Molecular characterization and phylogenetic analyses of Fasciola gigantica of buffaloes and goats in Punjab, Pakistan.

Fasciola gigantica is considered to be a major pathogen causing fasciolosis in the Indian subcontinent, resulting in production losses of millions of dollars in the livestock industry. Understading the dispersal origin and the patterns of spread of F. gigantica is important. A total of 53 Fasciola flukes collected from buffaloes and goats in Punjab, Pakistan between 2017 and 2018 were identified as F. gigantica based on the multiplex PCR for the phosphoenolpyruvate carboxykinase (pepck) and the PCR-restriction fragment length polymorphism (RFLP) for DNA polymerase delta (pold). A significant genetic difference between F. gigantica from buffaloes and goats was indicated by the genetic analyses of mitochondrial markers, NADH dehydrogenase subunit 1 (nad1) and cytochrome C oxidase subunit 1 (cox1). Phylogenetic analysis of the seventeen nad1 haplotypes of F. gigantica from Pakistan with those in neighbouring countries of the Indian subcontinent revealed that all the haplotypes identified in Pakistan were clustered in haplogroup A. fasciola gigantica with the eight haplotypes might be expanded in Pakistan from Indian origin, along with the migration of the domestic animals, since they were related to Indian haplotypes. In contrast, the remaining nine haplotypes were not shared with any neighbouring countries, suggesting independent origin, probably from neighbouring Middle East countries. However, cautious interpretation is required due to the very limited samples size of this study. Our study provides a proof of concept for a method that could be used to investigate the epidemiology of F. gigantica.

Identification and characterization of cytosolic malate dehydrogenase from the liver fluke Fasciola gigantica.

The liver fluke zoonoses, Fasciola spp. are parasitic helminths infecting humans and animals globally. Recent sequencing of the genome of Fasciola gigantica has provided a basis to understand the biochemistry of this parasite. Here, we identified the cytosolic malate dehydrogenase in F. gigantica (FgMDH) and characterized the enzyme biochemically and structurally. F. gigantica encodes a single cytosolic MDH, a key enzyme of the citric acid cycle. It catalyzes the reversible oxidation of malate to oxaloacetate using NAD+. The Fgmdh gene was amplified and cloned for expression of the recombinant protein. The purified protein showed a molecular weight of ~ 36 kDa that existed in a dimeric form in solution. The recombinant enzyme was catalytically active as it catalyzed both forward and reverse reactions efficiently. The kinetic parameters were determined for both directions. The structure of FgMDH and human MDH were modeled and validated. The superimposition of both the model structures showed overall structural similarity in the active site loop region, however, the conformation of the residues was different. Molecular docking elucidated the binding sites and affinities of the substrates and cofactors to the enzyme. Simulation of molecular dynamics and principal component analysis indicated the stability of the systems and collective motions, respectively. Understanding the structural and functional properties of MDH is important to better understand the roles of this enzyme in the biochemistry of the parasite.

Genome-wide identification and characterization of eukaryotic protein kinases.

The tropical liver fluke, Fasciola gigantica is a food-borne parasite responsible for the hepatobiliary disease fascioliasis. The recent completion of F. gigantica genome sequencing by our group has provided a platform for the systematic analysis of the parasite genome. Eukaryotic protein kinases (ePKs) are regulators of cellular phosphorylation. In the present study, we used various computational and bioinformatics tools to extensively analyse the ePKs in F. gigantica (FgePKs) genome. A total of 455 ePKs were identified that represent ~2% of the parasite genome. Out of these, 214 ePKs are typical kinases (Ser/Thr- and Tyr-specific ePKs), and 241 were other kinases. Several FgePKs were found to possess unusual domain architectures, which suggests the diverse nature of the proteins that can be exploited for designing novel inhibitors. 115 kinases showed <35% query coverage when compared to human ePKs highlighting significant divergences in their respective kinomes, further providing a platform for novel structure-based drug designing. This study provides a platform that may open new avenues into our understanding of helminth biochemistry and drug discovery.

Designing a vaccine for fascioliasis using immunogenic 24 kDa mu-class glutathione s-transferase.

