Exploring naphthyl derivatives as SARS-CoV papain-like protease (PLpro) inhibitors and its implications in COVID-19 drug discovery.

Novel coronavirus disease 2019 (COVID-19) emerges as a serious threat to public health globally. The rapid spreading of COVID-19, caused by severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2), proclaimed the multitude of applied research needed not only to save the human health but also for the environmental safety. As per the recent World Health Organization reports, the novel corona virus may never be wiped out completely from the world. In this connection, the inhibitors already designed against different targets of previous human coronavirus (HCoV) infections will be a great starting point for further optimization. Pinpointing biochemical events censorious to the HCoV lifecycle has provided two proteases: a papain-like protease (PLpro) and a 3C-like protease (3CLpro) enzyme essential for viral replication. In this study, naphthyl derivatives inhibiting PLpro enzyme were subjected to robust molecular modelling approaches to understand different structural fingerprints important for the inhibition. Here, we cover two main aspects such as (a) exploration of naphthyl derivatives by classification QSAR analyses to find important fingerprints that module the SARS-CoV PLpro inhibition and (b) implications of naphthyl derivatives against SARS-CoV-2 PLpro enzyme through detailed ligand-receptor interaction analysis. The modelling insights will help in the speedy design of potent broad spectrum PLpro inhibitors against infectious SARS-CoV and SARS-CoV-2 in the future.

Inhibition and disruption of amyloid formation by the antibiotic levofloxacin: A new direction for antibiotics in an era of multi-drug resistance.

Neurodegenerative diseases are a group of debilitating maladies involving protein aggregation. To this day, all advances in neurodegenerative disease therapeutics have helped symptomatically but have not prevented the root cause of the disease, i.e., the aggregation of involved proteins. Antibiotics are becoming increasingly obsolete due to the rising multidrug resistance strains of bacteria. Thus, antibiotics, if put to different use as therapeutics against other diseases, could pave a new direction to the world of antibiotics. Hence, we studied the antibiotic levofloxacin for its potential anti-amyloidogenic behavior using human lysozyme, a protein involved in non-systemic amyloidosis, as a model system. At the sub-stoichiometric level, levofloxacin was able to inhibit amyloid formation in human lysozyme as observed by various spectroscopic and microscopic methods, with IC50 values as low as 8.8 ± 0.1 µM. Levofloxacin also displayed a retarding effect on seeding phenomena by elongating the lag-phase (from 0 to 88 h) at lower concentration, and arresting lysozyme fibrillation at the lag stage in sub-stoichiometric concentrations. Structural and computational analyses provided mechanistic insight showing that levofloxacin stabilizes the lysozyme in the native state by binding to the aggregation-prone residues, and thereby inhibiting amyloid fibrillation. Levofloxacin also showed the property of disrupting amyloid fibrils into a smaller polymeric form of proteins which were less cytotoxic as confirmed by hemolytic assay. Therefore, we throw new light on levofloxacin as an amyloid inhibitor and disruptor which could pave way to utilization of levofloxacin as a potential therapeutic against non-systemic amyloidosis and neurodegenerative diseases.

Amino acid starvation sensing dampens IL-1ß production by activating riboclustering and autophagy.

Activation of the amino acid starvation response (AAR) increases lifespan and acute stress resistance as well as regulates inflammation. However, the underlying mechanisms remain unclear. Here, we show that activation of AAR pharmacologically by Halofuginone (HF) significantly inhibits production of the proinflammatory cytokine interleukin 1ß (IL-1ß) and provides protection from intestinal inflammation in mice. HF inhibits IL-1ß through general control nonderepressible 2 kinase (GCN2)-dependent activation of the cytoprotective integrated stress response (ISR) pathway, resulting in rerouting of IL-1ß mRNA from translationally active polysomes to inactive ribocluster complexes-such as stress granules (SGs)-via recruitment of RNA-binding proteins (RBPs) T cell-restricted intracellular antigen-1(TIA-1)/TIA-1-related (TIAR), which are further cleared through induction of autophagy. GCN2 ablation resulted in reduced autophagy and SG formation, which is inversely correlated with IL-1ß production. Furthermore, HF diminishes inflammasome activation through suppression of reactive oxygen species (ROS) production. Our study unveils a novel mechanism by which IL-1ß is regulated by AAR and further suggests that administration of HF might offer an effective therapeutic intervention against inflammatory diseases.

