Diet-induced induction of hepatic serine/threonine kinase STK38 triggers proinflammation and hepatic lipid accumulation.

Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases worldwide. Although the involvement of chronic overnutrition, systemic inflammation, and insulin resistance in the development of NAFLD is well-established, however, the associations among these remain to be elucidated. Several studies have reported that chronic overnutrition, such as excessive consumption of fats (high-fat diet, HFD), can cause insulin resistance and inflammation. However, the mechanisms by which HFD exerts inflammation and thereby promotes insulin resistance and intrahepatic fat accumulation remain poorly understood. Here, we show that HFD induces the expression of hepatic serine/threonine kinase 38 (STK38), which further induces systemic inflammation leading to insulin resistance. Notably, ectopic expression of STK38 in mouse liver leads to lean NAFLD phenotype with hepatic inflammation, insulin resistance, intrahepatic lipid accumulation, and hypertriglyceridemia in mice fed on a regular chow diet. Further, depletion of hepatic STK38 in HFD-fed mice remarkably reduces proinflammation, improves hepatic insulin sensitivity, and decreases hepatic fat accumulation. Mechanistically, two critical stimuli are elicited by STK38 action. For one stimulus, STK38 binds to Tank-Binding protein Kinase 1 and induces Tank-Binding protein Kinase 1 phosphorylation to promote NF-κß nuclear translocation that mobilizes the release of proinflammatory cytokines and eventually leads to insulin resistance. The second, stimulus involves intrahepatic lipid accumulation by enhanced de novo lipogenesis via reducing the AMPK-ACC signaling axis. These findings identify STK38 as a novel nutrient-sensitive proinflammatory and lipogenic factor in maintaining hepatic energy homeostasis, and it provides a promising target for hepatic and immune health.

Design, synthesis, and biological evaluation of a small molecule oral agonist of the glucagon-like-peptide-1 receptor.

An absolute or relative deficiency of pancreatic ß-cells mass and functionality is a crucial pathological feature common to type 1 diabetes mellitus and type 2 diabetes mellitus. Glucagon-like-peptide-1 receptor (GLP1R) agonists have been the focus of considerable research attention for their ability to protect ß-cell mass and augment insulin secretion with no risk of hypoglycemia. Presently commercially available GLP1R agonists are peptides that limit their use due to cost, stability, and mode of administration. To address this drawback, strategically designed distinct sets of small molecules were docked on GLP1R ectodomain and compared with previously known small molecule GLP1R agonists. One of the small molecule PK2 (6-((1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-6H-indolo[2,3-b]quinoxaline) displays stable binding with GLP1R ectodomain and induces GLP1R internalization and increasing cAMP levels. PK2 also increases insulin secretion in the INS-1 cells. The oral administration of PK2 protects against diabetes induced by multiple low-dose streptozotocin administration by lowering high blood glucose levels. Similar to GLP1R peptidic agonists, treatment of PK2 induces ß-cell replication and attenuate ß-cell apoptosis in STZ-treated mice. Mechanistically, this protection was associated with decreased thioredoxin-interacting protein expression, a potent inducer of diabetic ß-cell apoptosis and dysfunction. Together, this report describes a small molecule, PK2, as an orally active nonpeptidic GLP1R agonist that has efficacy to preserve or restore functional ß-cell mass.

Structural insights into SARS-CoV-2 main protease conformational plasticity.