Fascioliasis, caused by the liver fluke Fasciola gigantica, is a significant zoonotic disease of the livestock and human, causing substantial economic loss worldwide. Triclabendazole (TCBZ) is the only drug available for the management of the disease against which there is an alarming increase in drug resistance. No vaccine is available commercially for the protection against this disease. Increasing resistance to TCBZ and the lack of a successful vaccine against fascioliasis demands the development of vaccines. In the present study, a structural immunoinformatics approach was used to design a multi-epitope subunit vaccine using the glutathione S-transferase (GST) protein of Fasciola gigantica. The GST antigen is a safe, non-allergic, highly antigenic, and effective vaccine candidate against various parasitic flukes and worms. The cytotoxic T lymphocytes, helper T lymphocytes, and B-cell epitopes were selected for constructing the vaccine based on their immunogenic behavior and binding affinity. The physicochemical properties, allergenicity, and antigenicity of the designed vaccine were analyzed. To elucidate the tertiary structure of the vaccine, homology modeling was performed, followed by structure refinement and docking against the TLR2 immune receptor. Molecular dynamics simulations showed a stable interaction between the vaccine and the receptor complex. Finally, in silico cloning was performed to evaluate the expression and translation of the vaccine construct in the E. coli expression system. Further studies require experimental validation for the safety and immunogenic behavior of the designed vaccine.

Monoclonal antibody against Fasciola gigantica glutathione peroxidase and their immunodiagnosis potential for fasciolosis.

Glutathione peroxidases (GPx), major antioxidant enzymes, secreted by Fasciola spp., are important for the parasite evasion and protection against the host's immune responses. In the present study, a monoclonal antibody (MoAb) against recombinant F. gigantica glutathione peroxidase (rFgGPx) was produced by hybridoma technique using spleen cells from BALB/c mice immunized with rFgGPx. This MoAb (named 7B8) is IgG1 with κ light chains, and it reacted specifically with rFgGPx at a molecular weight 19 kDa as shown by immunoblotting, and reacted with the native FgGPx in the extracts of whole body (WB), metacercariae, newly excysted juveniles (NEJs), 4 week-old juveniles and adult F. gigantica as shown by indirect ELISA. It did not cross react with antigens in WB fractions from other adult trematodes, including Fischoederius cobboldi, Paramphistomum cervi, Setaria labiato-papillosa, Eurytrema pancreaticum, Gastrothylax crumenifer and Gigantocotyle explanatum. By immunolocalization, MoAb against rFgGPx reacted with the native protein in the tegument, vitelline cells, and eggs of adult F. gigantica. In addition, the sera from mice experimentally infected with F. gigantica were tested positive by this indirect sandwich ELISA. This result indicated that FgGPx is an abundantly expressed parasite protein that is secreted into the tegumental antigens (TA), therefore, FgGPx and its MoAb may be used for immunodiagnosis of both early and late fasciolosis gigantica in animals and humans.

Development of Cytochrome B, a new candidate gene for a high accuracy detection of Fasciola eggs in fecal specimens.

Fasciolosis among domestic ruminants has resulted in a decrease in the production of milk products and has occasionally led to the deaths of young ruminants due to of acute infections. This study aimed to discriminate between the eggs of Fasciola gigantica and other trematode eggs in samples collected from ruminant feces specimens using PCR-based methods with the new candidate gene Cytochrome B (CYTB). A species-specific primer was developed with a high degree of sensitivity (3.285 pg). The primer was able to amplify the F. gigantica genomic DNA and there were no positive results with the other related trematodes (Paramphistomum sp., Orthocoelium sp., Fischoederius sp., Calicophoron sp., Echinostoma revolutum, E. cinetorchis, E. ilocanum and Isthmiophora hortensis), freshwater snails (Lymnaea auricularia, Bithynia siamensis, Indoplanorbis exustus, Melanoides tuberculata, Tarebia granifera) or definitive hosts (Bos primigenius and Bubalus bubalis). The minimum concentration of DNA from eggs that could be give a positive result was 3.285 pg. Moreover, the results of the study confirmed the existence of F. gigantica in Nakhon Pathom Province with a high prevalence (28.57%) and revealed the area of infection through epidemiological mapping. Thus, the species-specific primer and epidemiological data in this study may be helpful for use in epidemiological studies, phylogenetic studies and veterinary studies in the future.

Conserved Arg451 residue is critical for maintaining the stability and activity of thioredoxin glutathione reductase.

