Thioredoxin Glutathione Reductase Research Articles

In silico Assessment of Efficacy Against the Zoonotic Parasite Echinococcus granulosus, Pharmacokinetic and Toxicity Predictions for the Siddha Formulation Murukkanvithai Mathirai

Background: Thioredoxin glutathione reductase (TGR) is essential for survival of the zoonotic tapeworm Echinococcus granulosus. Albendazole is the only promising drug for the treatment of cystic echinococcosis. To avoid the development of resistance to albendazole in the parasite, an herbal alternative to albendazole is the need-of-the-hour to combat the development of resistance. Aim: This study aimed to identify active compounds against the parasite Echinococcus granulosus from Murukkan Vithai Mathirai, an anthelmintic formulation used in Siddha practice. Objectives: To evaluate the efficacy of Murukkan Vithai Mathirai (MVM), a Siddha formulation against Echinococcus granulosus, a tapeworm of carnivores, by molecular docking, and to evaluate its ADMET properties using in silico tools. Materials and Methods: The 3D structure of Echinococcus granulosus-thioredoxin-glutathione systems (egTGR) and the phytoconstituents of Piper nigrum, Zingiber officinalis, Piper longum, Cuminum syminum, Coptis teeta, Butea monosperma, and Croton tiglium were obtained from a curated database of Indian Medicinal Plants, Phytochemistry and Therapeutics (IMPPAT). 12 promising phytocompounds were selected based on their physicochemical properties, drug-likeness, bioavailability scores, and medicinal chemistry properties. These phytochemicals were docked individually with the egTGR using Autodock vina and binding affinity (kcal/mol) was recorded for each compound and compared with albendazole, a positive control drug. The results of docking were read using the Biovia Discovery studio visualizer. The ADMET properties of the phytochemicals were analysed using pkCSM and VEGA-QSAR tools. The toxicity score for each phytochemical was calculated by assigning weightage to the toxicities predicted and the weighted average was calculated to compare with the albendazole's weighted average. The geometric mean of NOAELs predicted for MVM phytochemicals was compared with the predicted NOAEL of albendazole. Results: The binding affinity (G) scores of the selected phytocompounds were found to be higher than the albendazole and the phytocompounds with G > -8.0 are as follows, aristolodione, berberastine and pluviatilol of Piper longum, apigenin of Cuminum syminum and prunetin of Butea monosperma, whereas the G of albendazole was -6.7. The calculated toxicity score for MVM (0.56) was significantly lower than albendazole (1.08) and the predicted NOAELs was significantly higher (26.63 mg/kg) than albendazole (3.49), indicating that MVM is less toxic than albendazole. Conclusion: Murukkan Vithai Mathirai is a promising and safe anthelmintic in in silico studies, however, it needs to be validated by in vitro and in vivo studies.

In Silico Comparison of Bioactive Compounds Characterized from Azadirachta indica with an FDA-Approved Drug against Schistosomal Agents: New Insight into Schistosomiasis Treatment.

The burden of human schistosomiasis, a known but neglected tropical disease in Sub-Saharan Africa, has been worrisome in recent years. It is becoming increasingly difficult to tackle schistosomiasis with praziquantel, a drug known to be effective against all Schistosoma species, due to reports of reduced efficacy and resistance. Therefore, this study seeks to investigate the antischistosomal potential of phytochemicals from Azadirachta indica against proteins that have been implicated as druggable targets for the treatment of schistosomiasis using computational techniques. In this study, sixty-three (63) previously isolated and characterized phytochemicals from A. indica were identified from the literature and retrieved from the PubChem database. In silico screening was conducted to assess the inhibitory potential of these phytochemicals against three receptors (Schistosoma mansoni Thioredoxin glutathione reductase, dihydroorotate dehydrogenase, and Arginase) that may serve as therapeutic targets for schistosomiasis treatment. Molecular docking, ADMET prediction, ligand interaction, MMGBSA, and molecular dynamics simulation of the hit compounds were conducted using the Schrodinger molecular drug discovery suite. The results show that Andrographolide possesses a satisfactory pharmacokinetic profile, does not violate the Lipinski rule of five, binds with favourable affinity with the receptors, and interacts with key amino acids at the active site. Importantly, its interaction with dihydroorotate dehydrogenase, an enzyme responsible for the catalysis of the de novo pyrimidine nucleotide biosynthetic pathway rate-limiting step, shows a glide score and MMGBSA of -10.19 and -45.75 Kcal/mol, respectively. In addition, the MD simulation shows its stability at the active site of the receptor. Overall, this study revealed that Andrographolide from Azadirachta indica could serve as a potential lead compound for the development of an anti-schistosomal drug.

