Zinc fingers are the most abundant eukaryotic DNA-binding motifs, which recognize DNA triplets wi... more Zinc fingers are the most abundant eukaryotic DNA-binding motifs, which recognize DNA triplets with high efficacy and are used to develop chimeric enzymes for genome modification and site specific gene therapy. Considering the importance of zinc finger motifs and their recognition patterns we have developed a web interface with the incorporation of sequence based search algorithm named as Bind-Predict. This algorithm facilitates the identification of target sites of zinc finger proteins (ZFPs) in the given DNA sequences. This paper describes the concepts of the developed algorithm (scoring system) and its applications in predicting ZFP binding sites. Bind-Predict web interface contains dual applications such as a web server (Bind-Predict tool) and a database (Bind-PredictdB). In order to enhance its utility and to validate the prediction results, Bind-Predicttool is interconnected with Bind-PredictdB. The combination of sequence based pattern recognition followed by computing Bind-Predict scores is used to identify the ZFP binding sites in the direct (5′→3′), complementary (3′→5′), double strand DNA (5′→3′ and 3′→5′) and user-defined binding sites of the input sequence. Currently Bind-PredictdB contains 374 entries of zinc finger motifs comprising 91 engineered constructs, 250 naturally occurring and 33 zinc binuclear clusters. Bind-Predict web interface is freely accessible at http://zfp.bicpu.edu.in.
Background: Entry of HIV-1 into human lymphoid requires activities of viral envelope glycoprotein... more Background: Entry of HIV-1 into human lymphoid requires activities of viral envelope glycoproteins gp120 and gp41, and two host-cell proteins, the primary receptor CD4 and a chemokine co-receptor. In addition, a third cell-surface protein called protein disulfide isomerise (PDI) is found to play a major role in HIV-1 entry. PDI is capable of mediating thio-disulfide interchange reactions and could enable the reduction of gp120 disulfide bonds, which triggers the major conformational changes in gp120 and gp41 required for virus entry. In this scenario, inhibition of HIV-1 entry can be brought about by introducing agents that can block thiol-disulfide interchange reaction of cell surface PDI. There have been studies with agents that inhibit PDI activity, but the exact mode of binding remains to be elucidated; this might provide insights to develop new drugs to target PDI. This study attempts to perceive the mode of binding of dithionitrobenzoic acid (DTNB), and its structurally related compounds on PDI enzyme. Results: We performed molecular docking simulation with six different inhibitors (ligand), which includes DTNB, NSC695265, thionitrobenzoic acid, 2-nitro-5-thiocyanobenzoic acid, 2-nitro-5-sulfo-sulfonyl-benzoic acid and NSC517871 into the redox-active site [C37-G38-H39-C40] of the PDI enzyme and the activity was inferred by redox inhibitory models. All ligands showed favourable interactions and most of them seemed to bind to hydrophobic amino acids Ala34, Trp36, Cys37, Cys40, His39, Thr68 and Phe80. The redox inhibitory conformations were energetically and statistically favored and supported the evidence from wet laboratory experiments reported in the literature. Conclusion: We demonstrated that in silico docking experiment can be effectively carried out to recognize the redox inhibitory models of PDI with inhibitor molecules. Interestingly we found that number of docked clusters with each ligand varies in the range of five to eight and conveys that the binding specificity of each inhibitor varies for PDI. We also identified that Cys37 of the enzyme plays an important role in hydrogen bonding with inhibitors. This residue can be considered to being an active site for anti-HIV drug design. Therefore, by inhibiting PDI, one can, not only prevent the viral entry but also circumvent the problem of viral resistance
Background: Information on the occurrence of zinc finger protein motifs in genomes is crucial to ... more Background: Information on the occurrence of zinc finger protein motifs in genomes is crucial to the developing field of molecular genome engineering. The knowledge of their target DNA-binding sequences is vital to develop chimeric proteins for targeted genome engineering and site-specific gene correction. There is a need to develop a computational resource of zinc finger proteins (ZFP) to identify the potential binding sites and its location, which reduce the time of in vivo task, and overcome the difficulties in selecting the specific type of zinc finger protein and the target site in the DNA sequence.
Description: ZifBASE provides an extensive collection of various natural and engineered ZFP. It uses standard names and a genetic and structural classification scheme to present data retrieved from UniProtKB, GenBank, Protein Data Bank, ModBase, Protein Model Portal and the literature. It also incorporates specialized features of ZFP including finger sequences and positions, number of fingers, physiochemical properties, classes, framework, PubMed itations with links to experimental structures (PDB, if available) and modeled structures of natural zinc finger proteins. ZifBASE provides information on zinc finger proteins (both natural and engineered ones), the number of finger units in each of the zinc finger proteins (with multiple fingers), the synergy between the adjacent fingers and their positions. Additionally, it gives the individual finger sequence and their target DNA site to which it binds for better and clear understanding on the interactions of adjacent fingers. The current version of ZifBASE contains 139 entries of which 89 are engineered ZFPs, containing 3-7F totaling to 296 fingers. There are 50 natural zinc finger protein entries ranging from 2-13F, totaling to 307 fingers. It has sequences and structures from literature, Protein Data Bank, ModBase and Protein Model Portal. The interface is cross linked to other public databases like UniprotKB, PDB, ModBase and Protein Model Portal and PubMed for making it more informative.