The spread of different severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants underscores the need for insights into the structural properties of its structural and non-structural proteins. The highly conserved homo-dimeric chymotrypsin-like protease (3CL MPRO ), belonging to the class of cysteine hydrolases, plays an indispensable role in processing viral polyproteins that are involved in viral replication and transcription. Studies have successfully demonstrated the role of MPRO as an attractive drug target for designing antiviral treatments because of its importance in the viral life cycle. Herein, we report the structural dynamics of six experimentally solved structures of MPRO (i.e., 6LU7, 6M03, 6WQF, 6Y2E, 6Y84, and 7BUY including both ligand-free and ligand-bound states) at different resolutions. We have employed a structure-based balanced forcefield, CHARMM36m through state-of-the-art all-atoms molecular dynamics simulations at µ-seconds scale at room temperature (303K) and pH 7.0 to explore their structure-function relationship. The helical domain-III responsible for dimerization mostly contributes to the altered conformational states and destabilization of MPRO . A keen observation of the high degree of flexibility in the P5 binding pocket adjoining domain II-III highlights the reason for observation of conformational heterogeneity among the structural ensembles of MPRO . We also observe a differential dynamics of the catalytic pocket residues His41, Cys145, and Asp187, which may lead to catalytic impairment of the monomeric proteases. Among the highly populated conformational states of the six systems, 6LU7 and 7M03 forms the most stable and compact MPRO conformation with intact catalytic site and structural integrity. Altogether, our findings from this extensive study provides a benchmark to identify physiologically relevant structures of such promising drug targets for structure-based drug design and discovery of potent drug-like compounds having clinical potential.

Illuminating the structural basis of human neurokinin 1 receptor (NK1R) antagonism through classical all-atoms molecular dynamics simulations.

Advances in structural biology have bestowed insights into the pleiotropic effects of neurokinin 1 receptors (NK1R) in diverse patho-physiological processes, thereby highlighting the potential therapeutic value of antagonists directed against NK1R. Herein, we investigate the mode of antagonist recognition to discern the obscure atomic facets germane for the function and molecular determinants of NK1R. To commence discernment of potent antagonists and the conformational changes in NK1R, induced upon antagonist binding, state-of-the-art classical all-atoms molecular dynamics (MD) simulations in lipid mimetic bilayers have been utilized. MD simulations of structural ensembles reveals the involvement of TM5 and TM6 in tight anchoring of antagonists through a network of interhelical hydrogen-bonds, while, the extracellular loop 2 (ECL2) governs the overall size and nature of the pocket, thereby modulating NK1R. Consistent comparison between experiments and MD simulation results discerns the predominant role of TM3, TM4, and TM6 in lipid-NK1R interaction. Correlation between hydrophobic index and helicity of TM domains elucidates their importance in maintaining the structural stability in addition to regulating NK1R antagonism. Taken together, we anticipate that our computational study marks a comprehensive structural basis of NK1R antagonism in lipid bilayers, which may facilitate designing of new therapeutics against associated diseases targeting human neurokinin receptors.

Drug repurposing for Mpox: Discovery of small molecules as potential inhibitors against DNA-dependent RNA polymerase using molecular modeling approach.

Mpox (formerly Monkeypox), a zoonotic illness caused by the Mpox virus, belongs to the Orthopoxvirus genus in the family Poxviridae. To design and develop effective antiviral therapeutics against DNA viruses, the DNA-dependent RNA polymerase (DdRp) of poxviruses has emerged as a promising drug target. In the present study, we modeled the three-dimensional (3D) structure of DdRp using a template-based homology approach. After modeling, virtual screening was performed to probe the molecular interactions between 1755 Food and Drug Administration-approved small molecule drugs (≤500 molecular weight) and the DdRp of Mpox. Based on the binding affinity and molecular interaction patterns, five drugs, lumacaftor (-11.7 kcal/mol), conivaptan (-11.7 kcal/mol), betulinic acid (-11.6 kcal/mol), fluspirilene (-11.3 kcal/mol), and imatinib (-11.2 kcal/mol), have been ranked as the top drug compounds interacting with Mpox DdRp. Complexes of these shortlisted drugs with DdRp were further evaluated using state-of-the-art all-atoms molecular dynamics (MD) simulations on 200 nanoseconds followed by principal component analysis (PCA). MD simulations and PCA results revealed highly stable interactions of these small drugs with DdRp. After due validation in wet-lab using available in vitro and in vivo experiments, these repurposed drugs can be further utilized for the treatment of contagious Mpox virus. The outcome of this study may establish a solid foundation to screen repurposed and natural compounds as potential antiviral therapeutics against different highly pathogenic viruses.

