The association of small heat shock protein Hsp16.3 with the plasma membrane of Mycobacterium tuberculosis: dissociation of oligomers is a prerequisite

Hsp16.3, a small heat shock protein from Mycobacterium tuberculosis (MTB), was originally identified as an immuno-dominant antigen and later found to be a major membrane protein. In vitro studies show that Hsp16.3 exists as nonamers and undergoes dynamic dissociation/re-association equilibrium in solutions. Nevertheless, neither the details nor the physiological implications of the presence of Hsp16.3 in the plasma membrane have been studied. In this study, we demonstrated that the purified Hsp16.3 proteins were able to interact with the MTB plasma membrane in a specific and reversible manner, suggesting that there might be subunit exchange between membrane-bound Hsp16.3 and soluble Hsp16.3 oligomers. The dissociation of Hsp16.3 oligomers appears to be a prerequisite for its membrane binding, which is interesting in view that the dissociation of small heat shock protein oligomers was also found to be necessary for it to bind denaturing substrate proteins. Furthermore, the oligomeric structure of Hsp16.3 seems to be more dynamic and flexible when incubating with the mycobacterium lipids. The physiological implications of these observations for Hsp16.3, and small heat shock proteins in general, are discussed.     

Drug targeted virtual screening and molecular dynamics of LipU protein of Mycobacterium tuberculosis and Mycobacterium leprae

The lipolytic protein LipU was conserved in mycobacterium sp. including M. tuberculosis (MTB LipU) and M. leprae (MLP LipU). The MTB LipU was identified in extracellular fraction and was reported to be essential for the survival of mycobacterium. Therefore to address the problem of drug resistance in pathogen, LipU was selected as a drug target and the viability of finding out some FDA approved drugs as LipU inhibitors in both the cases was explored. Three-dimensional (3D) model structures of MTB LipU and MLP LipU were generated and stabilized through molecular dynamics (MD). FDA approved drugs were screened against these proteins. The result showed that the top-scoring compounds for MTB LipU were Diosmin, Acarbose and Ouabain with the Glide XP score of ?12.8, ?11.9 and ?11.7 kcal/mol, respectively, whereas for MLP LipU protein, Digoxin (?9.2 kcal/mol), Indinavir (?8.2 kcal/mol) and Travoprost (?8.2 kcal/mol) showed highest affinity. These drugs remained bound in the active site pocket of MTB LipU and MLP LipU structure and interaction grew stronger after dynamics. RMSD, RMSF and Rg were found to be persistent throughout the simulation period. Hydrogen bonds along with large number of hydrophobic interactions stabilized the complex structures. Binding free energies obtained through Prime/MM-GBSA were found in the significant range from ?63.85 kcal/mol to ?34.57 kcal/mol for MTB LipU and ?71.33 kcal/mol to ?23.91 kcal/mol for MLP LipU. The report suggested high probability of these drugs to demolish the LipU activity and could be probable drug candidates to combat TB and leprosy disease.     

Over-expression of a small heat shock protein,sHSP17.7, confers both heat tolerance and UV-B resistance to rice plants

Exposure of rice (Oryza sativa L.) seedlings to a high temperature (42°C) for 24 h resulted in a significant increase in resistance to UV-B damage. UV-B resistance was enhanced in parallel with the period of heat treatment. sHSP17.7 was isolated from heated rice seedlings, and the influence of rice sHSP17.7 expression on the viability of E. coli under heat-shock conditions was assessed. After heating, the survival rate of sHSP17.7 cells was 2-fold higher than that of the control cells. The molecular chaperone activity of sHSP17.7 was investigated using catalase as a substrate. Recombinant sHSP17.7 had heat-stable chaperone properties that were capable of protecting stressed catalase from precipitation. sHSP17.7 was overexpressed in the rice cultivar Hoshinoyume, by Agrobacterium-mediated transformation, under the control of a CaMV 35S promoter. Transgenic rice plants with increased levels of sHSP17.7 protein exhibited significantly increased thermotolerance compared to untransformed control plants. The level of increased thermotolerance was correlated with the level of increased sHSP17.7 protein in the transgenic plants. The transgenic rice plant with the highest constitutive expression of sHSP17.7 had significantly greater resistance to UV-B stress than untransformed control plants. Increase in the degree of resistance of transgenic plants to UV-B was accompanied by an increase in production of sHSP17.7 protein.     