Thioredoxin glutathione reductase (TGR), a potential anthelminthic drug target causes NADPH-dependent transfer of electrons to both thioredoxins and glutathione systems. In the present study, we showed that a single point mutation conserved at Arg451 position is critical for maintaining the structure-function of FgTGR. The current biochemical results showed that R451A mutation significantly decreases both oxidoreductase activities (glutathione reductase and thioredoxin reductase) of the enzyme. Computational analyses using molecular dynamics simulation provided an in-depth insight into the structural alterations caused as a result of the mutation. Furthermore, the different regions of the mutant FgTGR structure were found to be altered in flexibility/rigidity as a result of the mutation. This led to mutant-specific conformational alterations and dominant differential motions that contributed to the abrogated function of mutant FgTGR. These results were confirmed using GdnHCl-induced denaturation-based stability studies. Moreover, mutation reduced the free energy of stabilization of the protein, thereby destabilizing the mutant protein structure. Therefore, these findings displayed differential dynamics in the FgTGR structure and highlighted the relevance of residue-level interactions in the protein. Thus, the current study provided a basis for exploiting regions other than the active site of TGR for inhibitory effect and development of novel antihelminthics.

The cathepsin-like cysteine peptidases of trematodes of the genus Fasciola.

Fasciolosis caused by trematode parasites of the genus Fasciola is a global disease of livestock, particularly cattle, sheep, water buffalo and goats. It is also a major human zoonosis with reports suggesting that 2.4-17 million people are infected worldwide, and 91.1 million people currently living at risk of infection. A unique feature of these worms is their reliance on a family of developmentally-regulated papain-like cysteine peptidases, termed cathepsins. These proteolytic enzymes play central roles in virulence, infection, tissue migration and modulation of host innate and adaptive immune responses. The availability of a Fasciola hepatica genome, and the exploitation of transcriptomic and proteomic technologies to probe parasite growth and development, has enlightened our understanding of the cathepsin-like cysteine peptidases. Here, we clarify the structure of the cathepsin-like cysteine peptidase families and, in this context, review the phylogenetics, structure, biochemistry and function of these enzymes in the host-parasite relationship.

Identification and characterization of glyceraldehyde 3-phosphate dehydrogenase from Fasciola gigantica.

Fasciola gigantica is an important food-borne trematode responsible for the hepatobiliary disease, commonly known as fascioliasis. In F. gigantica, the glyceraldehyde 3-phosphate dehydrogenase (FgGAPDH) is a key enzyme of the glycolytic pathway and catalyzes the reversible oxidative phosphorylation of D-glyceraldehyde-3-phosphate (G-3-P) to 1,3-bisphosphoglycerate (1,3-BPG), with the simultaneous reduction of NAD+ to NADH. In the present study, we analyzed the sequence of FgGAPDH and investigated its structural, binding, and catalytic properties. Sequence alignment of FgGAPDH showed 100% identity with the sister fluke Fasciola hepatica GAPDH. The gapdh gene was cloned and expressed in Escherichia coli, and the recombinant protein was purified. The purified FgGAPDH exists as a homo-tetramer, composed of a ~ 37-kDa subunit under non-dissociating conditions at 300 mM salt concentration indicating that higher salt stabilizes the tetrameric state. The binding of the cofactor NAD+ caused a conformational rearrangement in the enzyme structure, leading to the stabilization of the enzyme. A homology model of FgGAPDH was constructed, the cofactor (NAD+) and substrate (G-3-P) were docked, and the binding sites were identified in a single chain. The inter-subunit cleft of GAPDH that has been exploited for structure-based drug design in certain protozoan parasites is closed in the case of FgGAPDH, similar to the human GAPDH. Thus, the conformation of FgGAPDH in this region is similar to the human enzyme. Therefore, GAPDH may not be a suitable target for drug discovery against fascioliasis. Still, the analysis of the structural and functional attributes of GAPDH will be significant in understanding the various roles of this enzyme in the parasite as well as provide new insights into the biochemistry of flukes.

Evaluation of a Commercial Enzyme-Linked Immunosorbent Assay Kit and In-House Fasciola gigantica Cysteine Proteinases-Based Enzyme-Linked Immunosorbent Assays for Diagnosis of Human Fascioliasis.