A Physiological Approach to Explore How Thioredoxin-Glutathione Reductase (TGR) and Peroxiredoxin (Prx) Eliminate H2O2 in Cysticerci of Taenia.

Peroxiredoxins (Prxs) and glutathione peroxidases (GPxs) are the main enzymes of the thiol-dependent antioxidant systems responsible for reducing the H2O2 produced via aerobic metabolism or parasitic organisms by the host organism. These antioxidant systems maintain a proper redox state in cells. The cysticerci of Taenia crassiceps tolerate millimolar concentrations of this oxidant. To understand the role played by Prxs in this cestode, two genes for Prxs, identified in the genome of Taenia solium (TsPrx1 and TsPrx3), were cloned. The sequence of the proteins suggests that both isoforms belong to the class of typical Prxs 2-Cys. In addition, TsPrx3 harbors a mitochondrial localization signal peptide and two motifs (-GGLG- and -YP-) associated with overoxidation. Our kinetic characterization assigns them as thioredoxin peroxidases (TPxs). While TsPrx1 and TsPrx3 exhibit the same catalytic efficiency, thioredoxin-glutathione reductase from T. crassiceps (TcTGR) was five and eight times higher. Additionally, the latter demonstrated a lower affinity (>30-fold) for H2O2 in comparison with TsPrx1 and TsPrx3. The TcTGR contains a Sec residue in its C-terminal, which confers additional peroxidase activity. The aforementioned aspect implies that TsPrx1 and TsPrx3 are catalytically active at low H2O2 concentrations, and the TcTGR acts at high H2O2 concentrations. These results may explain why the T. crassiceps cysticerci can tolerate high H2O2 concentrations.

Fragment library screening by X-ray crystallography and binding site analysis on thioredoxin glutathione reductase of Schistosoma mansoni.

Schistosomiasis is caused by parasites of the genus Schistosoma, which infect more than 200 million people. Praziquantel (PZQ) has been the main drug for controlling schistosomiasis for over four decades, butdespite that it is ineffective against juvenile worms and size and taste issues with its pharmaceutical forms impose challenges for treating school-aged children. It is also important to note that PZQ resistant strains can be generated in laboratory conditions and observed in the field, hence its extensive use in mass drug administration programs raises concerns about resistance, highlighting the need to search for new schistosomicidal drugs. Schistosomes survival relies on the redox enzyme thioredoxin glutathione reductase (TGR), a validated target for the development of new anti-schistosomal drugs. Here we report a high-throughput fragment screening campaign of 768 compounds against S. mansoni TGR (SmTGR) using X-ray crystallography. We observed 49 binding events involving 35 distinct molecular fragments which were found to be distributed across 16 binding sites. Most sites are described for the first time within SmTGR, a noteworthy exception being the "doorstop pocket" near the NADPH binding site. We have compared results from hotspots and pocket druggability analysis of SmTGR with the experimental binding sites found in this work, with our results indicating only limited coincidence between experimental and computational results. Finally, we discuss that binding sites at the doorstop/NADPH binding site and in the SmTGR dimer interface, should be prioritized for developing SmTGR inhibitors as new antischistosomal drugs.

Fasciola hepatica antioxidant and protease-inhibitor cocktail recombinant vaccines administered five times elicit potent and sustained immune responses in sheep but do not confer protection

