Structure-based in silico and in vitro Analysis Reveals Asiatic Acid as Novel Potential Inhibitor of Mycobacterium tuberculosis Maltosyl Transferase.

AIMS: The present study aimed to search for novel potent inhibitor(s) against the recently discovered maltosyltransferase (GlgE) target of M.tb. BACKGROUND: GlgE belongs to an α-amylase family and catalyzes the elongation of cytosolic branched α-glucan. Inactivation of M.tb. GlgE results in DNA damage and rapid death of M.tb. due to the accumulation of a toxic altosyl donor, maltose-1-phosphate (M1P), suggesting that GlgE is an intriguing target for inhibitor design. METHODS: 1000 natural compounds were compiled from public databases and literature through virtual screening, of which 25 compounds were found to satisfy all drug-likeness properties and ADME/ toxicity criteria, followed by molecular docking with GlgE. Compound(s) showing the lowest binding energy was further subjected to molecular dynamics simulation (MDS) and in vitro analysis. RESULTS: Molecular docking analysis allowed the selection of 5 compounds withsignificant binding affinity to GlgE targets. Amongst these compounds, asiatic acid exhibited the lowest binding energy (-12.61 kcal/mol). The results of 20-ns MDS showed that asiatic acid formed a stable complex with GlgE. Additionally, asiatic acid exhibited in vitro anti-mycobacterial activity against M.tb. H37Ra, M. bovis BCG, and M. smegmatis strains. CONCLUSION: The study reveals asiatic acid as a promising anti-mycobacterial agent that might emerge as a novel natural anti-TB lead molecule in the future.

Modulation of interaction of BRCA1-RAD51 and BRCA1-AURKA protein complexes by natural metabolites using as possible therapeutic intervention toward cardiotoxic effects of cancer drugs: an in-silico approach.

Breast cancer type 1 susceptibility protein (BRCA1) plays an important role in maintaining genome stability and is known to interact with several proteins involved in cellular pathways, gene transcription regulation and DNA damage response. More than 40% of inherited breast cancer cases are due to BRCA1 mutation. It is also a prognostic marker in non-small cell lung cancer patients as well as a gatekeeper of cardiac function. Interaction of mutant BRCA1 with other proteins is known to disrupt the tumor suppression mechanism. Two directly interacting proteins with BRCA1 namely, DNA repair protein RAD51 (RAD51) and Aurora kinase A (AURKA), known to regulate homologous recombination (HR) and G/M cell cycle transition, respectively, form protein complex with both wild and mutant BRCA1. To analyze the interactions, protein-protein complexes were generated for each pair of proteins. In order to combat the cardiotoxic effects of cancer drugs, pharmacokinetically screened natural metabolites derived from plant, marine and bacterial sources and along with FDA-approved cancer drugs as control, were subjected to molecular docking. Piperoleine B and dihydrocircumin were the best docked natural metabolites in both RAD51 and AURKA complexes, respectively. Molecular dynamics simulation (MDS) analysis and binding free energy calculations for the best docked natural metabolite and drug for both the mutant BRCA1 complexes suggested better stability for the natural metabolites piperolein B and dihydrocurcumin as compared to drug. Thus, both natural metabolites could be further analyzed for their role against the cardiotoxic effects of cancer drugs through wet lab experiments.Communicated by Ramaswamy H. Sarma.

Recombinant Expression and Characterization of Lemon (Citrus limon) Peroxidase.