Delineation of molecular interactions of plant growth promoting bacteria induced ß-1,3-glucanases and guanosine triphosphate ligand for antifungal response in rice: a molecular dynamics approach.

BACKGROUND: The plant growth is influenced by multiple interactions with biotic (microbial) and abiotic components in their surroundings. These microbial interactions have both positive and negative effects on plant. Plant growth promoting bacterial (PGPR) interaction could result in positive growth under normal as well as in stress conditions. METHODS: Here, we have screened two PGPR's and determined their potential in induction of specific gene in host plant to overcome the adverse effect of biotic stress caused by Magnaporthe grisea, a fungal pathogen that cause blast in rice. We demonstrated the glucanase protein mode of action by performing comparative modeling and molecular docking of guanosine triphosphate (GTP) ligand with the protein. Besides, molecular dynamic simulations have been performed to understand the behavior of the glucanase-GTP complex. RESULTS: The results clearly showed that selected PGPR was better able to induce modification in host plant at morphological, biochemical, physiological and molecular level by activating the expression of ß-1,3-glucanases gene in infected host plant. The docking results indicated that Tyr75, Arg256, Gly258, and Ser223 of glucanase formed four crucial hydrogen bonds with the GTP, while, only Val220 found to form hydrophobic contact with ligand. CONCLUSIONS: The PGPR able to induce ß-1,3-glucanases gene in host plant upon pathogenic interaction and ß-1,3-glucanases form complex with GTP by hydrophilic interaction for induction of defense cascade for acquiring resistance against Magnaporthe grisea.

Membrane dynamics of γ-secretase with the anterior pharynx-defective 1B subunit.

The four-subunit protease complex γ-secretase cleaves many single-pass transmembrane (TM) substrates, including Notch and ß-amyloid precursor protein to generate amyloid-ß (Aß), central to Alzheimer's disease. Two of the subunits anterior pharynx-defective 1 (APH-1) and presenilin (PS) exist in two homologous forms APH1-A and APH1-B, and PS1 and PS2. The consequences of these variations are poorly understood and could affect Aß production and γ-secretase medicine. Here, we developed the first complete structural model of the APH-1B subunit using the published cryo-electron microscopy (cryo-EM) structures of APH1-A (Protein Data Bank: 5FN2, 5A63, and 6IYC). We then performed all-atom molecular dynamics simulations at 303 K in a realistic bilayer system to understand both APH-1B alone and in γ-secretase without and with substrate C83-bound. We show that APH-1B adopts a 7TM topology with a water channel topology similar to APH-1A. We demonstrate direct transport of water through this channel, mainly via Glu84, Arg87, His170, and His196. The apo and holo states closely resemble the experimental cryo-EM structures with APH-1A, however with subtle differences: The substrate-bound APH-1B γ-secretase was quite stable, but some TM helices of PS1 and APH-1B rearranged in the membrane consistent with the disorder seen in the cryo-EM data. This produces different accessibility of water molecules for the catalytic aspartates of PS1, critical for Aß production. In particular, we find that the typical distance between the catalytic aspartates of PS1 and the C83 cleavage sites are shorter in APH-1B, that is, it represents a more closed state, due to interactions with the C-terminal fragment of PS1. Our structural-dynamic model of APH-1B alone and in γ-secretase suggests generally similar topology but some notable differences in water accessibility which may be relevant to the protein's existence in two forms and their specific function and location.

A computer-simulated mechanism of familial Alzheimer's disease: Mutations enhance thermal dynamics and favor looser substrate-binding to γ-secretase.