Fascioliasis, caused by Fasciola hepatica and Fasciola gigantica infection, is a major food-borne trematodiasis in many places of the world, with the central region of Vietnam being reported as a highly endemic area. Stool examination for Fasciola eggs is not a sensitive method, and immunodiagnostic methods are preferable. We investigated various enzyme-linked immunosorbent assays (ELISAs) to evaluate their efficacy for fascioliasis diagnosis. Test sera used are primarily screened using an ELISA kit produced in Vietnam (VN kit; Viet Sinh Chemical Producing & Trading Co. Ltd., Ho Chi Minh City, Vietnam): Seropositive individuals having symptoms compatible with fascioliasis were regarded as clinically diagnosed fascioliasis cases. A commercial Fasciola IgG ELISA kit from Diagnostic Automation/Cortez Diagnostics, Inc. (USA kit; Woodland Hills, CA), which has been commonly used in Vietnam, was assessed and compared with in-house ELISA systems, including a cystatin-capture (CC) ELISA using crude worm extract (CWE) and an indirect ELISA using a synthetic peptide Ac-TPTCHWECQVGYNKTYDEE-NHMe designed from the F. gigantica cathepsin B (FgCB5) molecule. The USA kit was suitable for routine diagnosis after recalibration of the manufacturer's suggested cutoff point. Cystatin-capture ELISA with CWE provided good sensitivity and specificity with perfect agreement to the results of the USA kit. In dot-blot ELISA, recombinant FgCB5 reacted more strongly with human antisera than did other F. gigantica antigens tested. Enzyme-linked immunosorbent assay using the synthetic peptide fragment of the FgCB5 exhibited nearly 80% sensitivity and specificity, but the test results showed low agreement with CC-ELISA or the USA kit. In conclusion, the commercially available Fasciola IgG ELISA kit from the United States and the in-house CC ELISA using CWE are suitable for practical diagnosis for fascioliasis.

Structural basis of urea-induced unfolding of Fasciola gigantica glutathione S-transferase.

Glutathione S-transferases (GSTs) are enzymes that are involved in the detoxification of harmful electrophilic endogenous and exogenous compounds by conjugating with glutathione (GSH). The liver fluke GSTs have multifunctional roles in the host-parasite interaction, such as general detoxification and bile acid sequestration to synthase activity. The GSTs have been highlighted as vaccine candidates towards parasitic flukes. In this study, we have thoroughly examined the urea-induced unfolding of a mu-class Fasciola gigantica GST1 (FgGST1) using spectroscopic techniques and molecular dynamic simulations. FgGST1 is a highly cooperative molecule, because during urea-induced equilibrium unfolding, a concurrent unfolding of the protein without stabilization of any folded intermediate was observed. The protein was stabilized with conformational free energy of about ~12.36 kcal/mol. The protein loses its activity with increasing urea concentration, as the GSH molecule is not able to bind to the protein. We also studied the fluorescence quenching of Trp residues and the obtained K SV data that provided additional information on the unfolding of FgGST1. Molecular dynamic trajectories simulated in different urea concentrations and temperatures indicated that urea destabilizes FgGST1 structure by weakening hydrophobic interactions and the hydrogen bond network. We observed a precise correlation between the in vitro and in silico studies.

Structure-function studies of the asparaginyl-tRNA synthetase from Fasciola gigantica: understanding the role of catalytic and non-catalytic domains.

The asparaginyl-tRNA synthetase (NRS) catalyzes the attachment of asparagine to its cognate tRNA during translation. NRS first catalyzes the binding of Asn and ATP to form the NRS-asparaginyl adenylate complex, followed by the esterification of Asn to its tRNA. We investigated the role of constituent domains in regulating the structure and activity of Fasciola gigantica NRS (FgNRS). We cloned the full-length FgNRS, along with its various truncated forms, expressed, and purified the corresponding proteins. Size exclusion chromatography indicated a role of the anticodon-binding domain (ABD) of FgNRS in protein dimerization. The N-terminal domain (NTD) was not essential for cognate tRNA binding, and the hinge region between the ABD and the C-terminal domain (CTD) was crucial for regulating the enzymatic activity. Molecular docking and fluorescence quenching experiments elucidated the binding affinities of the substrates to various domains. The molecular dynamics simulation of the modeled protein showed the presence of an unstructured region between the NTD and ABD that exhibited a large number of conformations over time, and further analysis indicated this region to be intrinsically disordered. The present study provides information on the structural and functional regulation, protein-substrate(s) interactions and dynamics, and the role of non-catalytic domains in regulating the activity of FgNRS.