Our laboratory’s vaccine development strategy against the livestock parasite Fasciola hepatica centres around disrupting key biological processes by combining groups of antigens with similar/complementary functional actions into a single vaccine cocktail. In this study the focus was on antioxidant protein vaccines and a protease inhibitor vaccine aimed at disrupting the parasite’s ability to defend against oxidative stress and protease-inhibitor balance, respectively. Two combinations of recombinantly expressed antioxidants were assessed, namely peroxiredoxin (rFhPrx), thioredoxin (rFhTrx) and thioredoxin-glutathione reductase (rFhTGR) (Group 1) and rFhPrx, rFhTrx, and two superoxide dismutases (rFhSOD1 and rFhSOD3) (Group 2). The protease inhibitor vaccine cocktail included representatives of each of the key secreted protease inhibitor families, namely a Kunitz-type inhibitor (rFhKT1), a serpin (rFhSrp1) and a stefin, (rFhStf1) (Group 3). The vaccine combinations were formulated in adjuvant Montanide 61VG administered at five timepoints; two before experimental challenge with 60 F. hepatica metacercariae and three after infection. The vaccine combinations did not reduce the liver fluke burden, and only Group 2 displayed a marginal reduction in egg viability (8.2%). Despite previous results showing an effect of liver fluke vaccines on overall weight gain in infected animals, no significant (P value >0.05) impact on weight gain was observed in this study. Antibodies were elicited against all the vaccine antigens within the cocktails and were maintained at high levels to the end of the trial, due to our strategy of continuing vaccine administration after infection. However, these responses were not boosted by the challenge F. hepatica infection. A comparative analysis with previous vaccine data using a protease inhibitor vaccine found no repeat of the promising outcomes associated with this vaccine, indicating that the addition of rFhSrp1 to the vaccine cocktail did not improve vaccine efficacy. Assessment of liver pathology across the two trials using a modified liver enzyme score (glutamate dehydrogenase to platelet ratio) at eight weeks post infection suggests an association with liver fluke burden above 45 flukes, which could be used to predict liver pathology in future trials. The results reported in this study highlight the ambiguousness in liver fluke vaccine development and the difficulty in obtaining consistent and repeatable protection. This work stresses the need for repetition of trials and the use of sufficiently sized groups to assess vaccine efficacy with adequate statistical power.

Assigning function to active site residues of Schistosoma mansoni thioredoxin/glutathione reductase from analysis of transient state reductive half-reactions with variant forms of the enzyme.

Thioredoxin/glutathione reductase (TGR) from the platyhelminthic parasitic worms has recently been identified as a drug target for the treatment of schistosomiasis. Schistosomes lack catalase, and so are heavily reliant on the regeneration of reduced thioredoxin (Trx) and glutathione (GSH) to reduce peroxiredoxins that ameliorate oxidative damage from hydrogen peroxide generated by the host immune response. This study focuses on the characterization of the catalytic mechanism of Schistosoma mansoni TGR (SmTGR). Variant forms of SmTGR were studied to assign the function of residues that participate in the electron distribution chain within the enzyme. Using anaerobic transient state spectrophotometric methods, redox changes for the FAD and NADPH were observed and the function of specific residues was defined from observation of charge transfer absorption transitions that are indicative of specific complexations and redox states. The C159S variant prevented distribution of electrons beyond the flavin and as such did not accumulate thiolate-FAD charge transfer absorption. The lack of this absorption facilitated observation of a new charge transfer absorption consistent with proximity of NADPH and FAD. The C159S variant was used to confine electrons from NADPH at the flavin, and it was shown that NADPH and FAD exchange hydride in both directions and come to an equilibrium that yields only fractional FAD reduction, suggesting that both have similar reduction potentials. Mutation of U597 to serine resulted in sustained thiolate-FAD charge transfer absorption and loss of the ability to reduce Trx, indicating that the C596-U597 disulfide functions in the catalytic sequence to receive electrons from the C154 C159 pair and distribute them to Trx. No kinetic evidence for a loss or change in function associated with the distal C28-C31 disulfide was observed when the C31S variant reductive half-reaction was observed. The Y296A variant was shown to slow the rate of but increase extent of reduction of the flavin, and the dissociation of NADP+. The H571 residue was confirmed to be the residue responsible for the deprotonation of the C159 thiol, increasing its reactivity and generating the prominent thiolate-FAD charge transfer absorption that accumulates with oxidation of the flavin.

Non-covalent inhibitors of thioredoxin glutathione reductase with schistosomicidal activity in vivo

Only praziquantel is available for treating schistosomiasis, a disease affecting more than 200 million people. Praziquantel-resistant worms have been selected for in the lab and low cure rates from mass drug administration programs suggest that resistance is evolving in the field. Thioredoxin glutathione reductase (TGR) is essential for schistosome survival and a validated drug target. TGR inhibitors identified to date are irreversible and/or covalent inhibitors with unacceptable off-target effects. In this work, we identify noncovalent TGR inhibitors with efficacy against schistosome infections in mice, meeting the criteria for lead progression indicated by WHO. Comparisons with previous in vivo studies with praziquantel suggests that these inhibitors outperform the drug of choice for schistosomiasis against juvenile worms.