The 4-subunit intramembrane protease complex γ-secretase cleaves many substrates including fragments of the ß-amyloid precursor protein (APP), leading to formation of Aß peptides, and Notch. Mutations in APP and the catalytic subunit of γ-secretase, presenilin, cause familial Alzheimer's disease (fAD). Mutations are assumed to change the substrate-binding and cleavage and thereby the Aß formed. Whereas a wild-type structure of substrate-bound γ-secretase became recently available from cryogenic electron microscopy (6IYC), the structure and dynamics of mutant proteins remain obscure. Here, we studied five prominent mutants of substrate-bound γ-secretase by explicit all-atom molecular dynamics in a phospholipid membrane model at physiological temperature using the experimental structure as template: The presenilin 1 mutants E280A, G384A, A434C, and L435F and the V717I mutant of APP. Our structures and dynamics provide the first atomic detail into how fAD-causing mutations affect substrate binding to γ-secretase. The pathogenic mutations tend to increase the space and variability in the substrate binding site, as seen e.g. from the distance from catalytic aspartate to substrate cleavage sites. We suggest that we have identified the molecular cause of the "imprecise cleavage" that leads to two trimming pathways in γ-secretase, consistent with the FIST model, which may rationalize the experimental Aß42/Aß40 ratios as a molecular basis for fAD.

Common mechanism of thermostability in small α- and ß-proteins studied by molecular dynamics.

Protein thermostability is important to evolution, diseases, and industrial applications. Proteins use diverse molecular strategies to achieve stability at high temperature, yet reducing the entropy of unfolding seems required. We investigated five small α-proteins and five ß-proteins with known, distinct structures and thermostability (Tm ) using multi-seed molecular dynamics simulations at 300, 350, and 400 K. The proteins displayed diverse changes in hydrogen bonding, solvent exposure, and secondary structure with no simple relationship to Tm . Our dynamics were in good agreement with experimental B-factors at 300 K and insensitive to force-field choice. Despite the very distinct structures, the native-state (300 + 350 K) free-energy landscapes (FELs) were significantly broader for the two most thermostable proteins and smallest for the three least stable proteins in both the α- and ß-group and with both force fields studied independently (tailed t-test, 95% confidence level). Our results suggest that entropic ensembles stabilize proteins at high temperature due to reduced entropy of unfolding, viz., ΔG = ΔH - TΔS. Supporting this mechanism, the most thermostable proteins were also the least kinetically stable, consistent with broader FELs, typified by villin headpiece and confirmed by specific comparison to a mesophilic ortholog of Thermus thermophilus apo-pyrophosphate phosphohydrolase. We propose that molecular strategies of protein thermostabilization, although diverse, tend to converge toward highest possible entropy in the native state consistent with the functional requirements. We speculate that this tendency may explain why many proteins are not optimally structured and why molten-globule states resemble native proteins so much.

Evaluating the immunomodulatory responses of LdODC-derived MHC Class-II restricted peptides against VL.

In a bid to develop a novel immunoprophylactic measure against visceral leishmaniasis (VL), MHC class-II-restricted epitopes of LdODC were identified by reverse vaccinology approach. Five consensus HLA-DRB1*0101-restricted epitopes were screened. The analysis revealed that the set of epitopes was presented by at least 54 diverse MHC class-II alleles. Based on in silico screening, followed by molecular dynamics simulation, population coverage analysis, and HLA cross-presentation ability, five best epitopes were evaluated. PBMCs isolated from treated VL subjects, when stimulated with synthetic peptide alone or as a cocktail of peptides, triggered a secretory IFN-γ, but not the IL-10 level. Support in this notion came from intracellular cytokine level with a considerable up-regulated IFN-γ produced by CD4+ T cells. Also, the enhanced IFN-γ seemed to be augmented with the activation of macrophages with prominent IL-12 production. Therefore, it can be concluded that the screened MHC class-II-restricted epitope hotspots derived from Leishmania ODC can trigger CD4+ T cells, which can skew macrophage functions towards protection. However, a detailed analysis can explore its potentiality as a vaccine candidate.

Cryo-temperature effects on membrane protein structure and dynamics.