Identification and structure–activity relationship study of carvacrol derivatives as Mycobacterium tuberculosis chorismate mutase inhibitors

Abstract

In the present study, we identified carvacrol, a major phenolic component of oregano oil as a novel small molecule inhibitor of Mycobacterium tuberculosis (MTB) chorismate mutase (CM) enzyme with IC₅₀ of 1.06?±?0.4?µM. Virtual screening of the BITS-Pilani in-house database using the crystal structure of the MTB CM bound transition state intermediate (PDB: 2FP2) as framework identified carvacrol as a potential lead. Further various carvacrol derivatives were evaluated in vitro for their ability to inhibit MTB CM enzyme, whole cell MTB and cytotoxicity as steps toward the derivation of structure–activity relationships (SAR) and lead optimization.     

Enhanced tolerance to drought stress in transgenic rice plants overexpressing a small heat-shock protein,sHSP17.7

Exposure of rice (Oryza sativa L.) seedlings to a high temperature (42°C) for 24 h resulted in a significant increase in tolerance to drought stress. To try to determine the mechanisms of acquisition of tolerance to drought stress by heat shock, the rice small heat-shock protein gene, sHSP17.7, the product of which was shown to act as molecular chaperones in vitro and in vivo in our previous study, was overexpressed in the rice cultivar “Hoshinoyume”. Western and Northern blot analyses showed higher expression levels of sHSP17.7 protein in three transgenic lines than in one transgenic line. Drought tolerance was assessed in these transgenic lines and wild-type plants by withholding water for 6 days for evaluation of the ability of plants to continue growth after water-stress treatments. Although no significant difference was found in water potential of seedlings between transgenic lines and wild-type plants at the end of drought treatments, only transgenic seedlings with higher expression levels of sHSP17.7 protein could regrow after rewatering. Similar results were observed in survival rates after treatments with 30% polyethylene glycol (PEG) 3640 for 3 days. These results suggest that overproduction of sHSP17.7 could increase drought tolerance in transgenic rice seedlings.     

Functional similarities between the small heat shock proteins Mycobacterium tuberculosis HSP 16.3 and human alphaB-crystallin.

Mycobacterium tuberculosis heat shock protein 16.3 (MTB HSP 16.3) accumulates as the dominant protein in the latent stationary phase of tuberculosis infection. MTB HSP 16.3 displays several characteristics of small heat shock proteins (sHsps): its expression is increased in response to stress, it protects against protein aggregation in vitro, and it contains the core 'alpha-crystallin' domain found in all sHsps. In this study we characterized the chaperone activity of recombinant MTB HSP 16.3 in several different assays and compared the results to those obtained with recombinant human alphaB-crystallin, a well characterized member of the sHsp family. Recombinant MTB HSP 16.3 was expressed in Escherichia coli and purified to apparent homogeneity. Similar to alphaB-crystallin, MTB HSP16.3 suppressed citrate synthase aggregation and in the presence of 3.5 mm ATP the chaperone activity was enhanced by twofold. ATP stabilized MTB HSP 16.3 against proteolysis by chymotrypsin, and no effect was observed with ATPgammaS, a nonhydrolyzable analog of ATP. Increased expression of MTB HSP 16.3 resulted in protection against thermal killing in E. coli at 48 degrees C. While the sequence similarity between human alphaB-crystallin and MTB HSP 16.3 is only 18%, these results suggest that the functional similarities between these proteins containing the core 'alpha-crystallin' domain are much closer.     

Synthesis of Ester-Linked Ursolic Acid-Based Hybrid Compounds: Potential Antibacterial and Anticancer Agents

The molecular hybridization of two or more drugs into a single molecule is an effective drug design approach to reduce pill burden and improve patient treatment adherence. Ursolic acid-based hybrid compounds were synthesized and characterized followed by molecular docking studies. In vitro studies against various bacterial strains and human cancer cells (MDA-MB-231, HeLa, and MCF-7) were performed. Compounds 14 – 19 , 21 , 34 , 31 , and 30 demonstrated significant antibacterial activities with MIC values of 15.625 μg/ml. Compounds 29 and 34 were more cytotoxic than ursolic acid, with IC₅₀ values of 46.99 and 48.18 μg/ml. Compounds 29 and 34 in the docking studies presented favourable binding interactions and better docking energy against the Epidermal Growth Factor Receptor (EGFR) than the parent compound, ursolic acid. The findings revealed that the ursolic acid scaffold is a promising precursor for the development of molecules with promising anticancer and antimicrobial activities. However, more studies are needed to fully understand their mode of action.     