BACKGROUND: Class III plant peroxidases play important role in a number of physiological processes in plants such as lignin biosynthesis, suberization, cell wall biosynthesis, reactive oxygen species metabolism and plant defense against pathogens. Peroxidases are also of significance in several industrial applications. In view of this, the production and identification of novel peroxidases having resistance towards temperature, pH, salts is desirable. OBJECTIVE: The objective of the present work was to clone and characterize a novel plant peroxidase suitable for industrial application. METHODS: A full length cDNA clone of lemon peroxidase was isolated using PCR and RACE approaches, characterized and heterologously expressed in Escherichia coli using standard protocols. The expressed peroxidase was purified using Ni-NTA agarose column and biochemically characterized using standard protocols. The peroxidase was also in-silico characterized at nucleotide as well as protein levels using standard protocols. RESULTS: A full length cDNA clone of lemon peroxidase was isolated and expressed heterologously in E. coli. The expressed recombinant lemon peroxidase (LPRX) was activated by in-vitro refolding and purified. The purified LPRX exhibited pH and temperature optima of pH 7.0 and 50°C, respectively. The LPRX was found to be activated by metal ions (Na+, Ca2+, Mg2+ and Mn2+) at lower concentration. The expressional analysis of the transcripts suggested involvement of lemon peroxidase in plant defense. The lemon peroxidase was in silico modelled and docked with the substrates guaiacol, and pyrogallol and shown the favourability of pyrogallol over guaiacol, which is in agreement with the in-vitro findings. The protein function annotation analyses suggested the involvement of lemon peroxidase in the phenylpropanoid biosynthesis pathway and plant defense mechanisms. CONCLUSION: Based on the biochemical characterization, the purified peroxidase was found to be resistant towards the salts and thus, might be a good candidate for industrial exploitation. The in-silico protein function annotation and transcript analyses highlighted the possible involvement of the lemon peroxidase in plant defense response.

Plant triterpenoid saponins: biosynthesis, in vitro production, and pharmacological relevance.

The saponins are a diverse class of natural products, with a broad scale distribution across different plant species. Chemically characterized as triterpenoid glycosides, they posses a 30C oxidosqualene precursor-based aglycone moiety (sapogenin), to which glycosyl residues are subsequently attached to yield the corresponding saponin. Based on the chemically distinct aglycone moieties, broadly, they are divided into triterpenoid saponins (dammaranes, ursanes, oleananes, lupanes, hopanes, etc.) and the sterol glycosides. This review aims to present in detail the biosynthesis patterns of the different aglycones from a common precursor and their glycosylation patterns to yield the functionally active glycoside. The review also presents recent advances in the pharmacological activities of these saponins, particularly as potent anti-neoplastic pharmacophores, antioxidants, or anti-viral/antibacterial agents. Since alternate production pedestals for these pharmacologically important triterpenes via cell and tissue cultures are an attractive option for their sustainable production, recent trends in the variety and scale of in vitro production of plant triterpenoids have also been discussed.

Discovering Innovative Drugs Targeting Both Cancer and Cardiovascular Disease by Shared Protein-Protein Interaction Network Analyses.

Cancer and cardiovascular disease (CVD) have a common co-occurrence. Both diseases display overlapping pathophysiology and risk factors, suggesting shared biological mechanisms. Conditions such as obesity, diabetes, hypertension, smoking, poor diet, and inadequate physical activity can cause both heart disease and cancer. The burgeoning field of onco-cardiology aims to develop diagnostics and innovative therapeutics for both diseases through targeting shared mechanisms and molecular targets. In this overarching context, this expert review presents an analysis of the protein-protein interaction (PPI) networks for onco-cardiology drug discovery. Several PPI complexes such as MDM2-TP53 and CDK4-pRB have been studied for their tumor-suppressive functions. In addition, XIAP-SMAC, RAC1-GEF, Sur-2ESX, and TP53-BRCA1 are other PPI complexes that offer potential breakthrough for onco-cardiology therapeutics innovation. As both cancer and CVD share biological mechanisms to a certain degree, the PPI network analyses for onco-cardiology drug discovery are promising for addressing comorbid diseases in the spirit of systems medicine. We discuss the emerging architecture of PPI networks in cancer and CVD and prospects and challenges for their exploitation toward therapeutics applications. Finally, we emphasize that PPIs that were once thought to be undruggable have become potential new class of innovative drug targets.

Protein Misfolding Diseases and Therapeutic Approaches.