Innovations in cryogenic electron microscopy (Cryo-EM) have led to high-quality structures of important proteins such as the ribosome and γ-secretase, the membrane protease that produces Aß involved in Alzheimer's disease. However, freezing may change protein structure and dynamics relative to the physiologically relevant "hot" state. To explore this, we studied substrate-bound γ-secretase (6IYC) by molecular dynamics as a hot, cold, and quickly cooled state in both membrane and water systems. We show that the experimental structure resembles the simulated cooled state, structurally between the hot and cold states and membrane and water systems, but with cold dynamics. We observe "cryo-contraction" in the membrane from 303 to 85 K, reducing radius of gyration (Rg) by 1% from 4.01 to 3.97 nm (6IYC = 3.95 nm). The hot state features an unwound C83-substrate with 10-14 α-helix residues (6IYC: 11) in equilibrium with an intact state with 16 helix residues not previously reported. The ß-sheet is weakened with temperature. Multiple hot conformations probably control the Aß42/Aß40 ratio. We thus propose that MD simulation protocols of hot, cold, and cooled states as applied here can correct cryo-EM coordinates. However, important frozen-out fast modes require specific supplementary hot simulations or experiments.

Molecular dynamics of C99-bound γ-secretase reveal two binding modes with distinct compactness, stability, and active-site retention: implications for Aß production.

The membrane protease γ-secretase cleaves the C99 fragment of the amyloid precursor protein, thus producing the Aß peptides central to Alzheimer's disease. Cryo-electron microscopy has provided the topology but misses the membrane and loop parts that contribute to substrate binding. We report here an essentially complete atomic model of C99 within wild-type γ-secretase that respects all the experimental constraints and additionally describes loop, helix, and C99 substrate dynamics in a realistic all-atom membrane. Our model represents the matured auto-cleaved state required for catalysis. From two independent 500-ns molecular dynamic simulations, we identify two conformation states of C99 in equilibrium, a compact and a loose state. Our simulations provide a basis for C99 processing and Aß formation and explain the production of longer and shorter Aß, as the compact state retains C99 for longer and thus probably trims to shorter Aß peptides. We expect pathogenic presenilin mutations to stabilize the loose over the compact state. The simulations detail the role of the Lys53-Lys54-Lys55 anchor for C99 binding, a loss of helicity of bound C99, and positioning of Thr48 and Leu49 leading to alternative trimming pathways on opposite sides of the C99 helix in three amino acid steps. The C99 binding topology resembles that of C83-bound γ-secretase without membrane but lacks a presenilin 1-C99 ß-sheet, which could be induced by C83's stronger binding. The loose state should be selectively disfavored by γ-secretase modulators to increase C99 trimming and reduce the formation of longer Aß, a strategy that is currently much explored but has lacked a structural basis.

Insights into the mode of flavin mononucleotide binding and catalytic mechanism of bacterial chromate reductases: A molecular dynamics simulation study.

Enzymes from natural sources protect the environment via complex biological mechanisms, which aid in reductive immobilization of toxic metals including chromium. Nevertheless, progress was being made in elucidating high-resolution crystal structures of reductases and their binding with flavin mononucleotide (FMN) to understand the underlying mechanism of chromate reduction. Therefore, herein, we employed molecular dynamics (MD) simulations, principal component analysis (PCA), and binding free energy calculations to understand the dynamics behavior of these enzymes with FMN. Six representative chromate reductases in monomeric and dimeric forms were selected to study the mode, dynamics, and energetic component that drive the FMN binding process. As evidenced by MD simulation, FMN prefers to bind the cervix formed between the catalytic domain surrounded by strong conserved hydrogen bonding, electrostatic, and hydrophobic contacts. The slight movement and reorientation of FMN resulted in breakage of some crucial H-bonds and other nonbonded contacts, which were well compensated with newly formed H-bonds, electrostatic, and hydrophobic interactions. The critical residues aiding in tight anchoring of FMN within dimer were found to be strongly conserved in the bacterial system. The molecular mechanics combined with the Poisson-Boltzmann surface area binding free energy of the monomer portrayed that the van der Waals and electrostatic energy contribute significantly to the total free energy, where, the polar solvation energy opposes the binding of FMN. The proposed proximity relationships between enzyme and FMN binding site presented in this study will open up better avenues to engineer enzymes with optimized chromate reductase activity for sustainable bioremediation of heavy metals.