Biochemical and structural insights into 6-phosphogluconate dehydrogenase from Leishmania donovani.

6-phosphogluconate dehydrogenase (6PGDH) participates in pentose phosphate pathway of glucose metabolism by catalyzing oxidative decarboxylation of 6-phsophogluconate (6PG) and its absence has been lethal for several eukaryotes. Despite being a validated drug target in many organisms like Plasmodium, the enzyme has not been explored in leishmanial parasites. In the present study, 6PGDH of Leishmania donovani (Ld6PGDH) is cloned and purified followed by its characterization using biochemical and structural approaches. Ld6PGDH lacks the glycine-serine-rich sequence at its C-terminal that is present in other eukaryotes including humans. Leishmanial 6PGDH possesses more affinity for substrate (6PG) and cofactor (NADP) in comparison to that of human. The enzymatic activity is inhibited by gentamicin and cefuroxime through competitive mode with functioning more potently towards leishmanial 6PGDH than its human counterpart. CD analysis has shown higher α-helical content in the secondary structure of Ld6PGDH, while fluorescence studies revealed that tryptophan residues are not completely accessible to solvent environment. The three-dimensional structure was generated through homology modelling and docked with substrate and cofactor. The docking studies demonstrated two separate binding pockets for 6PG and NADP with higher affinity for the cofactor binding, and Asn105 is interacting with substrate as well as the cofactor. Additionally, MD simulation has shown complexes of Ld6PGDH with 6PG and NADP to be more stable than its apo form. Altogether, the present study might provide the foundation to investigate this enzyme as potential target against leishmaniasis. KEY POINTS: • Ld6PGDH enzymatic activity is competitively inhibited by gentamicin and cefuroxime. • It displays more helical contents and all structural characteristics of 6PGDH family. • Interaction studies demonstrate higher affinity of cofactor than substrate for Ld6PGDH.

First structure-activity relationship analysis of SARS-CoV-2 virus main protease (Mpro) inhibitors: an endeavor on COVID-19 drug discovery.

Main protease (Mpro) of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) intervenes in the replication and transcription processes of the virus. Hence, it is a lucrative target for anti-viral drug development. In this study, molecular modeling analyses were performed on the structure activity data of recently reported diverse SARS-CoV-2 Mpro inhibitors to understand the structural requirements for higher inhibitory activity. The classification-based quantitative structure-activity relationship (QSAR) models were generated between SARS-CoV-2 Mpro inhibitory activities and different descriptors. Identification of structural fingerprints to increase or decrease in the inhibitory activity was mapped for possible inclusion/exclusion of these fingerprints in the lead optimization process. Challenges in ADME properties of protease inhibitors were also discussed to overcome the problems of oral bioavailability. Further, depending on the modeling results, we have proposed novel as well as potent SARS-CoV-2 Mpro inhibitors.

Protein phosphatase 1 of Leishmania donovani exhibits conserved catalytic residues and pro-inflammatory response.

Protein phosphorylation, governed by kinases and phosphatases, plays a pivotal role in enormous cellular signaling pathways. Although PPP family of serine/threonine phosphatases have been involved in multiplication and growth of trypanosomatid parasites, but comprehensive knowledge is still very limited. In the present study, protein phosphatase 1 from Leishmania donovani (LdPP1) was purified to homogeneity and its structural attributes were explored employing CD and fluorescence spectroscopy as well as bioinformatics methods. The CD analysis revealed an appropriate secondary structure with α-helices content outnumbering the ß-sheets, whereas intrinsic fluorescence study depicted about the buried positioning of tryptophan residues. The three-dimensional structure of LdPP1, determined by homology modeling, displayed all the characteristic features including similar position of metal as well as inhibitor binding site corresponding to the known PP1 structures. Furthermore, ELISA and qRT-PCR results showed that LdPP1 elicit the pro-inflammatory cytokines TNF-α and IL-6 at translated and transcriptional levels in THP1 macrophages. Subsequently, immune effector molecule nitric oxide and transcription factor NF-κB production was also found to be increased upon LdPP1 stimulation. Altogether, this is the first report on PPP phosphatase of trypanosomatid parasite that represents the structural highlights along with protein-mediated immunomodulation in human macrophages.