Conclusion: A database is established to maintain the information of the sequence features, including the class, framework, number of fingers, residues, position, recognition site and physio-chemical properties (molecular weight, isoelectric point) of both natural and engineered zinc finger proteins and dissociation constant of few. ZifBASE can provide more effective and efficient way of accessing the zinc finger protein sequences and their target binding sites with the links to their three-dimensional structures. All the data and functions are available at the advanced web-based search interface http://web.iitd.ac.in/~sundar/ zifbase.
GTP-bound mutant form H-Ras (Harvey-Ras) proteins are found in 30% of human tumors. Activation of... more GTP-bound mutant form H-Ras (Harvey-Ras) proteins are found in 30% of human tumors. Activation of H-Ras is due to point mutation at positions 12, 13, 59 and/or 61 codon. Mutant form of H-Ras proteins is continuously involved in signal transduction for cell growth and proliferation through interaction of downstream-regulated protein Raf. In this paper, we have reported the virtual screening of lead compounds for H-Ras P21 mutant protein from ChemBank and DrugBank databases using LigandFit and DrugBank-BLAST. The analysis resulted in 13 hits which were docked and scored to identify structurally active leads that make similar interaction to those of bound complex of H-Ras P21 mutant-Raf. This approach produced two different leads, 3-Aminopropanesulphonic acid (docked energy -3.014 kcal/mol) and Hydroxyurea (docked energy -0.009 kcal/mol) with finest Lipinski’s rule-of-five. Their docked energy scores were better than the complex structure of H-Ras P21 mutant protein bound with Raf (1.18 kcal/mol). All the leads were docked into effector region forming interaction with ILE36, GLU37, ASP38 and SER39.
HIV infected patients often take at least three anti-HIV drugs together in Highly Active Antiretr... more HIV infected patients often take at least three anti-HIV drugs together in Highly Active Antiretroviral Therapy (HAART) and/or Ritonavir-Boosted Protease Inhibitor Therapy (PI/r) to suppress the viral replications. The potential drug–drug interactions affect efficacy of anti-HIV treatment and major source of such interaction is competition for the drug metabolizing enzyme, cytochrome P450 (CYP). CYP3A4 isoform is the enzyme responsible for metabolism of currently available HIV-1 protease drugs. Hence administration of these drugs in HARRT or PI/r leads to increased toxicity and reduced efficacy in HIV treatment. We used computational molecular docking method to predict such interactions by which to compare experimentally measured metabolism of each HIV-1 protease drug. AutoDock 4.0 was used to carry out molecular docking of 10 HIV-protease drugs into CYP3A4 to explore sites of reaction and interaction energies (i.e., binding affinity) of the complexes. Arg105, Arg106, Ser119, Arg212, Ala370, Arg372, and Glu374 are identified as major drug binding residues, and consistent with previous data of site-directed mutagenesis, crystallography structure, modeling, and docking studies. In addition, our docking results suggested that phenylalanine clusters and heme are also participated in the binding to mediate drug oxidative metabolism. We have shown that HIV-1 protease drugs such as tipranavir, nelfinavir, lopinavir, and atazanavir differ in their binding modes on each other for metabolic clearance in CYP3A4, whereas ritonavir, amprenavir, indinavir, saquinavir, fosamprenavir, and darunavir share the same binding mode.
Natural rubber is an important polymer produced by plants and made up of isoprene units derived f... more Natural rubber is an important polymer produced by plants and made up of isoprene units derived from isopentenyl diphosphate (IPP). Although more than 2000 plant species are known to produce natural rubber, currently there are two important commercial sources, Hevea brasiliensis (the Brazilian rubber tree) and Parthenium argentatum Gray (guayule). Natural rubber biosynthesis requires three distinct biochemical processes such as (i) initiation, (ii) elongation and (iii) termination. Computational analyses of the enzymes farnesyl diphosphate (FPP) synthase in P. argentatum and cis-prenyl transferase (CPT) in H. brasiliensis that play a vital role in initiation and elongation stages for biosynthesis of cis-1,4-polyisoprene has been undertaken in this study. Amino acid sequence comparisons of FPP synthase and CPT to their identified similar sequences were carried out to understand the evolutionary relationship among different species. Homology modeling and binding pocket analysis aided in the understanding of structure-function relationship and enzyme-substrate interaction of FPP synthase and CPT. The structural templates farnesyl diphosphate synthase (Source: Gallus gallus) [PDB ID: 1UBX] for FPP synthase and undecaprenyl diphosphate synthase (Source: Micrococcus luteus B-P 26) [PDB ID: 1F75] for CPT were selected for homology modeling. The Ramachandran plots were developed for modeled structures of FPP synthase and CPT, which showed 95.9 and 92.6% of amino acid residues occurring in favored regions. These models were deposited into Protein Model Database [PMDB ID: PM0075218 and PM0075509]. The substrate and cofactor binding site residues of FPP synthase (R103, L149, A184, Y197, L211, H214, E223, T226, D332, K246, Y306, K313) and CPT (Y4, E7, R20, K21, G22, K154, K178, D193, E231, T232, R233) were identified by using binding pocket analysis, which is consistent with available X-ray crystal structure of both the templates. The computational analysis of initiation and elongation for cis-1,4-polyisoprene biosynthesis provided invaluable insights into the identification of putative initiation and elongation factors for FPP synthase and CPT.