Structural bioinformatics insights into the CARD-CARD interaction mediated by the mitochondrial antiviral-signaling protein of black carp.

The innate immune system offers the first line of defense against invading microbial pathogens through the recognition of conserved pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). The host innate immune system through PRRs, the sensors for PAMPs, induces the production of cytokines. Among different families of PRRs, the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), and its mitochondrial adaptor ie, the mitochondrial antiviral-signaling (MAVS) protein, are crucial for RLR-triggered interferon (IFN) antiviral immunity. Recent studies have shown that the N-terminal caspase recruitment domain (CARD) and transmembrane domain play a pivotal role in oligomerization of black carp MAVS (BcMAVS), crucial for the host innate immune response against viral invasion. In this study, we have used molecular modeling, docking, and molecular dynamics (MD) simulation approaches to shed molecular insights into the oligomerization mechanism of BcMAVSCARD . MD simulation and interaction analysis portrayed that the type-I surface patches of BcMAVS CARD  make the major contribution to the interaction. Moreover, the evidence from surface patches and critical residues involved in the said interaction is found to be similar to that of the human counterpart and requires further investigation for legitimacy. Altogether, our study provided crucial information on oligomerization of BcMAVS CARDs and might be helpful for clarifying the innate immune response against pathogens and downstream signaling in fishes.

Structural bioinformatics insights into ATP binding mechanism in zebrafish (Danio rerio) cyclin-dependent kinase-like 5 (zCDKL5) protein.

In mammalian systems, the conserved cyclin-dependent protein kinases (CDKs) control the process of cell division and curb the transcription mechanism in response to diverse signaling events that are essential for the catalytic activity. In zebrafish, zCDKL5 portrays differential expression profiling in several tissues and presumed to play a vital role in the neuronal development. In this present study, the sequence-structure relationship and mode of ATP binding in zCDKL5 was unveiled through theoretical modeling, molecular docking, and MD simulations. Like human CDKs, the modeled zCDKL5 was found to be bipartite in nature, where, ATP binds to the central cavity of the catalytic domain through a strong network of H-bonding, electrostatic, and hydrophobic interactions. MD simulation portrayed that conserved residues, viz, Ile10, Gly11, Glu12, Val18, Val64, Glu81, Cys143, and Asp144 were indispensable for tight anchoring of ATP and contribute to the stability of the zCDKL5-ATP complex. MM/PBSA binding free energy analysis displayed that van der Waal energy (ΔG vwd ) and Electrostatic energy (ΔG ele ) were the major contributors towards the overall binding free energy. Thus, the comparative structural bioinformatics approach has shed new insights into the dynamics and ATP binding mechanism of zCDKL5. The results from the study will help to undertake further research on the role of phosphorylated CDKL5 in the onset of neurodevelopmental disorders caused by mutations in higher eukaryotic systems.

Computing the Pathogenicity of Alzheimer's Disease Presenilin 1 Mutations.