Bioassay-Guided Fractionation and In Vitro Antiproliferative Effects of Fractions of Artemisia nilagirica on THP-1 cell line.

ABSTACT Artemisia nilagirica (Clarke) is a widely used medicinal herb in Indian traditional system of medicine. Therefore, the present study was designed to evaluate the effects of A. nilagirica extracts/fractions on inhibition of proliferation and apoptosis in a human monocytic leukemia (THP-1) cell line. The crude extracts (A. nilagirica ethyl acetate extract [ANE] and A. nilagirica methanolic extract [ANA]) showed cytotoxic activity toward THP-1 cells with the IC50 values of 38.21 ± 7.37 and 132.41 ± 7.19 µg/ml, respectively. However, the cytotoxic activity of active fractions (ANE-B and ANM-9) obtained after column chromatography was found to be much more pronounced than their parent extracts. The IC50 values of ANE-B and ANM-9 were found to be 27.04 ± 2.54 µg/ml and 12.70 ± 4.79 µg/ml, respectively, suggesting greater susceptibility of the malignant cells. Cell cycle analysis and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end-labeling (TUNEL) assay revealed that inhibition of cell growth by A. nilagirica fractions on THP-1 cells was mediated by apoptosis. Active fractions of A. nilagirica increased the expression levels of caspase-3, -7, and poly-ADP-ribose polymerase (PARP), a critical member of the apoptotic pathway. These results suggested that active fractions of A. nilagirica may play a promising role in growth suppression by inducing apoptosis in human monocytic leukemic cells via mitochondria-dependent and death receptor-dependent apoptotic pathways.

Comparative proteomics reveals differential induction of both biotic and abiotic stress response associated proteins in rice during Xanthomonas oryzae pv. oryzae infection.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight disease in rice and brutally affects the yield up to 50 % of total production. Here, we report a comparative proteomics analysis of total foliar protein isolated from infected rice leaves of susceptible Pusa Basmati 1 (PB1) and resistant Oryza longistaminata genotypes. Two-dimensional gel electrophoresis (2-DE) coupled with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) approaches identified 29 protein spots encoding unique proteins from both the genotypes. Identified proteins belonged to a large number of biological and molecular functions related to biotic and abiotic stress proteins which are potentially involved during Xoo infection. Biotic and abiotic stress-related proteins were induced during Xoo infection, indicating the activation of common stress pathway during bacterial blight infection. Candidate genes conferring tolerance against bacterial blight, which include germin-like protein, putative r40c1, cyclin-dependent kinase C, Ent-isokaur-15-ene synthase and glutathione-dependent dehydroascorbate reductase 1 (GSH-DHAR1), were also induced, with germin-like proteins induced only in the resistant rice genotype O. longistaminata. Energy, metabolism and hypothetical proteins were common among both the genotypes. Further, host defence/stress-related proteins were mostly expressed in resistant genotype O. longistaminata, indicating possible co-evolution of the pathogen and the wild rice, O. longistaminata.

Inhibition and structural insights of leishmanial glutamyl-tRNA synthetase for designing potent therapeutics.

Aminoacyl-tRNA synthetases (aaRSs), essential components of the protein synthesizing machinery, have been often chosen for devising therapeutics against parasitic diseases. Due to their relevance in drug development, the current study was designed to explore functional and structural aspects of Leishmania donovani glutamyl-tRNA synthetase (LdGluRS). Hence, LdGluRS was cloned into an expression vector and purified to homogeneity using chromatographic techniques. Purified protein showed maximum enzymatic activity at physiological pH, with more binding capacity towards its cofactor (Adenosine triphosphate, 0.06 ± 0.01 mM) than the cognate substrate (L-glutamate, 9.5 ± 0.5 mM). Remarkably, salicylate inhibited LdGluRS competitively with respect to L-glutamate and exhibited druglikeness with negligible effect on human macrophages. The protein possessed more α-helices (43 %) than ß-sheets (12 %), whereas reductions in thermal stability and cofactor-binding affinity, along with variation in mode of inhibition after mutation signified the role of histidine (H60) as a catalytic residue. LdGluRS could also generate a pro-inflammatory milieu in human macrophages by upregulating cytokines. The docking study demonstrated the placement of salicylate into LdGluRS substrate-binding site, and the complex was found to be stable during molecular dynamics (MD) simulation. Altogether, our study highlights the understanding of molecular inhibition and structural features of glutamyl-tRNA synthetase from kinetoplastid parasites.