Role of the glutaredoxin domain and FAD in the stabilization of thioredoxin glutathione reductase.

Thioredoxin glutathione reductase (TGRsec) is a multi-domain flavoprotein that plays a principal role in redox homeostasis maintenance. We have previously demonstrated the role of selenocysteine in maintaining TGRsec structure-function, but the role of the glutaredoxin (Grx) domain and FAD is still unclear. In the present study, the urea-induced unfolding of recombinant Fasciola gigantica TGRsec (FgTGRsec) and its N-terminal truncated variant (ΔNTD-FgTGRsec) were examined to understand the role of the Grx domain and FAD in the stabilization of FgTGRsec and ΔNTD-FgTGRsec. Our results showed that both proteins underwent unfolding in a three state manner. First, the protein undergoes a conformational transition rendering a near-native state with no FAD bound, and then full unfolding of the apo-dimer occurs without dissociation. The Grx domain stabilized the global FgTGRsec structure and positively regulated FgTGRsec activity, and alteration in the FAD microenvironment was directly proportional to the loss of thioredoxin reductase (TrxR) and glutathione reductase activities. Based on these results, we concluded that the Grx domain stabilizes the full-length FgTGRsec protein for efficient catalysis. Thus, we suggest that in platyhelminth parasites, during evolution, the Grx domain merged with the TrxR domain to confer higher catalytic activity and provide additional structural stability to the full-length TGR.

Expression and characterization of glutathione peroxidase of the liver fluke, Fasciola gigantica.

Glutathione peroxidase (GPx) is a key member of the family of antioxidant enzymes in trematode parasites including Fasciola spp. Because of its abundance and central role as an anti-oxidant that helps to protect parasites from damage by free radicals released from the host immune cells, it has both diagnostic as well as vaccine potential against fasciolosis. In this study, we have cloned, characterized, and detected the expression of the GPx protein in Fasciola gigantica (Fg). FgGPx (582 bp) was cloned by polymerase chain reaction (PCR) from complementary DNA (cDNA) from an adult fluke. Its putative peptide has no signal sequence and is composed of 168 amino acids, with a molecular weight of 19.1 kDa, and conserved sequences at NVACKUG, FPCNQFGGQ, and WNF. Phylogenetic analysis showed that GPx is present from protozoa to mammals and FgGPx was closely related to Fasciola hepatica GPx. A recombinant FgGPx (rFgGPx) was expressed in Escherichia coli BL21 (DE3) and used for immunizing mice to obtain polyclonal antibodies (anti-rFgGPx) for immunoblotting and immunolocalization. In immunoblotting analysis, the FgGPx was expressed in all stages of F. gigantica (eggs, metacercariae, newly excysted juveniles (NEJ), 4-week-old juveniles, and adults). This mouse anti-rFgGPx reacted with the native FgGPx at a molecular weight of 19.1 kDa in adult whole body (WB) and tegumental antigens (TA) as detected by immunoblotting. The FgGPx protein was expressed at a high level in the tegument, vitelline glands, and eggs of the parasite. Anti-rFgGPx exhibited no cross-reactivity with the other parasite antigens, including Eurytrema pancreaticum, Cotylophoron cotylophorum, Fischoederius cobboldi, Gastrothylax crumenifer, Paramphistomum cervi, and Setaria labiato papillosa. The possibility of using rFgGPx for immunodiagnosis and/or as a vaccine for fasciolosis in animals of economic importance will be explored in the future.

Design and synthesis of a new peptide derived from Fasciola gigantica cathepsin L1 with potential application in serodiagnosis of fascioliasis.