Mangroves are salt–tolerant forest ecosystems of tropical and subtropical intertidal regions of t... more Mangroves are salt–tolerant forest ecosystems of tropical and subtropical intertidal regions of the world. There may be no other group of plants with such highly developed morphological and physiological adaptations to extreme conditions. They are a rich source of steroids, triterpenes, saponins, flavonoids, alkaloids and tannins. The Pondicherry region in South India has no forest, but has scattered patches of mangroves and remnants of tropical dry evergreen forest in the form of sacred groves. The present study documents the directly observed diversity of true mangroves and their associates, in four geographical regions of Pondicherry in South India; namely Pondicherry, Karaikal, Mahe and Yanam. These places are far from one another and the mangrove species diversity varies from one place to another, due to factors such as climate, tidal factors and anthropogenic pressures. In addition, we have also developed a nowledgebase entitled “Mangroves of Pondicherry”, which contains information including botanical name, English name, local name (Tamil language), Family, description, images and common uses of true mangroves and their associates. This knowledge resource is available for open access at http://web.iitd.ac.in/~sundar/mangroves/.
Environmental toxicants, such as cadmium and bisphenol A (BPA) are endocrine disruptors. In utero... more Environmental toxicants, such as cadmium and bisphenol A (BPA) are endocrine disruptors. In utero, perinatal or neonatal exposure of BPA to rats affect the male reproductive function, such as the blood-testis barrier (BTB) integrity. This effect of BPA on BTB integrity in immature rats is likely mediated via a loss of gap junction function at the BTB, failing to coordinate tight junction and anchoring junction function at the site to maintain the immunological barrier integrity. This in turn activates the extracellular signal-regulated kinases 1/2 (Erk1/2) downstream and an increase in protein endocytosis, destabilizing the BTB. The cadmium-induced disruption of testicular dysfunction is mediated initially via its effects on the occludin/ZO-1/focal adhesion kinase (FAK) complex at the BTB, causing redistribution of proteins at the Sertoli-Sertoli cell interface, leading to the BTB disruption. The damaging effects of these toxicants to testicular function are mediated by mitogen-activated protein kinases (MAPK) downstream, which in turn perturbs the actin bundling and accelerates the actin-branching activity, causing disruption of the Sertoli cell tight junction (TJ)-barrier function at the BTB and perturbing spermatid adhesion at the apical ectoplasmic specialization (apical ES, a testis-specific anchoring junction type) that leads to premature release of germ cells from the testis. However, the use of specific inhibitors against MAPK was shown to block or delay the cadmium-induced testicular injury, such as BTB disruption and germ cell loss. These findings suggest that there may be a common downstream p38 and/or Erk1/2 MAPK-based signaling pathway involving polarity proteins and actin regulators that is shared between different toxicants that induce male reproductive dysfunction. As such, the use of inhibitors and/or antagonists against specific MAPKs can possibly be used to “manage” the illnesses caused by these toxicants and/or “protect” industrial workers being exposed to high levels of these toxicants in their work environment.
CYP2C19 is a polymorphic enzyme which metabolizes several clinically important drugs including pr... more CYP2C19 is a polymorphic enzyme which metabolizes several clinically important drugs including proguanil. Variation in the 5? regulatory region may influence CYP2C19 activity. This study evaluates the relationship between proguanil metabolic ratio and genetic variations of CYP2C19 in a South Indian population. Fifty unrelated healthy subjects were genotyped for CYP2C19 *2 and *3 alleles and the 5? flanking region of CYP2C19 was sequenced. Plasma concentrations of proguanil and cycloguanil were estimated by reverse phase HPLC after single oral doses (200 mg) of proguanil. In silico docking analysis of transcription factors binding to its sites in CYP2C19 5? regulatory region was performed. The mean metabolic ratios (proguanil/cycloguanil) were highest in *1/*2 or *1/*3 subjects and in *2/*2 or *2/*3 as compared to *1/*1 subjects. Subjects with promoter region variation －98TÀC showed decrease in the metabolic ratios irrespective of other variation, which may explain the deviation from the genotype-phenotype association of CYP2C19. In silico analysis predicted alteration in the interaction of transcription factors to their binding sites in the presence of variant alleles. The results of this study would be useful in predicting interindividual differences in the metabolism of substrates of CYP2C19.