Protein folding is the process by which a polypeptide chain acquires its functional, native 3D structure. Protein misfolding, on the other hand, is a process in which protein fails to fold into its native functional conformation. This misfolding of proteins may lead to precipitation of a number of serious diseases such as Cystic Fibrosis (CF), Alzheimer's Disease (AD), Parkinson's Disease (PD), and Amyotrophic Lateral Sclerosis (ALS) etc. Protein Quality-control (PQC) systems, consisting of molecular chaperones, proteases and regulatory factors, help in protein folding and prevent its aggregation. At the same time, PQC systems also do sorting and removal of improperly folded polypeptides. Among the major types of PQC systems involved in protein homeostasis are cytosolic, Endoplasmic Reticulum (ER) and mitochondrial ones. The cytosol PQC system includes a large number of component chaperones, such as Nascent-polypeptide-associated Complex (NAC), Hsp40, Hsp70, prefoldin and T Complex Protein-1 (TCP-1) Ring Complex (TRiC). Protein misfolding diseases caused due to defective cytosolic PQC system include diseases involving keratin/collagen proteins, cardiomyopathies, phenylketonuria, PD and ALS. The components of PQC system of Endoplasmic Reticulum (ER) include Binding immunoglobulin Protein (BiP), Calnexin (CNX), Calreticulin (CRT), Glucose-regulated Protein GRP94, the thiol-disulphide oxidoreductases, Protein Disulphide Isomerase (PDI) and ERp57. ER-linked misfolding diseases include CF and Familial Neurohypophyseal Diabetes Insipidus (FNDI). The components of mitochondrial PQC system include mitochondrial chaperones such as the Hsp70, the Hsp60/Hsp10 and a set of proteases having AAA+ domains similar to the proteasome that are situated in the matrix or the inner membrane. Protein misfolding diseases caused due to defective mitochondrial PQC system include medium-chain acyl-CoA dehydrogenase (MCAD)/Short-chain Acyl-CoA Dehydrogenase (SCAD) deficiency diseases, hereditary spastic paraplegia. Among therapeutic approaches towards the treatment of various protein misfolding diseases, chaperones have been suggested as potential therapeutic molecules for target based treatment. Chaperones have been advantageous because of their efficient entry and distribution inside the cells, including specific cellular compartments, in therapeutic concentrations. Based on the chemical nature of the chaperones used for therapeutic purposes, molecular, chemical and pharmacological classes of chaperones have been discussed.

Elucidation of marine fungi derived anthraquinones as mycobacterial mycolic acid synthesis inhibitors: an in silico approach.

Tuberculosis (TB) is a leading cause of mortality amongst infectious diseases. While the anti-TB drugs can cure TB, the non-compliance and rapidly increasing resistance is of serious concern. The study aimed to search novel potent inhibitor(s) against MabA and PKS18 targets of Mycobacterium tuberculosis (M.tb.) by virtual screening of anthraquinones from marine fungi. The target proteins MabA and PKS18 involved in M.tb. mycolic acid biosynthesis were retrieved from RCSB Protein Data Bank. Chemical structures of 100 marine fungal anthraquinones were retrieved from the PubChem database. These were filtered through Lipinski's rule of five (for druglikeness) and in silico ADME/Tox analysis (for pharmacokinetic properties) and subjected to molecular docking analysis using AutoDock 4.2. The molecular interaction revealed averufin to possess dual inhibitory potential against M.tb. MabA and PKS18 with binding energy of - 8.84 kcal/mol and - 8.23 kcal/mol, and Ki values of 1.79 and 3.12 µM respectively. Averufin exhibits improved drug-like properties, ADMET profile and binding affinity to both targets as compared to control drugs. Our study suggests that averufin a natural anthraquinone, satisfies all the in silico parameters tested and is expected to efficiently inhibit M.tb. mycolic acid pathway. It might therefore emerge as a promising dual-targeted, novel natural anti-TB lead in future.

Modulation in the conformational and stability attributes of the Alzheimer's disease associated amyloid-beta mutants and their favorable stabilization by curcumin: molecular dynamics simulation analysis.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive accumulation of amyloid-beta (Aß) peptides in brain. In the present study, two familial Aß42 mutations, namely A2V (harmful) and A2T (protective) have been analyzed and compared with the wild-type (WT) by performing all-atom molecular dynamics (MD) simulations in the absence and presence of curcumin, a well-known inhibitor of Aß plaque formation. Mutant A2V was found to exhibit highest stability followed by WT and mutant A2T in the absence of curcumin. This stability trend was found to be reversed in the presence of curcumin, suggesting a significant change in the conformational landscape of Aß42 folding. Due to significant differences in the folding and interaction patterns of the mutants A2V and A2T, curcumin exhibited higher binding affinity for mutant A2T as compared to that of A2V. To the best of our knowledge, this is the first report on the effect of curcumin binding on structural landscapes of the two contrasting point mutants providing an understanding of the basis of Aß plaque formation and its prevention by curcumin.

Natural Products as Anticancerous Therapeutic Molecules with Special Reference to Enzymatic Targets Topoisomerase, COX, LOX and Aromatase.