Discovery of 8-hydroxy-2-quinoline carbaldehyde derivatives as inhibitors for M1 aminopeptidase of Leishmania donovani.

M1 aminopeptidase is a metallopeptidase that plays a vital role in protein catabolism and has been identified as a validated drug target in various parasites; however, our understanding of this enzyme is restricted for leishmanial parasite. The present investigation involved the purification of Leishmania donovani M1 aminopeptidase (LdM1AP) to homogeneity by affinity chromatography. Purified LdM1AP was observed to be enzymatically active and displayed maximal activity in the presence of cobalt ions, whereas secondary structure analysis confirmed the dominance of α-helices. Intrinsic fluorescence and quenching studies of LdM1AP has revealed that tryptophan residues were predominantly concealed within the hydrophobic areas. The synthesized 8-hydroxy-2-quinoline carbaldehyde derivatives were screened, wherein HQ2 and HQ12 were found as potent inhibitors for LdM1AP that compete with the substrate and exhibit pharmacokinetic properties as well as no toxicity for macrophages. Moreover, structural insights of protein and ligand complexes demonstrated that lead compounds mostly interact via hydrophobic contacts into the substrate binding pocket of LdM1AP. Furthermore, lead compounds exhibited a greater affinity for LdM1AP compared to the substrate during in vitro and in silico studies. This report establishes the possibility of quinoline derivatives to target the LdM1AP activity and provide a platform to design the specific antileishmanial drugs.

Unraveling the peculiarities and development of novel inhibitors of leishmanial arginyl-tRNA synthetase.

Aminoacylation by tRNA synthetase is a crucial part of protein synthesis and is widely recognized as a therapeutic target for drug development. Unlike the arginyl-tRNA synthetases (ArgRSs) reported previously, here, we report an ArgRS of Leishmania donovani (LdArgRS) that can follow the canonical two-step aminoacylation process. Since a previously uncharacterized insertion region is present within its catalytic domain, we implemented the splicing by overlap extension PCR (SOE-PCR) method to create a deletion mutant (ΔIns-LdArgRS) devoid of this region to investigate its function. Notably, the purified LdArgRS and ΔIns-LdArgRS exhibited different oligomeric states along with variations in their enzymatic activity. The full-length protein showed better catalytic efficiency than ΔIns-LdArgRS, and the insertion region was identified as the tRNA binding domain. In addition, a benzothiazolo-coumarin derivative (Comp-7j) possessing high pharmacokinetic properties was recognized as a competitive and more specific inhibitor of LdArgRS than its human counterpart. Removal of the insertion region altered the mode of inhibition for ΔIns-LdArgRS and caused a reduction in the inhibitor's binding affinity. Both purified proteins depicted variances in the secondary structural content upon ligand binding and thus, thermostability. Apart from the trypanosomatid-specific insertion and Rossmann fold motif, LdArgRS revealed typical structural characteristics of ArgRSs, and Comp-7j was found to bind within the ATP binding pocket. Furthermore, the placement of tRNAArg near the insertion region enhanced the stability and compactness of LdArgRS compared to other ligands. This study thus reports a unique ArgRS with respect to catalytic as well as structural properties, which can be considered a plausible drug target for the derivation of novel anti-leishmanial agents.

Identification of structural fingerprints among natural inhibitors of HDAC1 to accelerate nature-inspired drug discovery in cancer epigenetics.

Histone deacetylase 1 (HDAC1), a class I HDAC enzyme, is crucial for histone modification. Currently, it is emerged as one of the important biological targets for designing small molecule drugs through cancer epigenetics. Along with synthetic inhibitors different natural inhibitors are showing potential HDAC1 inhibitions. In order to gain insights into the relationship between the molecular structures of the natural inhibitors and HDAC1, different molecular modelling techniques (Bayesian classification, recursive partitioning, molecular docking and molecular dynamics simulations) have been applied on a dataset of 155 HDAC1 nature-inspired inhibitors with diverse scaffolds. The Bayesian study showed acceptable ROC values for both the training set and test sets. The Recursive partitioning study produced decision tree 1 with 6 leaves. Further, molecular docking study was processed for generating the protein ligand complex which identified some potential amino acid residues such as F205, H28, L271, P29, F150, Y204 for the binding interactions in case of natural inhibitors. Stability of these HDAC1-natutal inhibitors complexes has been also evaluated by molecular dynamics simulation study. The current modelling study is an attempt to get a deep insight into the different important structural fingerprints among different natural compounds modulating HDAC1 inhibition.Communicated by Ramaswamy H. Sarma.