Inhibiting thioredoxin glutathione reductase is a promising approach to controlling Cryptocaryon irritans infection in fish

Marine cultured fish often suffer from Cryptocaryon irritans infection, which causes enormous mortality. C. irritans is resistant to oxidative damage induced by zinc. To develop an effective drug to control the parasite, a putative thioredoxin glutathione reductase (CiTGR) from C. irritans was cloned and characterized. CiTGR was designed as a target to screen for inhibitors by molecular docking. The selected inhibitors were tested both in vitro and in vivo. The results showed that CiTGR is located in the nucleus of the parasite, possesses a common pyridine-oxidoreductases redox active center, and lacks a glutaredoxin active site. Recombinant CiTGR exhibited high TrxR activity but low glutathione reductase activity. Shogaol was found to significantly suppress TrxR activity and enhance toxicity of zinc on C. irritans (P < 0.05). The abundance of C. irritans on the fish body decreased significantly after oral administration of shogaol (P < 0.05). These results implied that CiTGR could be used to screen for drugs that weaken the resistance of C. irritans to oxidative stress, which is critical for controlling the parasite in fish. This paper deepens the understanding of the interaction between ciliated parasites and oxidative stress.

Structure-based drug design of multi-targeting inhibitors of human pathogen fungi

The development of multi-targeting inhibitors is proposed to improve the antifungal effect. In a single chemical entity it is possible to inhibit two different targets. Thioredoxin reductase and glutathione reductase belong to the two main systems responsible for the maintenance of cell homeostasis. Herein, we describe the discovery and selection of dual-inhibitors targeting thioredoxin reductase and glutathione reductase proteins of Aspergillus fumigatus. Both proteins were modeled and molecular dynamics were performed to obtain different conformational frames to ensemble docking. After the independent virtual screening of each protein, a ranking containing molecules selected for both proteins were generated. Fourteen molecules were tested in vitro against A. fumigatus, Fusarium oxysporum, Paracoccidioides brasiliensis, Candida albicans and Cryptococcus neoformans by broth microdilution assay. Octenidine (OCT) was the most promising molecule which exhibited MIC < 0.5 µg/mL for all fungi tested. The literature describes the action of OCT in the cell membrane of gram-negative bacteria. Thus, we assume that the mechanism in which OCT acts on fungal cells is an association of disrupting the cell membrane and also the dual-inhibition of thioredoxin reductase and glutathione reductase which implies the increase of ROS and further fungal cell death.

Synthesis, virtual screening, and computational approach of 6-(4-metoxyphenyl)-4-phenyl-1,2-dihydropyrimidin-2-one as a potential target for thioredoxin glutathione reductase (TGR)

The three-component condensation reaction of 4-methoxybenzaldehyde with acetophenone and urea in the presence of CF3COOH was investigated and as a result, 6-(4-methoxyphenyl)-4-phenyl-1,2-dihydropyrimidine-2-one was synthesized. The structure of the synthesized compound was confirmed by the X-ray method. Both the DFT and TD-DFT computations for comprehensive analysis of spectral data, and electronic parameters were carried out as an add-on to synthetic research and well corroborated with the experimental results. NBO, NLO, and Hirshfeld surface and energy framework analysis were also used to segregate the different intermolecular hydrogen bonds involved in the molecular packing strength. All DFT findings imply that the synthesized molecule has enhanced non-linear optical properties increased charge transfer and is a highly stable, and proactive candidate for drugs. Furthermore, molecular docking studies were carried out to investigate the binding mechanism between 6-(4-metoxyphenyl)-4-phenyl-1,2-dihydropyrimidin-2-one and the thioredoxin glutathione reductase (TGR) complex. ADMET parameter analysis was also carried out for the compound to understand the compound's safety and compound efficiency for the drug candidate.