Recent studies have demonstrated the presence of a functional axis that coordinates the events of... more Recent studies have demonstrated the presence of a functional axis that coordinates the events of spermiation and blood-testis barrier (BTB) restructuring which take place simultaneously at opposite ends of the seminiferous epithelium at stage VIII of the epithelial cycle of spermatogenesis in the rat testis. In short, the disruption of the apical ectoplasmic specialization (apical ES) at the Sertoli cell-elongated spermatid interface, which facilitates the release of sperm at spermiation near the tubule lumen, is coordinated with restructuring at the BTB to accommodate the transit of preleptotene spermatocytes near the basement membrane. These two events are likely coordinated by a functional axis involving hemidesmosome at the Sertoli cell-basement membrane interface, and it was designated the apical ES-BTB-hemidesmosome axis. It was demonstrated that fragments of laminin chains (e.g., laminin β3 or γ3 chains) derived from the α6β1-integrin-laminin333 protein complex at the apical ES, which were generated via the action of MMP-2 (matrix metalloprotease-2, MMP2) prior to spermiation, acted as biologically active peptides to perturb the BTB permeability function by accelerating protein endocytosis (e.g., occludin) at the site, thereby destabilizing the BTB integrity to facilitate the transit of preleptotene spermatocytes. These laminin fragments also perturbed hemidesmosome function via their action on β1-integrin, a component of hemidesmosome in the testis, which in turn, sent a signal to further destabilize the BTB function. As such, the events of spermiation and BTB restructuring are coordinated via this functional axis. Recent studies using animal models treated with toxicants, such as mono-(2-ethylhexyl) phthalate (MEHP), or adjudin, a male contraceptive under investigation, have also supported the presence of this functional axis in the mouse. In this short review, we critically evaluate the role of this local functional axis in the seminiferous epithelium in spermatogenesis. We also provide molecular modeling information on the interactions between biologically active laminin fragments and β1-integrin, which will be important to assist in the design of more potent laminin-based peptides to disrupt this axis, thereby perturbing spermatogenesis for male contraception and to understand the underlying biology that coordinates spermiation and BTB restructuring during spermatogenesis
The blood-testis barrier (BTB) is a unique ultrastructure in the mammalian testis. Unlike other b... more The blood-testis barrier (BTB) is a unique ultrastructure in the mammalian testis. Unlike other blood-tissue barriers, such as the blood-brain barrier and the blood-ocular (or blood-retina) barrier which formed by tight junctions (TJ) between endothelial cells of the microvessels, the BTB is constituted by coexisting TJ, basal ectoplasmic specialization (basal ES), desmosomes and gap junctions between adjacent Sertoli cells near the basement membrane of the seminiferous tubule. The BTB also divides the seminiferous epithelium into the apical (or adluminal) and basal compartments so that meiosis I and II and post-meiotic germ cell development can all take place in a specialized microenvironment in the apical compartment behind the BTB. While the unusual anatomical features of the BTB have been known for decades, the physiological function of the coexisting junctions, in particular the desmosome and gap junction, that constitute the BTB was unknown until recently. Based on recently published findings, we critically evaluate the role of the desmosome and gap junction that serve as a signaling platform to coordinate the “opening” and “closing” of the TJ-permeability barrier conferred by TJ and basal ES during the seminiferous epithelial cycle of spermatogenesis. This is made possible by polarity proteins working in concert with nonreceptor protein tyrosine kinases, such as focal adhesion kinase (FAK) and c-Src, at the site to regulate endosome-mediated protein trafficking events (e.g., endocytosis, transcytosis, recycling or protein degradation). These events not only serve to destabilize the existing “old” BTB above preleptotene spermatocytes in transit in “clones” at the BTB, but also contribute to the assembly of “new” BTB below the transiting spermatocytes. Furthermore, hemidesmosomes at the Sertoli cell-basement membrane interface also contribute to the BTB restructuring events at stage VIII of the epithelial cycle. Additionally, the findings that a gap junction at the BTB provides a possible route for the passage of toxicants [e.g., bisphenol A (BPA)] and potential male contraceptives (e.g., adjudin) across the BTB also illustrate that these coexisting junctions, while helpful to maintain the immunological barrier integrity during the transit of spermatocytes, can be the “gateway” to making the BTB so vulnerable to toxicants and/or chemicals, causing male reproductive dysfunction.