Cancer, characterized by uncontrolled growth and proliferation of cells, is affecting millions of people every year and estimated as the second leading cause of death. Its successful treatment yet remains a challenge due to the lack of selectivity, toxicity and the development of multidrug resistant cells to the currently available drugs. Plant derived natural products hold great promise for discovery and development of new pharmaceuticals against cancer as evident by the fact that out of 121 drugs prescribed for cancer treatment till date, 90 are derived from plant sources. Furthermore, the plant derived therapeutic molecules are also considered as safer substitutes to those of synthetic ones. In this review, the therapeutic potentials of plant derived natural products belonging to secondary metabolites, namely alkaloids, flavonoids and terpenoids as anticancer molecules, involving various strategies of treatment, have been discussed with special reference to topoisomerases (Topo), cyclooxygenases (COX), lipoxygenase (LOX) and aromatase as enzymatic targets for various types of cancers. Furthermore, in view of the recent advances made in the field of computer aided drug design, the present review also discusses the use of computational approaches such as ADMET, molecular docking, molecular dynamics simulation and QSAR to assess and predict the safety, efficacy, potency and identification of such potent anticancerous therapeutic molecules.

Modulation of interaction of mutant TP53 and wild type BRCA1 by alkaloids: a computational approach towards targeting protein-protein interaction as a futuristic therapeutic intervention strategy for breast cancer impediment.

Protein-protein interactions (PPI) are a new emerging class of novel therapeutic targets. In order to probe these interactions, computational tools provide a convenient and quick method towards the development of therapeutics. Keeping this in view the present study was initiated to analyse interaction of tumour suppressor protein p53 (TP53) and breast cancer associated protein (BRCA1) as promising target against breast cancer. Using computational approaches such as protein-protein docking, hot spot analyses, molecular docking and molecular dynamics simulation (MDS), stepwise analyses of the interactions of the wild type and mutant TP53 with that of wild type BRCA1 and their modulation by alkaloids were done. Protein-protein docking method was used to generate both wild type and mutant complexes of TP53-BRCA1. Subsequently, the complexes were docked using sixteen different alkaloids, fulfilling ADMET and Lipinski's rule of five criteria, and were compared with that of a well-known inhibitor of PPI, namely nutlin. The alkaloid dicentrine was found to be the best docked alkaloid among all the docked alklaloids as well as that of nutlin. Furthermore, MDS analyses of both wild type and mutant complexes with the best docked alkaloid i.e. dicentrine, revealed higher stability of mutant complex than that of the wild one, in terms of average RMSD, RMSF and binding free energy, corroborating the results of docking. Results suggested more pronounced interaction of BRCA1 with mutant TP53 leading to increased expression of mutated TP53 thus showing a dominant negative gain of function and hampering wild type TP53 function leading to tumour progression.

Chitosan immobilized novel peroxidase from Azadirachta indica: Characterization and application.

In the present paper, a peroxidase was purified from the leaves of a medicinal tree, namely Azadirachta indica, to 45.2 folds with overall recovery of 61%. Based on the subunit size, the purified peroxidase was suggested to be a monomeric structure of size 50kDa and exhibited good thermostability as it was fully stable at 65°C for 1hr and also retained about 73% activity at 70°C till 30min. The substrate affinity was found to be in order of guaiacol>pyrogallol>o-dianisidine. The purified peroxidase was found to be insensitive towards high concentrations of Na+, Ca2+, Mg2+ and Mn2+. Heavy metals, namely Cs2+, Co2+ and Cd2+ activated the peroxidase while that of Hg2+ deactivated the peroxidase in concentration dependent manner. The purified peroxidase exhibited tolerance towards organic solvents in order of ethanol>butanol>isopropanol>acetone. Immobilization of purified peroxidase by entrapment into chitosan beads led to shift in its optimum pH from pH 5 to 7 and considerable enhancement in dye decolorization ability as compared to that of free enzyme. Thus, based on all the above properties, it may be suggested that the purified A. indica peroxidase is a promising candidate for industrial applications.

Lipoxygenase directed anti-inflammatory and anti-cancerous secondary metabolites: ADMET-based screening, molecular docking and dynamics simulation.