Functional expression, localization, and biochemical characterization of thioredoxin glutathione reductase from air-breathing magur catfish, Clarias magur

The glutathione (GSH) and thioredoxin (Trx) systems regulate cellular redox homeostasis and maintain antioxidant defense in most eukaryotes. We earlier reported the absence of gene coding for the glutathione reductase (GR) enzyme of the GSH system in the facultative air-breathing catfish, Clarias magur. Here, we identified three thioredoxin reductase (TrxR) genes, one of which was later confirmed as a thioredoxin glutathione reductase (TGR). We then characterized the novel recombinant TGR enzyme of C. magur (CmTGR). The tissue-specific expression of the txnrd genes and the tissue-specific activity of the TrxR enzyme were analyzed. The recombinant CmTGR is a dimer of ~133 kDa. The protein showed TrxR activity with 5,5′-diothiobis (2-nitrobenzoic acid) reduction assay with a Km of 304.40 μM and GR activity with a Km of 58.91 μM. Phylogenetic analysis showed that the CmTGR was related to the TrxRs of fishes and distantly related to the TGRs of platyhelminth parasites. The structural analysis revealed the conserved glutaredoxin active site and FAD- and NADPH-binding sites. To our knowledge, this is the first report of the presence of a TGR in any fish. This unusual presence of TGR in C. magur is crucial as it helps maintain redox homeostasis under environmental stressors-induced oxidative stress.

A novel thioredoxin glutathione reductase from evolutionary ancient metazoan Hydra

Thioredoxin (Trx) and glutathione disulfide (GSSG), are regenerated in reduced state by thioredoxin reductase (TrxR) and glutathione reductase (GR) respectively. A novel protein thioredoxin glutathione reductase (TGR) capable of reducing Trx as well as GSSG, linking two redox systems, has only been reported so far from parasitic flat worms and mammals. For the first time, we report a multifunctional antioxidant enzyme TGR from the nonparasitic, nonmammalian cnidarian Hydra vulgaris (HvTGR) which is a selenoprotein with unusual fusion of a TrxR domain with glutaredoxin (Grx) domain. We have cloned and sequenced HvTGR which encodes a polypeptide of 73 kDa. It contains conserved sequence CPYC of Grx domain, and CVNVGC and GCUG domains of thioredoxin reductase. Phylogenetic analysis revealed HvTGR to be closer to TGR from mammals rather than to TGR from parasitic helminths. We then subcloned HvTGR in plasmid pSelExpress-1 and expressed it in HEK293T cells to ensure selenocysteine incorporation. Purified HvTGR showed Grx, glutathione reductase and TrxR activities. Both thioredoxin and GSSG disulfide reductase activities were inhibited by 1-Chloro-2,4-dinitrobenzene (DNCB) supporting the existence of an essential selenocysteine residue. HvTGR expression was induced in response to H2O2 in Hydra. Interestingly, inhibition of HvTGR by DNCB, inhibited regeneration in Hydra indicating its involvement in other cellular processes.

Evaluating the effect of curcumin on the metacestode of Taenia crassiceps

Curcumin, a curcuminoid present in the rhizome of the plant Curcuma longa has multiple pharmacological effects including anticarcinogenic and anti-inflammatory properties. This work evaluates the anthelmintic effect of the curcumin molecule (98% pure) on Taenia crassiceps cysticerci viability in vitro. Cysticerci incubated in the presence of increasing concentrations of curcumin showed a dose-dependent mortality correlated with a significant increase in the production of reactive oxygen species and a partial inhibition of thioredoxin-glutathione reductase, the only disulfide reductase present in these parasites. At 500 μM curcumin, a 100% of cysticerci lethality was obtained after 2 h of treatment. These results suggest the curcumin-induced oxidative stress could be in the origin of the anthelminthic effect of curcumin. Mice with cysticerci were injected intraperitoneally with 20, 40, or 60 mM curcumin daily for 30 days. A decrease in the burden of cysticerci (46%) was observed with a 60 mM dose of curcumin, supporting this compound as a potential anthelmintic drug.