High level expression of recombinant Mycobacterium tuberculosis culture filtrate protein CFP32 in Pichia pastoris

Difficulty in obtaining large quantities of Mycobacterium tuberculosis (MTB) proteins remains a major obstacle in the development of subunit vaccines and diagnostic reagents for tuberculosis. A major reason is because Escherichia coli has not proven to be an optimal host for the expression of MTB genes. In this article, we used the yeast Pichia pastoris to express high levels of CFP32, a culture filtrate protein restricted to the MTB complex and a potential target antigen for serodiagnosis of tuberculosis in patients. Using shaker flasks, we generated a P. pastoris clone expressing CFP32 as a secreted protein fused to the myc-(His)₆ tag, at a yield of 0.5 g of purified protein per liter of culture. Recombinant CFP32 (rCFP32) produced in P. pastoris has a molecular weight of 35 kDa, which is slightly higher than that of the native protein We identified putative acylation and glycosylation sites in the CFP32 amino acid sequence that suggested post-translational modifications may contribute to the size difference. The NH₂-terminal peptide sequencing of rCFP32 showed that the signal peptide alpha factor is correctly excised. In addition, rCFP32 reacted with the sera of patients with tuberculosis. These data are the first to show that P. pastoris is a suitable host for high-yield production of good quality mycobacterium antigens, and especially culture filtrate proteins that have vaccine and diagnostic potential.     

In silico studies of Echinococcus granulosus FABPs

Fatty acid (FA) binding proteins are small intracellular proteins whose members exhibit great diversity and low similarity at the primary structure level, but a highly conserved three-dimensional structure. Characterised by a high-affinity non-covalent binding of hydrophobic ligands, these proteins have a molecular mass of 14–15?kDa with a characteristic β-barrel structure. Members of this family have been identified along the zoological scale, with Platyhelminthes being the more primitive organisms where they have been reported. Two FA binding proteins (FABPs), EgFABP1 and EgFABP2, with 88% similarity have been identified in Echinococcus granulosus. In an effort to understand why two such similar proteins are expressed by this organism, we performed an in silico analysis of the binding capabilities of both proteins. The crystallographic structure of EgFABP1 was utilised as a template to model EgFABP2, and both were docked against palmitate, oleate, linoleate and arachidonate. The docked structures were submitted to 4?ns molecular dynamics simulations, and their protein–ligand interaction energies were measured. The collected data demonstrated that linoleate and arachidonate had the higher interaction energies when bound to EgFABP1 and that palmitate and linoleate had the higher interaction energies when bound to EgFABP2. External and internal binding surfaces were analysed, showing differences at both levels. Internal surface compositions suggested that both proteins could have preferences for certain FAs. Comparisons of the holo and apo forms of each protein indicated that the ligand imposed subtle, but specific modifications that could trigger surface signals. The differences found between the proteins under study suggest that they could have functional uniqueness in the parasite's metabolism.     

Molecular chaperone activity of tomato (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis>) endoplasmic reticulum-located small heat shock protein

The gene encoding the small heat shock protein (sHSP), LeHSP21.5, has been previously cloned from tomato (GenBank accession no. AB026983). The deduced amino acid sequence of this tomato sHSP was most similar to that of other endoplasmic reticulum (ER)-localized sHSPs (ER-sHSP) and can be predicted to target the ER. We examined whether the gene product of LeHSP21.5 (probable ER-sHSP) can act as molecular chaperone. For functional analysis, LeHSP21.5 protein was expressed in Escherichia coli as His₆-tagged protein in the C-terminal and purified. We confirmed that ER-sHSP could provide thermal protection of soluble proteins in vitro. We compared the thermal stability of E. coli strain BL21 (DE3) transformed with pET-ER-sHSP with the control E. coli strain BL21(DE3) transformed with only the pET vector under heat shock and IPTG-induced conditions. Most of the protein extracts from E. coli cells expressing ER-sHSP were protected from heat-induced denaturation, whereas extracts from cells not expressing ER-sHSP were very heat-sensitive under these conditions. A similar protective effect was observed when purified ER-sHSP was added to an E. coli cell extract. ER-sHSP prevented the thermal aggregation and inactivation of citrate synthase. These collective findings indicate that ER-sHSP can function as a molecular chaperone in vitro. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Conformational changes in 2-<Emphasis Type="Italic">trans</Emphasis>-enoyl-ACP (CoA) reductase (InhA) from <Emphasis Type="Italic">M. tuberculosis</Emphasis> induced by an inorganic complex: a molecular dynamics simulation study

