sub-MLC concentration treatment for 1, 3, and 5 h with A. tsao-ko essential oil and geraniol.Discussion and conclusion: The study describes the first report on the activity and morphological changes of A. tsao-ko essential oil and geraniol against T. vaginalis. The results obtained herein presented new opportunities for antitrichomonal drugs.

Determination of the persistence of dimetridazole, metronidazole and ronidazole residues in black tiger shrimp (Penaeus monodon) tissue and their stability during cooking

The depletion of three banned nitroimidazole drugs – dimetridazole (DMZ), metronidazole (MNZ) and ronidazole (RNZ) – was investigated in black tiger shrimp (Penaeus monodon) following in-water medication. The highest concentrations of residues were measured immediately after the 24-h immersion (d0). At this time, MNZ and MNZ-OH residues were measured in shrimp tissue samples at concentrations ranging from 361 to 4189 and from 0.28 to 6.6 μg kg−1, respectively. DMZ and its metabolites HMMNI ranged in concentration between 31 509 and 37 780 and between 15.0 and 31.9 μg kg−1, respectively. RNZ and HMMNI concentrations ranged from 14 530 to 24 206 and from 25.0 to 55 μg kg−1, respectively. MNZ, DMZ and RNZ were the more persistent marker residues and can be detected for at least 8 days post-treatment. MNZ-OH was only detectable on d0 following treatment with MNZ. HMMNI residues were only detectable up to d1 (0.97–3.2 μg kg−1) or d2 (1.2–4.5 μg kg−1) following DMZ and RNZ treatment, respectively. The parent drugs MNZ, DMZ and RNZ were still measureable on d8 at 0.12–1.0, 40.5–55 and 8.8–18.7 μg kg−1, respectively. The study also investigated the stability of nitroimidazole residues under various cooking procedures (frying, grilling, boiling, and boiling followed by microwaving). The experiments were carried out in shrimp muscle tissue containing both high and low concentrations of these residues. Different cooking procedures showed the impact on nitroimidazole residue concentration in shrimp tissue. Residue concentration depleted significantly, but partially, by boiling and/or microwaving, but the compounds were largely resistant to conventional grilling or frying. Cooking cannot therefore be considered as a safeguard against harmful nitroimidazole residues in shrimp.

Effects of rifampin and ketoconazole on pharmacokinetics of morinidazole in healthy chinese subjects.

Morinidazole, a 5-nitroimidazole antimicrobial drug, has been approved for the treatment of amoebiasis, trichomoniasis, and anaerobic bacterial infections in China. It was reported that drug-drug interaction happened after the coadministration of ornidazole, an analog of morinidazole, and rifampin or ketoconazole. Therefore, we measured the plasma pharmacokinetics (PK) of morinidazole and its metabolites in the healthy Chinese volunteers prior to and following the administration of rifampin or ketoconazole using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The area under the concentration-time curve from time 0 to time t (AUC0-t) and maximum concentration in serum (Cmax) of morinidazole were decreased by 28% and 23%, respectively, after 6 days of exposure to 600 mg of rifampin once daily; the Cmaxs of N(+)-glucuronides were increased by 14%, while their AUC0-ts were hardly changed. After 7 days of exposure to 200 mg of ketoconazole once daily, the AUC0-t and Cmax of the parent drug were not affected significantly. Cmaxs of N(+)-glucuronides were decreased by 23%; AUC0-ts were decreased by 14%. The exposure of sulfate conjugate was hardly changed after the coadministration of rifampin or ketoconazole. Using recombinant enzyme of UGT1A9 and human hepatocytes, the mechanism of the altered PK behaviors of morinidazole and its metabolites was investigated. In human hepatocytes, ketoconazole dose dependently inhibited the formation of N(+)-glucuronides (50% inhibitory concentration [IC50], 1.5 μM), while rifampin induced the mRNA level of UGT1A9 by 28% and the activity of UGT1A9 by 53%. In conclusion, the effects of rifampin and ketoconazole on the plasma exposures of morinidazole and N(+)-glucuronide are less than 50%; therefore, rifampin and ketoconazole have little clinical significance in the pharmacokinetics of morinidazole.

Cu(I)Cl-promoted synthesis of novel N-alkylated isatin analogs with an extension toward isatin-4-aminoquinoline conjugates: in vitro analysis against Trichomonas vaginalis

Trichomonas vaginalis, the causative agent of trichomonosis, is the most common non-viral sexually transmitted disease. Infection with this protozoan may have serious consequences, especially for women. Currently, 5-nitroimidazole drugs are the treatment of choice for trichomonosis, but the emergence of drug resistant isolates has limited the effectiveness of this therapy. In this context, a library of isatin-mannich adducts and 4-aminoquinoline-isatin mannich conjugates was synthesized following the Cu-promoted Mannich reaction and evaluated for their preliminary in vitro analysis against T. vaginalis at 50 μM. The preliminary evaluation data revealed that the introduction of 4-aminoquinoline ring enhanced the activity profiles with compounds viz. 6a and 6c exhibiting percentage growth inhibition of 92.13 and 100 %, respectively. Compounds with high levels of potency were further analyzed to determine their IC50 values as well as cytotoxicity profiles against mammalian cells. The most active compound in the synthesized conjugates displayed an IC50 value of 23 μM against cultured G3 strain in TYM Diamond’s media of T. vaginalis and was non-toxic to cultured mammalian HeLa cells at the same concentration. Synthesis of N-alkylated isatin analogs, 4-aminoquinoline isatin hybrids, in vitro evaluation against trichomoniasis and cytotoxicities against HeLa cells.