Alzheimer's disease (AD) is one of the major global health challenges of the 21st century. More than 200 distinct mutations in presenilin 1 (PSEN1) cause severe early-onset familial AD (FAD) and are thus of central interest to the etiology of AD. PSEN1 is the catalytic subunit of γ-secretase that produces ß-amyloid peptide (Aß), and the mutations tend to increase the produced Aß42/Aß40 ratio. The molecular reasons for the pathogenesis of these mutations are unknown. We studied a close-to-complete data set of PSEN1 mutations using 21 different computational methods hypothesized to reproduce pathogenesis, using both sequence- and structure-based methods with the full γ-secretase complex as input. First, we tested whether pathogenicity can be estimated accurately using all possible mutations in PSEN1 as a direct control. Several methods predict the pathogenicity of the mutations (pathogenic vs all other possible mutations) well, with accuracies approaching 90%. We then designed a stricter test for predicting the severity of the mutations estimated by the average clinical age of symptom onset for mutation carriers. Surprisingly, we can predict the clinical age of symptom onset at 95% confidence or higher with several methods. Accordingly, our results show that simple biochemical properties of the amino acid changes rationalize an important part of the pathogenicity of FAD-causing PSEN1 mutations. Although pathogenic mutations generally destabilize γ-secretase, all of the tested protein stability methods failed to predict pathogenicity. Thus, either the static cryogenic-electron-microscopy-derived molecular-dynamics-equilibrated structures used as input fail to capture the stability effect of mutated side chains or protein stability is simply not a key factor in the pathogenicity. Our findings suggest that the chemical causes of FAD may be modeled and lend promise to the development of a semiquantitative model predicting the age of onset of mutation carriers that could eventually become of care-strategic value.

Molecular dynamics insights into the structure, function, and substrate binding mechanism of mucin desulfating sulfatase of gut microbe Bacteroides fragilis.

The complex and dynamic consortia of microbiota that harbors the human gastrointestinal tract contributes ominously to the maintenance of health, the onset and progression of diverse spectrum of disorders. The capability of these enteric microbes to bloom within the gut mucosal milieu is often associated to the glycan metabolism of mucin-degrading bacteria. Accruing evidences suggests that the desulfation of mucin is a rate-limiting step in mucin degradation mechanism by colonic bacterial mucin-desulfating sulfatase enzymes (MDS) enzymes. Till date no experimental evidence is available on how conformational flexibility influences structure and substrate specificity by MDS of gut microbe Bacteroides fragilis. Henceforth, to gain deep insights into the missing but very imperative mechanism, we performed a comprehensive molecular dynamics study, principal component analysis and MM/PBSA binding free energies to gain insights into (i) the domain architecture and mode of substrate binding (ii) conformational dynamics and flexibility that influence the orientation of substrate, (iii) energetic contribution that plays very decisive role to the overall negative binding free energy and stabilities of the complexes (iv) critical residues of active site which influence binding and aid in substrate recognition. This is the first ever report, depicting the molecular basis of recognition of substrates and provides insights into the mode of catalysis by mucin desulfating sulfatase enzymes in gut microbiota. Overall, our study shed new insights into the unmapped molecular mechanisms underlying the recognition of various substrates by mucin desulfating sulfatase, which could be of great relevance in therapeutic implications in human gut microbiota associated disorders.

Molecular docking and simulation study for synthesis of alternative dapsone derivative as a newer antileprosy drug in multidrug therapy.

Leprosy (causative, Mycobacterium leprae) continues to be the persisting public health problem with stable incidence rates, owing to the emergence of dapsone resistance that being the principal drug in the ongoing multidrug therapy. Hence, to overcome the drug resistance, structural modification through medicinal chemistry was used to design newer dapsone derivative(s) (DDs), against folic acid biosynthesis pathway. The approach included theoretical modeling, molecular docking, and molecular dynamic (MD) simulation as well as binding free energy estimation for validation of newly designed seven DDs, before synthesis. Theoretical modeling, docking, and MD simulation studies were used to understand the mode of binding and efficacy of DDs against the wild-type and mutant dihydropteroate synthases (DHPS). Principal component analysis was performed to understand the conformational dynamics of DHPS-DD complexes. Furthermore, the overall stability and negative-binding free energy of DHPS-DD complexes were deciphered using Molecular Mechanics/Poisson-Boltzmann Surface Area technique. Molecular mechanics study revealed that DD3 possesses higher binding free energy than dapsone against mutant DHPS. Energetic contribution analysis portrayed that van der Waals and electrostatic energy contributes profoundly to the overall negative free energy, whereas polar solvation energy opposes the binding. Finally, DD3 was synthesized and characterized using Fourier-transform infrared spectroscopy, UV, liquid chromatography-mass spectrometry, and proton nuclear magnetic resonance techniques. This study suggested that DD3 could be further promoted as newer antileprosy agent. The principles of medicinal chemistry and bioinformatics tools help to locate effective therapeutics to minimize resources and time in current drug development modules.