InhA, the NADH-dependent 2-trans-enoyl-ACP reductase enzyme from Mycobacterium tuberculosis (MTB), is involved in the biosynthesis of mycolic acids, the hallmark of mycobacterial cell wall. InhA has been shown to be the primary target of isoniazid (INH), one of the oldest synthetic antitubercular drugs. INH is a prodrug which is biologically activated by the MTB catalase-peroxidase KatG enzyme. The activation reaction promotes the formation of an isonicotinyl-NAD adduct which inhibits the InhA enzyme, resulting in reduction of mycolic acid biosynthesis. As a result of rational drug design efforts to design alternative drugs capable of inhibiting MTB’s InhA, the inorganic complex pentacyano(isoniazid)ferrate(II) (PIF) was developed. PIF inhibited both wild-type and INH-resistant Ile21Val mutants of InhA and this inactivation did not require activation by KatG. Since no three-dimensional structure of the InhA-PIF complex is available to confirm the binding mode and to assess the molecular interactions with the protein active site residues, here we report the results of molecular dynamics simulations of PIF interaction with InhA. We found that PIF strongly interacts with InhA and that these interactions lead to macromolecular instabilities reflected in the long time necessary for simulation convergence. These instabilities were mainly due to perturbation of the substrate binding loop, particularly the partial denaturation of helices α6 and α7. We were also able to correlate the changes in the SASAs of Trp residues with the recent spectrofluorimetric investigation of the InhA-PIF complex and confirm their suggestion that the changes in fluorescence are due to InhA conformational changes upon PIF binding. The InhA-PIF association is very strong in the first 20.0 ns, but becomes very week at the end of the simulation, suggesting that the PIF binding mode we simulated may not reflect that of the actual InhA-PIF complex.     

The structural insights of 16.3 kDa heat shock protein (HSP16.3) from Mycobacterium tuberculosis via in silico study

Mycobacterium tuberculosis (Mtb) is capable of surviving in dormancy before developing to tuberculosis (TB). One of the major challenges of TB management is the identification of patients, making TB diagnosis critical for disease management. This study focuses on the 16 kDa heat shock protein (HSP16.3; a potential biomarker for latent TB infection) that is expressed during the latent phase of Mtb growth. In order to explore the dynamics and interactions of HSP16.3, the 3-D structure of HSP16.3 was built via comparative modelling. The predicted structure shows a predominantly beta-sheet dodecamer with alpha-helical folds at its N-terminal. A known protein-hydrophobic probe (1,1′-Bi(4-anilino)naphthalene-5,5′-disulfonic acid; bisANS) was docked to the HSP16.3 model. Interacting residues predicted from docking and MD simulations are in good accordance with experimental data reported in the literature. MMPBSA calculation from MD simulation also showed favourable binding free energy of ?29.90 kcal/mol, driven mainly by van der waals and non-polar solvation energies. The statistical evaluation and results from the computational study on HSP16.3 indicate the reliability of the built model, which is potentially useful for further structural studies of HSP16.3 for latent TB diagnostics.     

AsHSP17, a creeping bentgrass small heat shock protein modulates plant photosynthesis and ABA‐dependent and independent signalling to attenuate plant response to abiotic stress

Heat shock proteins (HSPs) are molecular chaperones that accumulate in response to heat and other abiotic stressors. Small HSPs (sHSPs) belong to the most ubiquitous HSP subgroup with molecular weights ranging from 12 to 42 kDa. We have cloned a new sHSP gene, AsHSP17 from creeping bentgrass (Agrostis stolonifera) and studied its role in plant response to environmental stress. AsHSP17 encodes a protein of 17 kDa. Its expression was strongly induced by heat in both leaf and root tissues, and by salt and abscisic acid (ABA) in roots. Transgenic Arabidopsis plants constitutively expressing AsHSP17 exhibited enhanced sensitivity to heat and salt stress accompanied by reduced leaf chlorophyll content and decreased photosynthesis under both normal and stressed conditions compared to wild type. Overexpression of AsHSP17 also led to hypersensitivity to exogenous ABA and salinity during germination and post‐germinative growth. Gene expression analysis indicated that AsHSP17 modulates expression of photosynthesis‐related genes and regulates ABA biosynthesis, metabolism and ABA signalling as well as ABA‐independent stress signalling. Our results suggest that AsHSP17 may function as a protein chaperone to negatively regulate plant responses to adverse environmental stresses through modulating photosynthesis and ABA‐dependent and independent signalling pathways.     