Analysis of nucleotide diversity among alleles of the major bacterial blight resistance gene Xa27 in cultivars of rice (Oryza sativa) and its wild relatives.

Xa27 is one of the important R-genes, effective against bacterial blight disease of rice caused by Xanthomonas oryzae pv. oryzae (Xoo). Using natural population of Oryza, we analyzed the sequence variation in the functionally important domains of Xa27 across the Oryza species. DNA sequences of Xa27 alleles from 27 rice accessions revealed higher nucleotide diversity among the reported R-genes of rice. Sequence polymorphism analysis revealed synonymous and non-synonymous mutations in addition to a number of InDels in non-coding regions of the gene. High sequence variation was observed in the promoter region including the 5'UTR with 'π' value 0.00916 and 'θ w ' = 0.01785. Comparative analysis of the identified Xa27 alleles with that of IRBB27 and IR24 indicated the operation of both positive selection (Ka/Ks > 1) and neutral selection (Ka/Ks ≈ 0). The genetic distances of alleles of the gene from Oryza nivara were nearer to IRBB27 as compared to IR24. We also found the presence of conserved and null UPT (upregulated by transcriptional activator) box in the isolated alleles. Considerable amino acid polymorphism was localized in the trans-membrane domain for which the functional significance is yet to be elucidated. However, the absence of functional UPT box in all the alleles except IRBB27 suggests the maintenance of single resistant allele throughout the natural population.

Development of a multi-epitope vaccine candidate for leishmanial parasites applying immunoinformatics and in vitro approaches.

Leishmaniasis is a neglected tropical disease, and its severity necessitates the development of a potent and efficient vaccine for the disease; however, no human vaccine has yet been approved for clinical use. This study aims to design and evaluate a multi-epitope vaccine against the leishmanial parasite by utilizing helper T-lymphocyte (HTL), cytotoxic T-lymphocyte (CTL), and linear B-lymphocyte (LBL) epitopes from membrane-bound acid phosphatase of Leishmania donovani (LdMAcP). The designed multi-epitope vaccine (LdMAPV) was highly antigenic, non-allergenic, and non-toxic, with suitable physicochemical properties. The three-dimensional structure of LdMAPV was modeled and validated, succeeded by molecular docking and molecular dynamics simulation (MDS) studies that confirmed the high binding affinity and stable interactions between human toll-like receptors and LdMAPV. In silico disulfide engineering provided improved stability to LdMAPV, whereas immune simulation displayed the induction of both immune responses, i.e., antibody and cell-mediated immune responses, with a rise in cytokines. Furthermore, LdMAPV sequence was codon optimized and cloned into the pET-28a vector, followed by its expression in a bacterial host. The recombinant protein was purified using affinity chromatography and subjected to determine its effect on cytotoxicity, cytokines, and nitric oxide generation by mammalian macrophages. Altogether, this report provides a multi-epitope vaccine candidate from a leishmanial protein participating in parasitic virulence that has shown its potency to be a promising vaccine candidate against leishmanial parasites.

Aminoacyl tRNA Synthetases: Implications of Structural Biology in Drug Development against Trypanosomatid Parasites.

The ensemble of aminoacyl tRNA synthetases is regarded as a key component of the protein translation machinery. With the progressive increase in structure-based studies on tRNA synthetase-ligand complexes, the detailed picture of these enzymes is becoming clear. Having known their critical role in deciphering the genetic code in a living system, they have always been chosen as one of the important targets for development of antimicrobial drugs. Later on, the role of aminoacyl tRNA synthetases (aaRSs) on the survivability of trypanosomatids has also been validated. It became evident through several gene knockout studies that targeting even one of these enzymes affected parasitic growth drastically. Such successful studies have inspired researchers to search for inhibitors that could specifically target trypanosomal aaRSs, and their never-ending efforts have provided fruitful results. Taking all such studies into consideration, these macromolecules of prime importance deserve further investigation for the development of drugs that cure spectrum of infections caused by trypanosomatids. In this review, we have compiled advancements of over a decade that have taken place in the pursuit of devising drugs by using trypanosomatid aaRSs as a major target of interest. Several of these inhibitors work on an exemplary low concentration range without posing any threat to the mammalian cells which is a very critical aspect of the drug discovery process. Advancements have been made in terms of using structural biology as an important tool to analyze the architecture of the trypanosomatids aaRSs and concoction of inhibitors with augmented specificities toward their targets. Some of the inhibitors that have been tested on other parasites successfully but their efficacy has so far not been validated against these trypanosomatids have also been appended.