Lipoxygenases (LOXs), key enzymes involved in the biosynthesis of leukotrienes, are well known to participate in the inflammatory and immune responses. With the recent reports of involvement of 5-LOX (one of the isozymes of LOX in human) in cancer, there is a need to find out selective inhibitors of 5-LOX for their therapeutic application. In the present study, plant-derived 300 anti-inflammatory and anti-cancerous secondary metabolites (100 each of alkaloids, flavonoids and terpenoids) have been screened for their pharmacokinetic properties and subsequently docked for identification of potent inhibitors of 5-LOX. Pharmacokinetic analyses revealed that only 18 alkaloids, 26 flavonoids, and 9 terpenoids were found to fulfill all the absorption, distribution, metabolism, excretion, and toxicity descriptors as well as those of Lipinski's Rule of Five. Docking analyses of pharmacokinetically screened metabolites and their comparison with a known inhibitor (drug), namely zileuton revealed that only three alkaloids, six flavonoids and three terpenoids were found to dock successfully with 5-LOX with the flavonoid, velutin being the most potent inhibitor among all. The results of the docking analyses were further validated by performing molecular dynamics simulation and binding energy calculations for the complexes of 5-LOX with velutin, galangin, chrysin (in order of LibDock scores), and zileuton. The data revealed stabilization of all the complexes within 15 ns of simulation with velutin complex exhibiting least root-mean-square deviation value (.285 ± .007 nm) as well as least binding energy (ΔGbind = -203.169 kJ/mol) as compared to others during the stabilization phase of simulation.

Plant derived anti-cancerous secondary metabolites as multipronged inhibitor of COX, Topo, and aromatase: molecular modeling and dynamics simulation analyses.

In the present study, 300 plant derived secondary metabolites (100 each of alkaloid, flavonoid, and terpenoid), have been screened for their anti-cancerous activity through inhibition of selected key enzymatic targets, namely cyclooxygenases (COXs), topoisomerases (Topos), and aromatase by molecular docking approach. Furthermore, the stability of the complexes of top hits, from each class of secondary metabolites, with their respective enzymatic targets was analyzed using molecular dynamics (MD) simulation analyses and binding free energy calculations. Analysis of the results of the docking in light of the pharmacokinetically screened 18 alkaloids, 26 flavonoids, and 9 terpenoids, revealed that the flavonoid, curcumin, was the most potent inhibitor for all the selected enzymatic targets. The stability of the complexes of COX-1, COX-2, Topo I, Topo IIß and aromatase with the most potent inhibitor curcumin and those of the respective drugs, namely ibuprofen, aspirin, topotecan, etoposide, and exemestane were also analyzed through MD simulation analyses which revealed better stability of curcumin complexes than those of respective drugs. Binding energy calculations of the complexes of the curcumin with all the targets, except those of Topos, exhibited lower binding energies for the curcumin complexes than those of respective drugs which corroborated with the results of molecular docking analyses. Thus, the present study affirms the versatile and multipronged nature of curcumin, the traditionally used herbal medicine, as anti-cancer molecule directed against these enzymatic targets.

Citrus Functional Genomics and Molecular Modeling in Relation to Citrus sinensis (Sweet Orange) Infection with Xylella fastidiosa (Citrus Variegated Chlorosis).

Citrus are among the economically most important fruit tree crops in the world. Citrus variegated chlorosis (CVC), caused by Xylella fastidiosa infection, is a serious disease limiting citrus production at a global scale. With availability of citrus genomic resources, it is now possible to compare citrus expressed sequence tag (EST) data sets and identify single-nucleotide polymorphisms (SNPs) within and among different citrus cultivars that can be exploited for citrus resistance to infections, citrus breeding, among others. We report here, for the first time, SNPs in the EST data sets of X. fastidiosa-infected Citrus sinensis (sweet orange) and their functional annotation that revealed the involvement of eight C. sinensis candidate genes in CVC pathogenesis. Among these genes were xyloglucan endotransglycosylase, myo-inositol-1-phosphate synthase, and peroxidase were found to be involved in plant cell wall metabolism. These have been further investigated by molecular modeling for their role in CVC infection and defense. Molecular docking analyses of the wild and the mutant (SNP containing) types of the selected three enzymes with their respective substrates revealed a significant decrease in the binding affinity of substrates for the mutant enzymes, thus suggesting a decrease in the catalytic efficiency of these enzymes during infection, thereby facilitating a favorable condition for infection by the pathogen. These findings offer novel agrigenomics insights in developing future molecular targets and strategies for citrus fruit cultivation in ways that are resistant to X. fastidiosa infection, and by extension, with greater harvesting efficiency and economic value.