Insights into the structure–function relationship of both wild and mutant zinc transporter ZnT8 in human: a computational structural biology approach

Abstract

Polymorphism rs13266634 in SLC30A8 causes abnormal synthesis, maturation and secretion of insulin, resulting in decrease in efficiency of glucose metabolism and diabetes. SLC30A8 encodes Zinc transporter 8 protein (ZnT8). Due to lack of NMR/crystal structures of complete ZnT8 transporter, insights into the structure, function and its interaction with different drugs is still not known. Therefore, in-silico methods were adopted in the present studies for predicting three-dimensional structure of ZnT8 transporter via comparative modelling approach and studying the impact of mutation (p.ARG325TRP) on architecture and function via simulation studies. Wild-type protein comprises 15 α-helix and 3 β-strands, while mutant consists of 12 α-helix and 2 β-strands, respectively. Interaction studies of mutant ZnT8 transporter with phytochemicals/drugs screened the best phytochemicals, which can retain the wild-type property. Molecular docking studies reveal that mutant proteins have better binding energy with ligands of LY-2608204, Roseoside, and Luzonoid B. Further molecular dynamic simulation analysis exhibited a strong binding of these ligands with mutant protein and displaying similar behaviour as that of wild type. ALA79, ILE80, and ARG215 are the common interacting amino acids with ligand in all three complexes. As the ligands passed ADMET tests, these may be utilized as anti-diabetic drugs in near future. Although earlier studies have reported anti-diabetic property of LY-2608204 and Roseoside, for the first time, this study reporting Luzonoid B may have anti-diabetic property besides elucidating the structure and functions of ZnT8 transporter.     

Zinc finger protein binding to DNA: an energy perspective using molecular dynamics simulation and free energy calculations on mutants of both zinc finger domains and their specific DNA bases

Energy calculations based on MM-GBSA were employed to study various zinc finger protein (ZF) motifs binding to DNA. Mutants of both the DNA bound to their specific amino acids were studied. Calculated energies gave evidence for a relationship between binding energy and affinity of ZF motifs to their sites on DNA. ΔG values were ?15.82(12), ?3.66(12), and ?12.14(11.6) kcal/mol for finger one, finger two, and finger three, respectively. The mutations in the DNA bases reduced the value of the negative energies of binding (maximum value for ΔΔG = 42Kcal/mol for F1 when GCG mutated to GGG, and ΔΔG = 22 kcal/mol for F2, the loss in total energy of binding originated in the loss in electrostatic energies upon mutation (r = .98). The mutations in key amino acids in the ZF motif in positions-1, 2, 3, and 6 showed reduced binding energies to DNA with correlation coefficients between total free energy and electrostatic was .99 and with Van der Waal was .93. Results agree with experimentally found selectivity which showed that Arginine in position-1 is specific to G, while Aspartic acid (D) in position 2 plays a complicated role in binding. There is a correlation between the MD calculated free energies of binding and those obtained experimentally for prepared ZF motifs bound to triplet bases in other reports (), our results may help in the design of ZF motifs based on the established recognition codes based on energies and contributing energies to the total energy.     

Phytocompounds as potential inhibitors of SARS-CoV-2 Mpro and PLpro through computational studies

The inhibition of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) and papain-like protease (PLpro) prevents viral multiplications; these viral enzymes have been recognized as one of the most favorable targets for drug discovery against SARS-CoV-2. In the present study, we screened 225 phytocompounds present in 28 different Indian spices to identify compounds as potential inhibitors of SARS-CoV-2 Mpro and PLpro. Molecular docking, molecular dynamics simulation, molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) binding free energy calculations, and absorption, distribution, metabolism, excretion and toxicity (ADMET) studies were done. Based on binding affinity, dynamics behavior, and binding free energies, the present study identifies pentaoxahexacyclo-dotriacontanonaen-trihydroxybenzoate derivative (PDT), rutin, and dihyroxy-oxan-phenyl-chromen-4-one derivative (DOC), luteolin-7-glucoside-4′-neohesperidoside as promising inhibitors of SARS-CoV-2 Mpro and PLpro, respectively.     