Exploration of seryl tRNA synthetase to identify potent inhibitors against leishmanial parasites.

Aminoacyl-tRNA synthetases are crucial enzymes for cellular protein metabolism and have been considered as an attractive target for development of new antimicrobials. In the current study, seryl tRNA synthetase of Leishmania donovani (LdSerRS) and its mutants were purified and characterized through biochemical and structural methods. Purified LdSerRS was found to be enzymatically active and exhibited more alpha helices in secondary structure. The enzymatic activity of purified protein was observed as highest near physiological temperature and pH. Mutation in ATP binding residues (R295 and E297) demonstrated reduction in the affinity for cofactor with no significant deviation in secondary structure. In vitro inhibition studies with ureidosulfocoumarin derivatives helped to identify Comp 5l as a specific inhibitor for leishmanial SerRS that showed lesser potency towards purified HsSerRS. The identified compound presented competitive mode of inhibition for LdSerRS and also revealed druglikeness along with very low toxicity for human macrophages. Structural analysis of protein and ligand complex depicted the binding of Comp 5l into the cofactor binding site of LdSerRS with high affinity succeeded by validation employing molecular dynamics simulations. Altogether, our study presents a promising scaffold to explore small molecules to target the enzymatic activity of leishmanial SerRS to develop the specific therapeutics.

Multi-epitope vaccine against SARS-CoV-2 applying immunoinformatics and molecular dynamics simulation approaches.

COVID-19, caused by SARS-CoV-2, is severe respiratory illnesses leading to millions of deaths worldwide in very short span. The high case fatality rate and the lack of medical counter measures emphasize for an urgent quest to develop safe and effective vaccine. Receptor-binding domain (RBD) of spike protein of SARS-CoV-2 binds to the ACE2 receptor on human host cell for the viral attachment and entry, hence considered as a key target to develop vaccines, antibodies and therapeutics. In this study, immunoinformatics approach was employed to design a novel multi-epitope vaccine using RBD of SARS-CoV-2 spike protein. The potential B- and T-cell epitopes were selected from RBD sequence using various bioinformatics tools to design the vaccine construct. The in silico designed multi-epitope vaccine encompasses 146 amino acids with an adjuvant (human beta-defensin-2), which was further computationally evaluated for several parameters including antigenicity, allergenicity and stability. Subsequently, three-dimensional structure of vaccine construct was modelled and then docked with various toll-like receptors. Molecular dynamics (MD) study of docked TLR3-vaccine complex delineated it to be highly stable during simulation time and the stabilization of interaction was majorly contributed by electrostatic energy. The docked complex also showed low deformation and increased rigidity in motion of residues during dynamics. Furthermore, in silico cloning of the multi-epitope vaccine was carried out to generate the plasmid construct for expression in a bacterial system. Altogether, our study suggests that the designed vaccine candidate containing RBD region could provide the specific humoral and cell-mediated immune responses against SARS-CoV-2.Communicated by Ramaswamy H. Sarma.

Identification of potent inhibitors against chorismate synthase of Toxoplasma gondii using molecular dynamics simulations.