Determination of nitroimidazole residues in aquaculture tissue using ultra high performance liquid chromatography coupled to tandem mass spectrometry.

An UHPLC-MS/MS method was developed for the quantitative confirmatory analysis of residues of nitroimidazole drugs (dimetridazole, ipronidazole, metronidazole, ornidazole and ronidazole) and their corresponding hydroxy metabolites (HMMNI, ipronidazile-OH and metronidazole-OH) in aquaculture tissue. Samples were extracted by shaking in acetonitrile, water, MgSO4 and NaCl before being defatted with n-hexane pre-saturated with acetonitrile and concentrated under nitrogen. Nitroimidazole residues were determined by UHPLC-MS/MS operating in positive electrospray ionisation mode using a reversed phase BEH C18 column. The method was validated according to the EU Commission Decision 2002/657/EC guidelines. The following performance studies were carried out: specificity, selectivity, linearity, within laboratory repeatability (WLr)/reproducibility (WLR), accuracy, precision, decision limit (CCα), detection capability (CCβ), absolute recovery and stability. The analytical range of the method is 0.1-20 μg kg(-1). Accuracy and precision of the method, under within-laboratory reproducibility conditions, ranged from 83 to 105% and 2.3 to 14.0%, respectively. CCα were 0.07-1.0 μg kg(-1) depending on analyte and matrix. A total of 50 samples can be analysed in a single day using the assay. The method has been extensively evaluated through application to real test samples.

Trypanosoma cruzi chemical proteomics using immobilized benznidazole

Benznidazole (Bzn) is a nitroimidazole drug currently used as first line treatment against Chagas disease, a neglected tropical disease caused by the flagellated protozoan Trypanosoma cruzi. Although the drug has been used since the late 1960s, its mechanism of action is not fully understood. In an attempt to study Bzn mode of action, a structurally modified derivative of the drug was synthesized and immobilized into a solid matrix. This allowed enrichment of T. cruzi proteins capable of binding immobilized Bzn, which were subsequently analysed by mass spectrometry. The proteins identified as specific non-covalent Bzn interactors were a homologue of the bacterial YjeF proteins, a Sec23A orthologue and the aldo–ketoreductase family member TcAKR. TcAKR is closely related to other enzymes previously associated with Bzn reductive activation such as NTRI and TcOYE. Thus, our untargeted search for Bzn binding partners allowed us to encounter proteins that could be related to drug reductive activation and/or resistance mechanisms.

An Improved Kilogram-Scale Synthesis of 2-Bromo-4-nitro-1H-imidazole: A Key Building Block of Nitroimidazole Drugs

An efficient two-step method for the synthesis of 2-bromo-4-nitroimidazole, 6, a key building block for nitroimidazole drugs, has been developed. The synthesis involves dibromination of 4-nitroimidazole 10 followed by selective debromination using in situ reductive deiodination strategy. The reactions are facile, safe, and easy to scale up. The large-scale applicability of this improved method was tested by conducting the reactions on kilogram scale to produce the desired product in high yield and quality.

Novel solid phase extraction method for the analysis of 5-nitroimidazoles and metabolites in milk samples by capillary electrophoresis

A new sample treatment has been developed for the extraction of 5-nitroimidazole (5-NDZ) drugs in milk samples previous to their determination by micellar electrokinetic chromatography (MEKC). Fat removing and protein precipitation were simultaneously carried out by the addition of trichloroacetic acid (TCA) and subsequent centrifugation. Clean-up and off-line concentration were achieved by a novel solid-phase extraction (SPE) method employing mixed cation exchange (MCX) cartridges, obtaining an off-line concentration factor of 18. Analyses were performed in less than 18min employing 20mM phosphate buffer (pH 6.5) and 150mM SDS as background electrolyte (BGE). During the separation procedure a temperature of 20°C and a voltage of 25kV (normal mode) were applied. Due to sweeping effects, an on-line concentration was achieved for all the studied compounds and detection limits lower than 1.8μgL−1 were obtained. This method has been successfully applied to milk samples of different origins, including raw ewe milk.