Fascioliasis is a global parasitic disease that affects domestic animals and causes considerable economic losses in the process of domestic animal breeding in endemic regions. The cause of the disease involves a liver trematode of the genus Fasciola, which secretes materials into a host's body (mainly proteins) in order to protect it from the host's immune system. These materials can be involved in the migration, growth, and nutrition of the parasite. Among the expressive proteins of Fasciola, proteases have been introduced as the appropriate targets for diagnosis, treatment, and vaccination against parasites. Cathepsin L (CL) is a member of cysteine proteases; it is widely expressed in the Fasciola species. The aim of this study was to evaluate two synthetic peptides from F. gigantica CL1 for improving serological diagnosis of the Fasciola infection. Therefore, the potential diagnostic value of the surface epitopes of CL1 was assessed using ELISA. In the current study, bioinformatics tools were applied to select two appropriate epitopes of Fasciola Cathepsin L1 as synthetic antigens. Their diagnostic values were evaluated by two methods of indirect ELISA and dot blot analysis. The findings revealed that the first peptide at a dilution ratio of 1:400 and the second peptide at a dilution ratio of 1:100 had the best results and the best concentration of antigens was introduced at 4 µg/ml. Moreover, 191 sera samples were analyzed by both peptides by using the ELISA method, including fascioliasis sera, other parasitic sera and negative sera. The sensitivity of the peptides 1-ELISA and peptide 2-ELISA for the diagnosis of the various cases was 100%. The specificity of the first peptide was 87.3% and its efficacy was determined to be 93.65%. The specificity and the efficacy of the second peptide were 79% and 89.5%, respectively. The positive predictive values of the first and second peptides were obtained to be 86.27% and 79.27% respectively, and the negative predictive values of both peptides was calculated as 100%. In conclusion, the results of this study indicated that the peptide 1 from CL1 may be used as an appropriate antigen for the diagnosis of fascioliasis if the findings are backed up by using other serodiagnostic methods for checking serological cross-reactivity linked to other parasites.

Biochemical and thermodynamic comparison of the selenocysteine containing and non-containing thioredoxin glutathione reductase of Fasciola gigantica.

The thiol-disulfide redox metabolism in platyhelminth parasites depends entirely on a single selenocysteine (Sec) containing flavoenzyme, thioredoxin glutathione reductase (TGR) that links the classical thioredoxin (Trx) and glutathione (GSH) systems. In the present study, we investigated the catalytic and structural properties of different variants of Fasciola gigantica TGR to understand the role of Sec. The recombinant full-length Sec containing TGR (FgTGRsec), TGR without Sec (FgTGR) and TGRsec without the N-terminal glutaredoxin (Grx) domain (∆NTD-FgTGRsec) were purified to homogeneity. Biochemical studies revealed that Sec597 is responsible for higher thioredoxin reductase (TrxR) and glutathione reductase (GR) activity of FgTGRsec. The N-terminal Grx domain was found to positively regulate the DTNB-based TrxR activity of FgTGRsec. The FgTGRsec was highly sensitive to inhibition by auranofin (AF). The structure of FgTGR was modeled, and the inhibitor AF was docked, and binding sites were identified. Unfolding studies suggest that all three proteins are highly cooperative molecules since during GdnHCl-induced denaturation, a monophasic unfolding of the proteins without stabilization of any intermediate is observed. The Cm for GdnHCl induced unfolding of FgTGR was higher than FgTGRsec and ∆NTD-FgTGRsec suggesting that FgTGR without Sec was more stable in solution than the other protein variants. The free energy of stabilization for the proteins was also determined. To our knowledge, this is also the first report on unfolding and stability analysis of any TGR.

Soluble expression and purification of a full-length asparaginyl tRNA synthetase from Fasciola gigantica.

We report the molecular cloning, expression, and single-step homogeneous purification of a full-length asparaginyl tRNA synthetase (NRS) from Fasciola gigantica (FgNRS). Fasciola gigantica is a parasitic liver fluke of the class Trematoda. It causes fascioliasis that infects the liver of various mammals, including humans. Aminoacyl tRNA synthetases (AARS) catalyze the first step of protein synthesis. They attach an amino acid to its cognate tRNA, forming an amino acid-tRNA complex. The gene that codes for FgNRS was generated by amplification by polymerase chain reaction. It was then inserted in the expression vector pQE30 under the transcriptional control of the bacteriophage T5 promoter and lac operator. M15 Escherichia coli strain transformed with the FgNRS expression vector pQE30-NRS accumulates large amounts of a soluble protein of about 61 kDa. The protein was purified to homogeneity using immobilized metal affinity chromatography. The recombinant protein was further confirmed by immunoblotting with anti-His antibody. Following size exclusion chromatography, the FgNRS was stable and observed to be a dimeric protein. In this study, the expression and purification procedures have provided a simple and efficient method to obtain full-length FgNRS in large quantities. This will provide an opportunity to study the structure, dynamics and function of NRS.