Molecular docking is a reliable method with which to identify the binding conformations of substr... more Molecular docking is a reliable method with which to identify the binding conformations of substrates, inducers and inhibitors of cytochrome P450 (CYP) enzymes. We used the docking method to explore possible binding modes of an entry inhibitor (maraviroc) and nonnucleoside reverse transcriptase inhibitors (delavirdine, efavirenz and etravirine) to cytochrome P450 3A4 (CYP3A4). In addition, docking results were compared with the binding conformations of HIV protease drugs to infer the binding site residues and potential drug–drug interaction profiles for combination therapy in the treatment of AIDS. We observed that efavirenz and etravirine induce metabolism of co-administered drugs by binding to a unique position in the active site of CYP3A4. Dosage adjustment is required for delavirdine and maraviroc when combined with HIV protease drugs. The present results are in good agreement with experimental data from drug interaction profiles. The information provided in this paper will be helpful in furthering our understanding the functions of CYP3A4, and could aid in the design of new drugs that would be metabolized easily without having any drug–drug interaction profile.
Zinc fingers are the most abundant eukaryotic DNA-binding motifs, which recognize DNA triplets wi... more Zinc fingers are the most abundant eukaryotic DNA-binding motifs, which recognize DNA triplets with high efficacy and are used to develop chimeric enzymes for genome modification and site specific gene therapy. Considering the importance of zinc finger motifs and their recognition patterns we have developed a web interface with the incorporation of sequence based search algorithm named as Bind-Predict. This algorithm facilitates the identification of target sites of zinc finger proteins (ZFPs) in the given DNA sequences. This paper describes the concepts of the developed algorithm (scoring system) and its applications in predicting ZFP binding sites. Bind-Predict web interface contains dual applications such as a web server (Bind-Predict tool) and a database (Bind-PredictdB). In order to enhance its utility and to validate the prediction results, Bind-Predicttool is interconnected with Bind-PredictdB. The combination of sequence based pattern recognition followed by computing Bind-Predict scores is used to identify the ZFP binding sites in the direct (5′→3′), complementary (3′→5′), double strand DNA (5′→3′ and 3′→5′) and user-defined binding sites of the input sequence. Currently Bind-PredictdB contains 374 entries of zinc finger motifs comprising 91 engineered constructs, 250 naturally occurring and 33 zinc binuclear clusters. Bind-Predict web interface is freely accessible at http://zfp.bicpu.edu.in.
Background: Entry of HIV-1 into human lymphoid requires activities of viral envelope glycoprotein... more Background: Entry of HIV-1 into human lymphoid requires activities of viral envelope glycoproteins gp120 and gp41, and two host-cell proteins, the primary receptor CD4 and a chemokine co-receptor. In addition, a third cell-surface protein called protein disulfide isomerise (PDI) is found to play a major role in HIV-1 entry. PDI is capable of mediating thio-disulfide interchange reactions and could enable the reduction of gp120 disulfide bonds, which triggers the major conformational changes in gp120 and gp41 required for virus entry. In this scenario, inhibition of HIV-1 entry can be brought about by introducing agents that can block thiol-disulfide interchange reaction of cell surface PDI. There have been studies with agents that inhibit PDI activity, but the exact mode of binding remains to be elucidated; this might provide insights to develop new drugs to target PDI. This study attempts to perceive the mode of binding of dithionitrobenzoic acid (DTNB), and its structurally related compounds on PDI enzyme. Results: We performed molecular docking simulation with six different inhibitors (ligand), which includes DTNB, NSC695265, thionitrobenzoic acid, 2-nitro-5-thiocyanobenzoic acid, 2-nitro-5-sulfo-sulfonyl-benzoic acid and NSC517871 into the redox-active site [C37-G38-H39-C40] of the PDI enzyme and the activity was inferred by redox inhibitory models. All ligands showed favourable interactions and most of them seemed to bind to hydrophobic amino acids Ala34, Trp36, Cys37, Cys40, His39, Thr68 and Phe80. The redox inhibitory conformations were energetically and statistically favored and supported the evidence from wet laboratory experiments reported in the literature. Conclusion: We demonstrated that in silico docking experiment can be effectively carried out to recognize the redox inhibitory models of PDI with inhibitor molecules. Interestingly we found that number of docked clusters with each ligand varies in the range of five to eight and conveys that the binding specificity of each inhibitor varies for PDI. We also identified that Cys37 of the enzyme plays an important role in hydrogen bonding with inhibitors. This residue can be considered to being an active site for anti-HIV drug design. Therefore, by inhibiting PDI, one can, not only prevent the viral entry but also circumvent the problem of viral resistance
Background: Information on the occurrence of zinc finger protein motifs in genomes is crucial to ... more Background: Information on the occurrence of zinc finger protein motifs in genomes is crucial to the developing field of molecular genome engineering. The knowledge of their target DNA-binding sequences is vital to develop chimeric proteins for targeted genome engineering and site-specific gene correction. There is a need to develop a computational resource of zinc finger proteins (ZFP) to identify the potential binding sites and its location, which reduce the time of in vivo task, and overcome the difficulties in selecting the specific type of zinc finger protein and the target site in the DNA sequence.