Immobilization of papaya laccase in chitosan led to improved multipronged stability and dye discoloration.

A purified papaya laccase was immobilized in chitosan beads using entrapment approach and its physico-chemical properties were investigated and compared with that of free enzyme. Increase in properties of the laccase such as optimum temperature (by 10 °C), thermostability (by 3-folds) and optimum pH (from 8.0 to 10.0) was observed after immobilization. Immobilization led to increased tolerance of enzyme to a number of metal ions (including heavy metals) and organic solvents namely, ethanol, isopropanol, methanol, benzene and DMF. The catalytic efficiency (Kcat/Km) of the immobilized enzyme was found to increase more than ten folds, in comparison to that of the free enzyme, with hydroquinone as substrate. Immobilization of laccase also led to improvement in dye decolorization such that the synthetic dye indigo carmine (50 µg/ml) was completely decolorized within 8h of incubation as compared to that of the free laccase which decolorized the same dye to only 56% under similar conditions. Thus, immobilization of laccase into chitosan beads led to tremendous improvement in various useful attributes of this enzyme thereby making it more versatile for its industrial exploitation.

Alzheimer's disease: An overview of amyloid beta dependent pathogenesis and its therapeutic implications along with in silico approaches emphasizing the role of natural products.

Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder characterized by amyloid beta (Aß) deposition in brain with subsequent formation of neuritic plaques leading to dementia. A number of therapeutic strategies targeted against Aß depositions have been rigorously explored which provided successful results corresponding to the existing symptomatic treatments. However, at the same time, several failures corresponding to the disease altering therapies and drugs have also been observed due to potential drawbacks in understanding of the pathogenesis of the disease, development of drug candidates and subsequent designing of clinical trials. Preclinical research, along with experimental and clinical studies, is continuously providing novel information which may reveal multi-target directed ligands and combination therapies for targeting Aß. Thus, in view of the estimated increase in the number of AD patients globally, the present review attempts to summarize the available evidence dealing with various therapeutic approaches targeting Aß, focusing specifically on pharmaceutical compounds under various stages of clinical trials. Furthermore, in view of a number of computational advances having significant impact in the field of computer aided drug design, we have also presented results of analysis of natural compounds as potential therapeutic molecules in preventing Aß plaque formation using in silico approaches.

DNA topoisomerase-directed anticancerous alkaloids: ADMET-based screening, molecular docking, and dynamics simulation.

Topoisomerases (Topo I and II) have been looked as crucial targets against various types of cancers. In the present paper, 100 anticancerous alkaloids were subjected to in silico absorption, distribution, metabolism, excretion, and toxicity (ADMET) analyses to investigate their pharmacokinetic properties. Out of 100 alkaloids, only 18 were found to fulfill all the ADMET descriptors and obeyed the Lipinski's rule of five. All the 18 alkaloids were found to dock successfully within the active site of both Topo I and II. A comparison of the inhibitory potential of 18 screened alkaloids with those of selected drugs revealed that four alkaloids (oliveroline, coptisine, aristolactam, and piperine) inhibited Topo I, whereas six alkaloids (oliveroline, aristolactam, anonaine, piperine, coptisine, and liriodenine) inhibited Topo II more strongly than those of their corresponding drugs, topotecan and etoposide, respectively, with oliveroline being the outstanding. The stability of the complexes of Topo I and II with the best docked alkaloid, oliveroline, was further analyzed using 10 nSec molecular dynamics simulation and compared with those of the respective drugs, namely, topotecan and etoposide, which revealed stabilization of these complexes within 5 nSec of simulation with better stability of Topo II complex than that of Topo I.

Treponema pallidum putative novel drug target identification and validation: rethinking syphilis therapeutics with plant-derived terpenoids.