Toxoplasmosis, caused by Toxoplasma gondii, affects about 20-30% of the human population every year globally. The emergence of severe side effects of current chemotherapeutics and drug-resistant strains emphasize upon finding new therapeutics to treat toxoplasmosis. Chorismate synthase (CS) is a vital enzyme of shikimate pathway and responsible for formation of chorismate, which acts as a precursor for production of several aromatic compounds important for virulence and survival in many bacteria and protozoans. In this study, comparative modeling was employed to predict the three-dimensional structure of T. gondii chorismate synthase (TgCS) followed by its refinement and validation using various computational tools. The modeled structure of TgCS monomer shows all the conserved features of CS, particularly the beta-alpha-beta sandwich fold. Molecular docking studies has displayed that 5-enolpyruvylshikimate-3-phosphate (EPSP, substrate) and flavin mononucleotide (FMN, cofactor) bind into the active site of TgCS and all the structures (apo, binary, and ternary) were observed to be stable during molecular dynamics (MD) simulation. Subsequently, structure-based virtual screening using TgCS has inferred two of each benzofuran and EPSP analogs as the best hits on the basis of RCS, molecular interactions, ADME properties, and MD simulations. The MD data of resultant protein-ligand complex structures was subjected to calculate the binding energy through MMPBSA method, which highlights that the EPSP analogs have higher binding affinity for the substrate-binding site of TgCS in comparison to benzofuran derivatives as well as substrate. Altogether, our study could pave the way for designing and development of next generation chemotherapeutic molecules against toxoplasmosis.

Pyridoxal Kinase of Disease-causing Human Parasites: Structural and Functional Insights to Understand its Role in Drug Discovery.

Human parasites cause several diseased conditions with high morbidity and mortality in a large section of the population residing in various geographical areas. Nearly three billion people suffer from either one or many parasitic infections globally, with almost one million deaths annually. In spite of extensive research and advancement in the medical field, no effective vaccine is available against prominent human parasitic diseases that necessitate identification of novel targets for designing specific inhibitors. Vitamin B6 is an important ubiquitous co-enzyme that participates in several biological processes and plays an important role in scavenging ROS (reactive oxygen species) along with providing resistance to oxidative stress. Moreover, the absence of the de novo vitamin B6 biosynthetic pathway in human parasites makes this pathway indispensable for the survival of these pathogens. Pyridoxal kinase (PdxK) is a crucial enzyme for vitamin B6 salvage pathway and participates in the process of vitamers B6 phosphorylation. Since the parasites are dependent on pyridoxal kinase for their survival and infectivity to the respective hosts, it is considered a promising candidate for drug discovery. The detailed structural analysis of PdxK from disease-causing parasites has provided insights into the catalytic mechanism of this enzyme as well as significant differences from their human counterpart. Simultaneously, structure-based studies have identified small lead molecules that can be exploited for drug discovery against protozoan parasites. The present review provides structural and functional highlights of pyridoxal kinase for its implication in developing novel and potent therapeutics to combat fatal parasitic diseases.

Biophysical and Structural Characterization of Ribulose-5-phosphate Epimerase from Leishmania donovani.

Pentose phosphate pathway (PPP) plays a crucial role in the maintenance of NADPH/NADP+ homeostasis and provides protection against oxidative stress through detoxification of the reactive oxygen species. Ribulose-5-phosphate epimerase (RPE) participates in catalysis of the interconversion of ribulose-5-phosphate (Ru5P) to xylulose-5-phosphate (Xu5P) during PPP, however the structural attributes of this enzyme are still underexplored in many human pathogens including leishmanial parasites. The present study focuses upon cloning, purification and characterization of RPE of Leishmania donovani (LdRPE) using various biophysical and structural approaches. Sequence analysis has shown the presence of trypanosomatid-specific insertions at the N-terminus that are absent in humans and other eukaryotes. Gel filtration chromatography indicated recombinant LdRPE to exist as a dimer in the solution. Circular dichroism studies revealed a higher alpha helical content at physiological pH and temperature that comparatively varies with changing these parameters. Additionally, intrinsic fluorescence and quenching studies of LdRPE have depicted that tryptophan residues are mainly buried in the hydrophobic regions, and the recombinant enzyme is moderately tolerant to urea. Moreover, homology modeling was employed to generate the three-dimensional structure of LdRPE followed by molecular docking with the substrate, product, and substrate analogues. The modeled structure of LdRPE unravelled the presence of conserved active site residues as well as a single binding pocket for the substrate and product, while an in silico study suggested binding of substrate analogues into a similar pocket with more affinity than the substrate. Additionally, molecular dynamics simulation analysis has deciphered complexes of LdRPE with most of the ligands exhibiting more stability than its apo form and lesser fluctuations in active site residues in the presence of ligands. Altogether, our study presents structural insights into leishmanial RPE that could provide the basis for its implication to develop potent antileishmanials.