Description: ZifBASE provides an extensive collection of various natural and engineered ZFP. It uses standard names and a genetic and structural classification scheme to present data retrieved from UniProtKB, GenBank, Protein Data Bank, ModBase, Protein Model Portal and the literature. It also incorporates specialized features of ZFP including finger sequences and positions, number of fingers, physiochemical properties, classes, framework, PubMed itations with links to experimental structures (PDB, if available) and modeled structures of natural zinc finger proteins. ZifBASE provides information on zinc finger proteins (both natural and engineered ones), the number of finger units in each of the zinc finger proteins (with multiple fingers), the synergy between the adjacent fingers and their positions. Additionally, it gives the individual finger sequence and their target DNA site to which it binds for better and clear understanding on the interactions of adjacent fingers. The current version of ZifBASE contains 139 entries of which 89 are engineered ZFPs, containing 3-7F totaling to 296 fingers. There are 50 natural zinc finger protein entries ranging from 2-13F, totaling to 307 fingers. It has sequences and structures from literature, Protein Data Bank, ModBase and Protein Model Portal. The interface is cross linked to other public databases like UniprotKB, PDB, ModBase and Protein Model Portal and PubMed for making it more informative.
Conclusion: A database is established to maintain the information of the sequence features, including the class, framework, number of fingers, residues, position, recognition site and physio-chemical properties (molecular weight, isoelectric point) of both natural and engineered zinc finger proteins and dissociation constant of few. ZifBASE can provide more effective and efficient way of accessing the zinc finger protein sequences and their target binding sites with the links to their three-dimensional structures. All the data and functions are available at the advanced web-based search interface http://web.iitd.ac.in/~sundar/ zifbase.
GTP-bound mutant form H-Ras (Harvey-Ras) proteins are found in 30% of human tumors. Activation of... more GTP-bound mutant form H-Ras (Harvey-Ras) proteins are found in 30% of human tumors. Activation of H-Ras is due to point mutation at positions 12, 13, 59 and/or 61 codon. Mutant form of H-Ras proteins is continuously involved in signal transduction for cell growth and proliferation through interaction of downstream-regulated protein Raf. In this paper, we have reported the virtual screening of lead compounds for H-Ras P21 mutant protein from ChemBank and DrugBank databases using LigandFit and DrugBank-BLAST. The analysis resulted in 13 hits which were docked and scored to identify structurally active leads that make similar interaction to those of bound complex of H-Ras P21 mutant-Raf. This approach produced two different leads, 3-Aminopropanesulphonic acid (docked energy -3.014 kcal/mol) and Hydroxyurea (docked energy -0.009 kcal/mol) with finest Lipinski’s rule-of-five. Their docked energy scores were better than the complex structure of H-Ras P21 mutant protein bound with Raf (1.18 kcal/mol). All the leads were docked into effector region forming interaction with ILE36, GLU37, ASP38 and SER39.
HIV infected patients often take at least three anti-HIV drugs together in Highly Active Antiretr... more HIV infected patients often take at least three anti-HIV drugs together in Highly Active Antiretroviral Therapy (HAART) and/or Ritonavir-Boosted Protease Inhibitor Therapy (PI/r) to suppress the viral replications. The potential drug–drug interactions affect efficacy of anti-HIV treatment and major source of such interaction is competition for the drug metabolizing enzyme, cytochrome P450 (CYP). CYP3A4 isoform is the enzyme responsible for metabolism of currently available HIV-1 protease drugs. Hence administration of these drugs in HARRT or PI/r leads to increased toxicity and reduced efficacy in HIV treatment. We used computational molecular docking method to predict such interactions by which to compare experimentally measured metabolism of each HIV-1 protease drug. AutoDock 4.0 was used to carry out molecular docking of 10 HIV-protease drugs into CYP3A4 to explore sites of reaction and interaction energies (i.e., binding affinity) of the complexes. Arg105, Arg106, Ser119, Arg212, Ala370, Arg372, and Glu374 are identified as major drug binding residues, and consistent with previous data of site-directed mutagenesis, crystallography structure, modeling, and docking studies. In addition, our docking results suggested that phenylalanine clusters and heme are also participated in the binding to mediate drug oxidative metabolism. We have shown that HIV-1 protease drugs such as tipranavir, nelfinavir, lopinavir, and atazanavir differ in their binding modes on each other for metabolic clearance in CYP3A4, whereas ritonavir, amprenavir, indinavir, saquinavir, fosamprenavir, and darunavir share the same binding mode.