Redox regulation by TXNRD3 during epididymal maturation underlies capacitation-associated mitochondrial activity and sperm motility in mice

During epididymal transit, redox remodeling protects mammalian spermatozoa, preparing them for survival in the subsequent journey to fertilization. However, molecular mechanisms of redox regulation in sperm development and maturation remain largely elusive. In this study, we report that thioredoxin-glutathione reductase (TXNRD3), a thioredoxin reductase family member particularly abundant in elongating spermatids at the site of mitochondrial sheath formation, regulates redox homeostasis to support male fertility. Using Txnrd3−/− mice, our biochemical, ultrastructural, and live cell imaging analyses revealed impairments in sperm morphology and motility under conditions of TXNRD3 deficiency. We find that mitochondria develop more defined cristae during capacitation in wildtype sperm. Furthermore, we show that absence of TXNRD3 alters thiol redox status in both the head and tail during sperm maturation and capacitation, resulting in defective mitochondrial ultrastructure and activity under capacitating conditions. These findings provide insights into molecular mechanisms of redox homeostasis and bioenergetics during sperm maturation, capacitation, and fertilization.

First In Silico Screening of Insect Molecules for Identification of Novel Anti-Parasitic Compounds.

Schistosomiasis is a neglected tropical disease caused by blood flukes of the genus Schistosoma. In silico screenings of compounds for the identification of novel anti-parasitic drug candidates have received considerable attention in recent years, including the screening of natural compounds. For the first time, we investigated molecules from insects, a rather neglected source in drug discovery, in an in silico screening approach to find novel antischistosomal compounds. Based on the Dictionary of Natural Products (DNP), we created a library of 1327 insect compounds suitable for molecular docking. A structure-based virtual screening against the crystal structure of a known druggable target in Schistosoma mansoni, the thioredoxin glutathione reductase (SmTGR), was performed. The top ten compounds predominantly originated from beetles and were predicted to interact particularly with amino acids in the doorstop pocket of SmTGR. For one compound from a jewel beetle, buprestin H, we tested and confirmed antischistosomal activity against adult and juvenile parasites in vitro. At concentrations with anti-parasitic activity, we could also exclude any unspecific cytotoxic activity against human HepG2 cells. This study highlights the potential of insect molecules for the identification of novel antischistosomal compounds. Our library of insect-derived molecules could serve not only as basis for future in silico screenings against additional target proteins of schistosomes, but also of other parasites.

Deep Neural Networks Predict Inhibitors ofSchistosoma MansoniThioredoxin Glutathione Reductase (SmTGR)

Over 200 million people worldwide are affected annually by schistosomiasis with debilitating socio-economic effects. Praziquantel remains the main chemotherapy against this neglected tropical disease but there are reports of drug resistance. Therefore, necessitating the need to identify potential biotherapeutic molecules. The study describes the first deep learning (DL)-based computational models for predicting inhibitors of Schistosoma mansoni Thioredoxin glutathione reductase (SmTGR), which is an essential protein for the survival of the helminths in the host. The state-of-the-art performance of DL in similar applications makes it ideal to deploy on bioactive datasets of the SmTGR drug target. Cost-sensitive deep neural network classifiers were trained using the binary classification approach. Based on the area under curve (AUC) of the receiver operating characteristic (ROC) Curve scores (86.3–86.5%), the five best models generated were able to classify inhibitors with high accuracy (85–90%). Additionally, the DL classifier outperformed random forest by far. This is a proof of concept that deep neural networks can efficiently and robustly classify active schistosomal molecules from inactive. The generated models could be used to screen large-scale compound libraries to prioritize potential inhibitors for experimental characterization.

Ailanthus excelsa leaf extract: Chemical characterization, antischistosomal activity, and in silico study of isolated phenolic compounds as promising thioredoxin glutathione reductase inhibitors

Ailanthus excelsa leaf extract: Chemical characterization, antischistosomal activity, and in silico study of isolated phenolic compounds as promising thioredoxin glutathione reductase inhibitors

Probing the Surface of a Parasite Drug Target Thioredoxin Glutathione Reductase Using Small Molecule Fragments.

Fragment screening is a powerful drug discovery approach particularly useful for enzymes difficult to inhibit selectively, such as the thiol/selenol-dependent thioredoxin reductases (TrxRs), which are essential and druggable in several infectious diseases. Several known inhibitors are reactive electrophiles targeting the selenocysteine-containing C-terminus and thus often suffering from off-target reactivity in vivo. The lack of structural information on the interaction modalities of the C-terminus-targeting inhibitors, due to the high mobility of this domain and the lack of alternative druggable sites, prevents the development of selective inhibitors for TrxRs. In this work, fragments selected from actives identified in a large screen carried out against Thioredoxin Glutathione Reductase from Schistosoma mansoni (SmTGR) were probed by X-ray crystallography. SmTGR is one of the most promising drug targets for schistosomiasis, a devastating, neglected disease. Utilizing a multicrystal method to analyze electron density maps, structural analysis, and functional studies, three binding sites were characterized in SmTGR: two sites are close to or partially superposable with the NADPH binding site, while the third one is found between two symmetry related SmTGR subunits of the crystal lattice. Surprisingly, one compound bound to this latter site stabilizes, through allosteric effects mediated by the so-called guiding bar residues, the crucial redox active C-terminus of SmTGR, making it finally visible at high resolution. These results further promote fragments as small molecule probes for investigating functional aspects of the target protein, exemplified by the allosteric effect on the C-terminus, and providing fundamental chemical information exploitable in drug discovery.