Structural insights into natural compounds as inhibitors of Fasciola gigantica thioredoxin glutathione reductase.

Fascioliasis is caused by the helminth parasites of genus Fasciola. Thioredoxin glutathione reductase (TGR) is an important enzyme in parasitic helminths and plays an indispensable role in its redox biology. In the present study, we conducted a structure-based virtual screening of natural compounds against the Fasciola gigantica TGR (FgTGR). The compounds were docked against FgTGR in four sequential docking modes. The screened ligands were further assessed for Lipinski and ADMET prediction so as to evaluate drug proficiency and likeness property. After refinement, three potential inhibitors were identified that were subjected to 50 ns molecular dynamics simulation and free energy binding analyses to evaluate the dynamics of protein-ligand interaction and the stability of the complexes. Key residues involved in the interaction of the selected ligands were also determined. The results suggested that three top hits had a negative binding energy greater than GSSG (-91.479 KJ · mol-1 ), having -152.657, -141.219, and -92.931 kJ · mol-1 for compounds with IDs ZINC85878789, ZINC85879991, and ZINC36369921, respectively. Further analysis showed that the compound ZINC85878789 and ZINC85879991 displayed substantial pharmacological and structural properties to be a drug candidate. Thus, the present study might prove useful for the future design of new derivatives with higher potency and specificity.

Comprehensive analysis of the catalytic and structural properties of a mu-class glutathione s-transferase from Fasciola gigantica.

Glutathione S‒transferases (GSTs) play an important role in the detoxification of xenobiotics. They catalyze the nucleophilic addition of glutathione (GSH) to nonpolar compounds, rendering the products water-soluble. In the present study, we investigated the catalytic and structural properties of a mu-class GST from Fasciola gigantica (FgGST1). The purified recombinant FgGST1 formed a homodimer composed of 25 kDa subunit. Kinetic analysis revealed that FgGST1 displays broad substrate specificity and shows high GSH conjugation activity toward 1-chloro-2,4-dinitrobenzene, 4-nitroquinoline-1-oxide, and trans-4-phenyl-3-butene-2-one and peroxidase activity towards trans-2-nonenal and hexa-2,4-dienal. The FgGST1 was highly sensitive to inhibition by cibacron blue. The cofactor (GSH) and inhibitor (cibacron blue) were docked, and binding sites were identified. The molecular dynamics studies and principal component analysis indicated the stability of the systems and the collective motions, respectively. Unfolding studies suggest that FgGST1 is a highly cooperative molecule because, during GdnHCl-induced denaturation, a simultaneous unfolding of the protein without stabilization of any partially folded intermediate is observed. The protein is stabilized with a conformational free energy of about 10 ± 0.3 kcal mol-1. Additionally, the presence of conserved Pro-53 and structural motifs such as N-capping box and hydrophobic staple, further aided in the stability and proper folding of FgGST1.

Fasciola gigantica thioredoxin glutathione reductase: Biochemical properties and structural modeling.

Platyhelminth thioredoxin glutathione reductase (TGR) is a multifunctional enzyme that crosstalk between the conventional thioredoxin (Trx) and glutathione (GSH) system. It has been validated as a potential drug target in blood flukes. In the present study, we have performed a biochemical study on Fasciola gigantica TGR with substrates DTNB and GSSG. The Michaelis constant (Km) with DTNB was found to be 4.34±0.12µM while it was 61.15±1.50µM with GSSG. The kinetic results were compared with the TGR activities of other helminths. FgTGR showed typical hysteretic behavior with GSSG as other TGRs. We also described a homology-based structure of FgTGR. The cofactors (NADPH and FAD) and substrates (GSSG and DTNB) were docked, and two possible binding sites for substrates were identified in a single chain. The substrates were found to bind more favorably in the second site of TrxR domains. We also presented the first report on binding interaction of DTNB with a TGR. DTNB forms H-bond with His204 and Arg450 of chain A, Sec597, and Gly598 from chain B, salt-bridge with Lys124, and numerous other hydrophobic interactions. Helminth TGR represents an important enzyme in the redox and antioxidant system; hence, its inhibition can be used as an effective strategy against liver flukes.