Natural rubber is an important polymer produced by plants and made up of isoprene units derived f... more Natural rubber is an important polymer produced by plants and made up of isoprene units derived from isopentenyl diphosphate (IPP). Although more than 2000 plant species are known to produce natural rubber, currently there are two important commercial sources, Hevea brasiliensis (the Brazilian rubber tree) and Parthenium argentatum Gray (guayule). Natural rubber biosynthesis requires three distinct biochemical processes such as (i) initiation, (ii) elongation and (iii) termination. Computational analyses of the enzymes farnesyl diphosphate (FPP) synthase in P. argentatum and cis-prenyl transferase (CPT) in H. brasiliensis that play a vital role in initiation and elongation stages for biosynthesis of cis-1,4-polyisoprene has been undertaken in this study. Amino acid sequence comparisons of FPP synthase and CPT to their identified similar sequences were carried out to understand the evolutionary relationship among different species. Homology modeling and binding pocket analysis aided in the understanding of structure-function relationship and enzyme-substrate interaction of FPP synthase and CPT. The structural templates farnesyl diphosphate synthase (Source: Gallus gallus) [PDB ID: 1UBX] for FPP synthase and undecaprenyl diphosphate synthase (Source: Micrococcus luteus B-P 26) [PDB ID: 1F75] for CPT were selected for homology modeling. The Ramachandran plots were developed for modeled structures of FPP synthase and CPT, which showed 95.9 and 92.6% of amino acid residues occurring in favored regions. These models were deposited into Protein Model Database [PMDB ID: PM0075218 and PM0075509]. The substrate and cofactor binding site residues of FPP synthase (R103, L149, A184, Y197, L211, H214, E223, T226, D332, K246, Y306, K313) and CPT (Y4, E7, R20, K21, G22, K154, K178, D193, E231, T232, R233) were identified by using binding pocket analysis, which is consistent with available X-ray crystal structure of both the templates. The computational analysis of initiation and elongation for cis-1,4-polyisoprene biosynthesis provided invaluable insights into the identification of putative initiation and elongation factors for FPP synthase and CPT.
Mangroves are salt–tolerant forest ecosystems of tropical and subtropical intertidal regions of t... more Mangroves are salt–tolerant forest ecosystems of tropical and subtropical intertidal regions of the world. There may be no other group of plants with such highly developed morphological and physiological adaptations to extreme conditions. They are a rich source of steroids, triterpenes, saponins, flavonoids, alkaloids and tannins. The Pondicherry region in South India has no forest, but has scattered patches of mangroves and remnants of tropical dry evergreen forest in the form of sacred groves. The present study documents the directly observed diversity of true mangroves and their associates, in four geographical regions of Pondicherry in South India; namely Pondicherry, Karaikal, Mahe and Yanam. These places are far from one another and the mangrove species diversity varies from one place to another, due to factors such as climate, tidal factors and anthropogenic pressures. In addition, we have also developed a nowledgebase entitled “Mangroves of Pondicherry”, which contains information including botanical name, English name, local name (Tamil language), Family, description, images and common uses of true mangroves and their associates. This knowledge resource is available for open access at http://web.iitd.ac.in/~sundar/mangroves/.
Environmental toxicants, such as cadmium and bisphenol A (BPA) are endocrine disruptors. In utero... more Environmental toxicants, such as cadmium and bisphenol A (BPA) are endocrine disruptors. In utero, perinatal or neonatal exposure of BPA to rats affect the male reproductive function, such as the blood-testis barrier (BTB) integrity. This effect of BPA on BTB integrity in immature rats is likely mediated via a loss of gap junction function at the BTB, failing to coordinate tight junction and anchoring junction function at the site to maintain the immunological barrier integrity. This in turn activates the extracellular signal-regulated kinases 1/2 (Erk1/2) downstream and an increase in protein endocytosis, destabilizing the BTB. The cadmium-induced disruption of testicular dysfunction is mediated initially via its effects on the occludin/ZO-1/focal adhesion kinase (FAK) complex at the BTB, causing redistribution of proteins at the Sertoli-Sertoli cell interface, leading to the BTB disruption. The damaging effects of these toxicants to testicular function are mediated by mitogen-activated protein kinases (MAPK) downstream, which in turn perturbs the actin bundling and accelerates the actin-branching activity, causing disruption of the Sertoli cell tight junction (TJ)-barrier function at the BTB and perturbing spermatid adhesion at the apical ectoplasmic specialization (apical ES, a testis-specific anchoring junction type) that leads to premature release of germ cells from the testis. However, the use of specific inhibitors against MAPK was shown to block or delay the cadmium-induced testicular injury, such as BTB disruption and germ cell loss. These findings suggest that there may be a common downstream p38 and/or Erk1/2 MAPK-based signaling pathway involving polarity proteins and actin regulators that is shared between different toxicants that induce male reproductive dysfunction. As such, the use of inhibitors and/or antagonists against specific MAPKs can possibly be used to “manage” the illnesses caused by these toxicants and/or “protect” industrial workers being exposed to high levels of these toxicants in their work environment.