Autonomous Non Antioxidant Roles for Fasciola hepatica Secreted Thioredoxin-1 and Peroxiredoxin-1.

Trematode parasites of the genus Fasciola are the cause of liver fluke disease (fasciolosis) in humans and their livestock. Infection of the host involves invasion through the intestinal wall followed by migration in the liver that results in extensive damage, before the parasite settles as a mature egg-laying adult in the bile ducts. Genomic and transcriptomic studies revealed that increased metabolic stress during the rapid growth and development of F. hepatica is balanced with the up-regulation of the thiol-independent antioxidant system. In this cascade system thioredoxin/glutathione reductase (TGR) reduces thioredoxin (Trx), which then reduces and activates peroxiredoxin (Prx), whose major function is to protect cells against the damaging hydrogen peroxide free radicals. F. hepatica expresses a single TGR, three Trx and three Prx genes; however, the transcriptional expression of Trx1 and Prx1 far out-weighs (>50-fold) other members of their family, and both are major components of the parasite secretome. While Prx1 possesses a leader signal peptide that directs its secretion through the classical pathway and explains why this enzyme is found freely soluble in the secretome, Trx1 lacks a leader peptide and is secreted via an alternative pathway that packages the majority of this enzyme into extracellular vesicles (EVs). Here we propose that F. hepatica Prx1 and Trx1 do not function as part of the parasite’s stress-inducible thiol-dependant cascade, but play autonomous roles in defence against the general anti-pathogen oxidative burst by innate immune cells, in the modulation of host immune responses and regulation of inflammation.

Synthesis of oxadiazole-2-oxide derivatives as potential drug candidates for schistosomiasis targeting SjTGR

BackgroundSchistosomiasis is a chronic parasitic disease that affects millions of people’s health worldwide. Because of the increasing drug resistance to praziquantel (PZQ), which is the primary drug for schistosomiasis, developing new drugs to treat schistosomiasis is crucial. Oxadiazole-2-oxides have been identified as potential anti-schistosomiasis reagents targeting thioredoxin glutathione reductase (TGR).MethodsIn this work, one of the oxadiazole-2-oxides derivatives furoxan was used as the lead compound to exploit a series of novel furoxan derivatives for studying inhibitory activity against both recombinant Schistosoma japonicum TGR containing selenium (rSjTGR-Sec) and soluble worm antigen protein (SWAP) containing wild-type Schistosoma japonicum TGR (wtSjTGR), in order to develop a new leading compound for schistosomiasis. Thirty-nine novel derivatives were prepared to test their activity toward both enzymes. The docking method was used to detect the binding site between the active molecule and SjTGR. The structure–activity relationship (SAR) of these novel furoxan derivatives was preliminarily analyzed.ResultsIt was found that several new derivatives, including compounds 6a–6d, 9ab, 9bd and 9be, demonstrated greater activity toward rSjTGR-Sec or SWAP containing wtSjTGR than did furoxan. Interestingly, all intermediates bearing hydroxy (6a–6d) showed excellent inhibitory activity against both enzymes. In particular, compound 6d with trifluoromethyl on a pyridine ring was found to have much higher inhibition toward both rSjTGR-Sec (half-maximal inhibitory concentration, IC50,7.5nM) and SWAP containing wtSjTGR (IC50 55.8nM) than furoxan. Additionally, the docking method identified the possible matching sites between 6d and Schistosoma japonicum TGR (SjTGR), which theoretically lends support to the inhibitory activity of 6d.ConclusionThe data obtained herein showed that 6d with trifluoromethyl on a pyridine ring could be a valuable leading compound for further study.Graphical