Targeting carbonic anhydrase IX by nitroimidazole based sulfamides enhances the therapeutic effect of tumor irradiation: A new concept of dual targeting drugs

Background and purposeCarbonic anhydrase IX (CAIX) plays an important role in pH regulation processes critical for tumor cell growth and metastasis. We hypothesize that a dual targeting bioreductive nitroimidazole based anti-CAIX sulfamide drug (DH348) will reduce tumor growth and sensitize tumors to irradiation in a CAIX dependent manner. Material and methodsThe effect of the dual targeting anti-CAIX (DH348) and its single targeting control drugs on extracellular acidification and radiosensitivity was examined in HT-29 colorectal carcinoma cells. Tumor growth and time to reach 4× start volume (T4×SV) was monitored for animals receiving DH348 (10mg/kg) combined with tumor single dose irradiation (10Gy). ResultsIn vitro, DH348 reduced hypoxia-induced extracellular acidosis, but did not change hypoxic radiosensitivity. In vivo, DH348 monotherapy decreased tumor growth rate and sensitized tumors to radiation (enhancement ratio 1.50) without systemic toxicity only for CAIX expressing tumors. ConclusionsA newly designed nitroimidazole and sulfamide dual targeting drug reduces hypoxic extracellular acidification, slows down tumor growth at nontoxic doses and sensitizes tumors to irradiation all in a CAIX dependent manner, suggesting no “off-target” effects. Our data therefore indicate the potential utility of a dual drug approach as a new strategy for tumor-specific targeting.

Drug library screening against metronidazole-sensitive and metronidazole-resistant Trichomonas vaginalis isolates

ObjectivesMetronidazole and tinidazole are effective treatments for most patients with trichomoniasis but not for individuals who are infected with very resistant strains of Trichomonas vaginalis or persons with hypersensitivity to...

Determination and confirmation of metronidazole, dimetridazole, ronidazole and their metabolites in bovine muscle by LC-MS/MS

Nitroimidazoles are a class of veterinary drugs used for the treatment and prevention of certain bacterial and protozoal diseases in poultry, swine dysentery and genital trichomoniasis in cattle. Since the safety assessment of nitroimidazoles showed them to be genotoxic, carcinogenic and mutagenic, a number of nitroimidazoles have been banned for therapeutic purposes in different countries. Moreover, nitroimidazoles have also been extensively used as growth promoters in food-producing animals. Due to their efficacious improvement in meat production and feed conversion, deliberate use still exists. Therefore, the illegal use of nitroimidazoles in animal husbandry must be monitored. A sensitive method based on LC-MS/MS for the simultaneous determination and confirmation of five banned nitroimidazole drugs including metronidazole, ronidazole, dimetridazole, metronidazole-OH (metabolite of metronidazole), and 2-hydroxymethyl-1-methyl-5-nitroimidazole (metabolite of ronidazole and dimetridazole) in bovine muscle, using ronidazole-d3 as an internal standard, was developed and validated. After extraction with ethyl acetate and evaporation, the nitroimidazoles were reconstituted in petroleum ether and purified, and LC-MS/MS analysis was performed. The method was validated according to Brazilian Regulation 24/2009 (equivalent to European Union Decision 2002/657/EC). Parameters such as decision limit (CCα), detection capability (CCβ), precision, accuracy, uncertaincy and ruggedness were determined. Average accuracy of the five nitroimidazoles from bovine muscle fortified at 5 levels (0.5, 1.0, 1.5, 2.0 and 2.5 μg kg–1) ranged from 96% to 103%. The calculated CCα ranged from 0.0 to 0.17 μg kg–1; CCβ ranged from 0.08 to 0.41 μg kg–1. A complete statistical analysis was performed and the results indicate that the method is robust when subjected to day-to-day analytical variations.

Entamoeba histolytica: identification of thioredoxin-targeted proteins and analysis of serine acetyltransferase-1 as a prototype example

Entamoeba histolytica, the causative agent of amoebiasis, possesses the dithiol-containing redox proteins Trx (thioredoxin) and TrxR (Trx reductase). Both proteins were found to be covalently modified and inactivated by metronidazole, a 5-nitroimidazole drug that is commonly used to treat infections with microaerophilic protozoan parasites in humans. Currently, very little is known about enzymes and other proteins participating in the Trx-dependent redox network of the parasite that could be indirectly affected by metronidazole treatment. On the basis of the disulfide/dithiol-exchange mechanism we constructed an active-site mutant of Trx, capable of binding interacting proteins as a stable mixed disulfide intermediate to screen the target proteome of Trx in E. histolytica. By applying Trx affinity chromatography, two-dimensional gel electrophoresis and MS, peroxiredoxin and 15 further potentially redox-regulated proteins were identified. Among them, EhSat1 (E. histolytica serine acetyltransferase-1), an enzyme involved in the L-cysteine biosynthetic pathway, was selected for detailed analysis. Binding of Trx to EhSat1 was verified by Far-Western blot analysis. Trx was able to restore the activity of the oxidatively damaged EhSat1 suggesting that the TrxR/Trx system protects sensitive proteins against oxidative stress in E. histolytica. Furthermore, the activity of peroxiredoxin, which is dependent on a functioning TrxR/Trx system, was strongly reduced in metronidazole-treated parasites.