The systematic modeling studies and free energy calculations of the phenazine compounds as anti-tuberculosis agents

Abstract

Phenazine compounds have good activity against Mycobacterium tuberculosis (MTB). Based on the reported activities that were obtained in MTB H37Rv, a three-dimensional quantitative structure–activity relationship (3D-QSAR) model was built to design novel compounds against MTB. A fivefold cross-validation method and external validation were used to analyze the accuracy of forecasting. The model has a cross-validation coefficient q²=0.7 and a non-cross-validation coefficient r² = 0.903, indicating that the model has good predictive possibility. The design of anti-pneumococcus MTB compounds was guided by the obtained 3D-QSAR model, and several compounds with better activity were obtained. To test the activity of these compounds, molecular docking, molecular dynamics simulation, and post-simulation analysis of the already reported drug targets in MTB were carried out. Among the total 15 drug targets, only three targets (Rv2361c, Rv2965c, and Rv3048c) were selected based on the docking results. Initial results reported that these compounds possessed good inhibition activity for Rv2361c. The top nine complexes of Rv2361 ligands were only subjected to MD simulation which resulted in a stable dynamics of the structures and showed a residual fluctuation in inhibitors binding pocket. Free energy reported that overall, the derivatives hold strong energy against the protein target. Energetic contribution results showed that residues, Asp76, Arg80, Asn124, Arg127, Arg244, and Arg250, play a major role in total energy. Systems biology approach validates shortlisted drug effect on the entire system which might be useful to predict potential drug in wet lab as well.

Communicated by Ramaswamy H. Sarma     

Insights into the structure–function relationship of brown plant hopper resistance protein,Bph14 of rice plant: a computational structural biology approach

Brown plant hopper (BPH) is one of the major destructive insect pests of rice, causing severe yield loss. Thirty-two BPH resistance genes have been identified in cultivated and wild species of rice Although, molecular mechanism of rice plant resistance against BPH studied through map-based cloning, due to non-existence of NMR/crystal structures of Bph14 protein, recognition of leucine-rich repeat (LRR) domain and its interaction with different ligands are poorly understood. Thus, in the present study, in silico approach was adopted to predict three-dimensional structure of LRR domain of Bph14 using comparative modelling approach followed by interaction study with jasmonic and salicylic acids. LRR domain along with LRR-jasmonic and salicylic acid complexes were subjected to dynamic simulation using GROMACS, individually, for energy minimisation and refinement of the structure. Final binding energy of jasmonic and salicylic acid with LRR domain was calculated using MM/PBSA. Free-energy landscape analysis revealed that overall stability of LRR domain of Bph14 is not much affected after forming complex with jasmonic and salicylic acid. MM/PBSA analysis revealed that binding affinities of LRR domain towards salicylic acid is higher as compared to jasmonic acid. Interaction study of LRR domain with salicylic acid and jasmonic acid reveals that THR987 of LRR form hydrogen bond with both complexes. Thus, THR987 plays active role in the Bph14 and phytochemical interaction for inducing resistance in rice plant against BPH. In future, Bph14 gene and phytochemicals could be used in BPH management and development of novel resistant varieties for increasing rice yield.     

Phytochemicals in Morinda citrifolia fruit selectively modulate age-associated immunity and antioxidant enzyme activities through ERK pathway in splenic lymphocytes of male F344 rats

Context/objective: The mechanisms of immunomodulatory effects of Morinda citrifolia (Noni) were examined through intracellular signaling pathways in the splenocytes and their modulation by phytochemicals using bioinformatics tools. Materials and methods: Noni fruit juices without seeds (NSL) and with seeds (NWS) were co-incubated in vitro with splenocytes from young, middle-aged and old F344 male rats and proliferation of lymphocytes, cytokine production, antioxidant enzyme activities and intracellular signaling markers were measured. Results: NSL decreased lymphoproliferation in early middle-aged rats, and IL-2 and IFN-γ production in old rats. In contrast, NWS enhanced lymphoproliferation in young and old rats, IL-2 and IFN-γ production in middle-aged and old rats. The activities of antioxidant enzymes were augmented by NWS and NSL in old rats. NWS reversed age-related increase in lipid peroxidation in all age-groups, while NSL increased lipid peroxidation in old rats. NSL increased p-ERK in old rats and decreased p-CREB in young and middle-aged rats. In contrast, NWS decreased p-ERK in all age groups and increased p-CREB in old rats. Both NSL and NWS increased p-Akt expression in middle-aged and old rats. Both NSL and NWS suppressed p-NF-κB expression in middle-aged and old rats. Docking studies demonstrated that Noni phytochemicals, damnacanthal, myricetin and ursolic acid, are potent inhibitors of ERK with binding sites in the catalytic and phosphorylation sites of the molecule. Conclusions: These results suggest that Noni fruit juices with or without seeds modulate cell-mediated immunity and antioxidant enzyme activities based on the phytochemicals that may differentially influence cell signaling and therefore, age-associated immunity.