Syphilis, a slow progressive and the third most common sexually transmitted disease found worldwide, is caused by a spirochete gram negative bacteria Treponema pallidum. Emergence of antibiotic resistant T. pallidum has led to a search for novel drugs and their targets. Subtractive genomics analyses of pathogen T. pallidum and host Homo sapiens resulted in identification of 126 proteins essential for survival and viability of the pathogen. Metabolic pathway analyses of these essential proteins led to discovery of nineteen proteins distributed among six metabolic pathways unique to T. pallidum. One hundred plant-derived terpenoids, as potential therapeutic molecules against T. pallidum, were screened for their drug likeness and ADMET (absorption, distribution, metabolism, and toxicity) properties. Subsequently the resulting nine terpenoids were docked with five unique T. pallidum targets through molecular modeling approaches. Out of five targets analyzed, D-alanine:D-alanine ligase was found to be the most promising target, while terpenoid salvicine was the most potent inhibitor. A comparison of the inhibitory potential of the best docked readily available natural compound, namely pomiferin (flavonoid) with that of the best docked terpenoid salvicine, revealed that salvicine was a more potent inhibitor than that of pomiferin. To the best of our knowledge, this is the first report of a terpenoid as a potential therapeutic molecule against T. pallidum with D-alanine:D-alanine ligase as a novel target. Further studies are warranted to evaluate and explore the potential clinical ramifications of these findings in relation to syphilis that has public health importance worldwide.

A ripening associated peroxidase from papaya having a role in defense and lignification: heterologous expression and in-silico and in-vitro experimental validation.

Fruit ripening associated full length cDNA of a peroxidase from papaya was cloned and heterologously expressed. The expressed peroxidase was activated by in-vitro re-folding in the presence of hemin and calcium. The purified recombinant peroxidase exhibited broad substrate affinity in the order of o-dianisidine>pyrogallol>guaiacol and was found to be a homotetramer of 155kDa with each subunit having a size of 38kDa. The basis of the distinctive preferences for various substrates was investigated through in-silico molecular modeling approaches. Thus, when the modeled papaya peroxidase-heme complex was docked with these substrates, the in-silico binding efficiency was found to be in agreement with those of wet lab results with the involvement of Arg37, Phe40, His41, Pro137, Asn138, His139, His167, and Phe239 as the common interacting residues in all the cases. However, the binding of the different substrates were found to be associated with conformational changes in the peroxidase. Thus, in the case of o-dianisidine (the most efficient substrate), the protein was folded in the most compact fashion when compared to guaiacol (the least efficient substrate). Protein function annotation analyses revealed that the papaya peroxidase may have biological roles in oxidation-reduction processes, stresses, defense responses etc. In order to further validate its role in lignifications, the papaya peroxidase was compared with a lignin biosynthetic peroxidase from Leucaena leucocephala, a tree legume. Thus, based on 3D structure superimposition and docking, both peroxidases exhibited a great extent of similarity suggesting the papaya peroxidase having a role in lignification (defense response) too. The predicted functions of papaya peroxidase in defense response and lignification were further validated experimentally using qRT-PCR analyses and measurement of oxidation of coniferyl alcohol.

Molecular docking and dynamics simulation analyses unraveling the differential enzymatic catalysis by plant and fungal laccases with respect to lignin biosynthesis and degradation.

Laccase, widely distributed in bacteria, fungi, and plants, catalyzes the oxidation of wide range of compounds. With regards to one of the important physiological functions, plant laccases are considered to catalyze lignin biosynthesis while fungal laccases are considered for lignin degradation. The present study was undertaken to explain this dual function of laccases using in-silico molecular docking and dynamics simulation approaches. Modeling and superimposition analyses of one each representative of plant and fungal laccases, namely, Populus trichocarpa and Trametes versicolor, respectively, revealed low level of similarity in the folding of two laccases at 3D levels. Docking analyses revealed significantly higher binding efficiency for lignin model compounds, in proportion to their size, for fungal laccase as compared to that of plant laccase. Residues interacting with the model compounds at the respective enzyme active sites were found to be in conformity with their role in lignin biosynthesis and degradation. Molecular dynamics simulation analyses for the stability of docked complexes of plant and fungal laccases with lignin model compounds revealed that tetrameric lignin model compound remains attached to the active site of fungal laccase throughout the simulation period, while it protrudes outwards from the active site of plant laccase. Stability of these complexes was further analyzed on the basis of binding energy which revealed significantly higher stability of fungal laccase with tetrameric compound than that of plant. The overall data suggested a situation favorable for the degradation of lignin polymer by fungal laccase while its synthesis by plant laccase.