Mining the Proteome of Leishmania donovani for the Development of Novel MHC Class I Restricted Epitope for the Control of Visceral Leishmaniasis.

Although, the precise host defence mechanism(s) is not completely understood, T cell-mediated immune responses is believed to play a pivotal role in controlling parasite infection. Here we target the stage dependent over expressed gene. Here, the consensus based computational approach was adopted for the screening of potential major histocompatibility complex class I restricted epitopes. Based on the computational analysis and previously published report, a set 19 antigenic proteins derived from Leishmania donovani were screened for further characterization as vaccine candidates. A total of 49 epitopes were predicted, which revealed a comprehensive binding affinity to the 40 different MHC class I supertypes. Based on the population coverage and HLA cross presentation, nine highly promiscuous epitopes such as LTYDDVWTV (P1), FLFPQRTAL(P2), FLFSNGAVV (P3), YIYNFGIRV (P4), YMTAAFAAL (P5), KLLRPFAPL (P6), FMLGWIVTI (P7), SLFERNKRV (P8), and SVWNRIFTL (P9) which have either a high or an intermediate TAP binding affinity were selected for further analysis. Theoretical population coverage analysis of polytope vaccine (P1-P9) revealed more than 92% population. Stimulation with the cocktail of peptide revealed a proliferative CD8+ T cell response and increased IFN-γ production. An upregulated NF-κB activity is thought to be play a pivotal role in T cell proliferation against the selected peptide. The Th1-type cytokine profile (presence of IFN-γ and absence of IL-10) suggests the potentiality of the cocktail of epitope as a subunit vaccine against leishmaniasis. However, the efficiency of these epitopes to trigger other Th1 cytokines and chemokines in a humanized mice model could explore its plausibility as a vaccine candidate. J. Cell. Biochem. 119: 378-391, 2018. © 2017 Wiley Periodicals, Inc.

Mining and comparative survey of EST-SSR markers among members of Euphorbiaceae family.

Euphorbiaceae represents flowering plants family of tropical and sub-tropical region rich in secondary metabolites of economic importance. To understand and assess the genetic makeup among the members, this study was undertaken to characterize and compare SSR markers from publicly available ESTs and GSSs of nine selected species of the family. Mining of SSRs was performed by MISA, primer designing by Primer3, while functional annotation, gene ontology (GO) and enrichment analysis were performed by Blast2GO. A total 12,878 number of SSRs were detected from 101,701 number of EST sequences. SSR density ranged from 1 SSR/3.22 kb to 1 SSR/15.65 kb. A total of 1873 primer pairs were designed for the annotated SSR-Contigs. About 77.07% SSR-ESTs could be assigned a significant match to the protein database. 3037 unique SSR-FDM were assigned and IPR003657 (WRKY Domain) was found to be the most dominant FDM among the members. 1810 unique GO terms obtained were further subjected to enrichment analysis to obtain 513 statistically significant GO terms mapped to the SSR containing ESTs. Most frequent enriched GO terms were, GO:0003824 for molecular function, GO:0006350 for biological process and GO:0005886 for cellular component, justifying the richness of defensive secondary metabolites and phytomedicine within the family. The results from this study provides tangible insight to genetic make-up and distribution of SSRs. Functional annotation corresponded many genes of unknown functions which may be considered as novel genes or genes responsible for stress specific secondary metabolites. Further studies are required to understand stress specific genes accountable for leveraging the synthesis of secondary metabolites.