CYP2C19 is a polymorphic enzyme which metabolizes several clinically important drugs including pr... more CYP2C19 is a polymorphic enzyme which metabolizes several clinically important drugs including proguanil. Variation in the 5? regulatory region may influence CYP2C19 activity. This study evaluates the relationship between proguanil metabolic ratio and genetic variations of CYP2C19 in a South Indian population. Fifty unrelated healthy subjects were genotyped for CYP2C19 *2 and *3 alleles and the 5? flanking region of CYP2C19 was sequenced. Plasma concentrations of proguanil and cycloguanil were estimated by reverse phase HPLC after single oral doses (200 mg) of proguanil. In silico docking analysis of transcription factors binding to its sites in CYP2C19 5? regulatory region was performed. The mean metabolic ratios (proguanil/cycloguanil) were highest in *1/*2 or *1/*3 subjects and in *2/*2 or *2/*3 as compared to *1/*1 subjects. Subjects with promoter region variation －98TÀC showed decrease in the metabolic ratios irrespective of other variation, which may explain the deviation from the genotype-phenotype association of CYP2C19. In silico analysis predicted alteration in the interaction of transcription factors to their binding sites in the presence of variant alleles. The results of this study would be useful in predicting interindividual differences in the metabolism of substrates of CYP2C19.
Recent studies have demonstrated the presence of a functional axis that coordinates the events of... more Recent studies have demonstrated the presence of a functional axis that coordinates the events of spermiation and blood-testis barrier (BTB) restructuring which take place simultaneously at opposite ends of the seminiferous epithelium at stage VIII of the epithelial cycle of spermatogenesis in the rat testis. In short, the disruption of the apical ectoplasmic specialization (apical ES) at the Sertoli cell-elongated spermatid interface, which facilitates the release of sperm at spermiation near the tubule lumen, is coordinated with restructuring at the BTB to accommodate the transit of preleptotene spermatocytes near the basement membrane. These two events are likely coordinated by a functional axis involving hemidesmosome at the Sertoli cell-basement membrane interface, and it was designated the apical ES-BTB-hemidesmosome axis. It was demonstrated that fragments of laminin chains (e.g., laminin β3 or γ3 chains) derived from the α6β1-integrin-laminin333 protein complex at the apical ES, which were generated via the action of MMP-2 (matrix metalloprotease-2, MMP2) prior to spermiation, acted as biologically active peptides to perturb the BTB permeability function by accelerating protein endocytosis (e.g., occludin) at the site, thereby destabilizing the BTB integrity to facilitate the transit of preleptotene spermatocytes. These laminin fragments also perturbed hemidesmosome function via their action on β1-integrin, a component of hemidesmosome in the testis, which in turn, sent a signal to further destabilize the BTB function. As such, the events of spermiation and BTB restructuring are coordinated via this functional axis. Recent studies using animal models treated with toxicants, such as mono-(2-ethylhexyl) phthalate (MEHP), or adjudin, a male contraceptive under investigation, have also supported the presence of this functional axis in the mouse. In this short review, we critically evaluate the role of this local functional axis in the seminiferous epithelium in spermatogenesis. We also provide molecular modeling information on the interactions between biologically active laminin fragments and β1-integrin, which will be important to assist in the design of more potent laminin-based peptides to disrupt this axis, thereby perturbing spermatogenesis for male contraception and to understand the underlying biology that coordinates spermiation and BTB restructuring during spermatogenesis
The blood-testis barrier (BTB) is a unique ultrastructure in the mammalian testis. Unlike other b... more The blood-testis barrier (BTB) is a unique ultrastructure in the mammalian testis. Unlike other blood-tissue barriers, such as the blood-brain barrier and the blood-ocular (or blood-retina) barrier which formed by tight junctions (TJ) between endothelial cells of the microvessels, the BTB is constituted by coexisting TJ, basal ectoplasmic specialization (basal ES), desmosomes and gap junctions between adjacent Sertoli cells near the basement membrane of the seminiferous tubule. The BTB also divides the seminiferous epithelium into the apical (or adluminal) and basal compartments so that meiosis I and II and post-meiotic germ cell development can all take place in a specialized microenvironment in the apical compartment behind the BTB. While the unusual anatomical features of the BTB have been known for decades, the physiological function of the coexisting junctions, in particular the desmosome and gap junction, that constitute the BTB was unknown until recently. Based on recently published findings, we critically evaluate the role of the desmosome and gap junction that serve as a signaling platform to coordinate the “opening” and “closing” of the TJ-permeability barrier conferred by TJ and basal ES during the seminiferous epithelial cycle of spermatogenesis. This is made possible by polarity proteins working in concert with nonreceptor protein tyrosine kinases, such as focal adhesion kinase (FAK) and c-Src, at the site to regulate endosome-mediated protein trafficking events (e.g., endocytosis, transcytosis, recycling or protein degradation). These events not only serve to destabilize the existing “old” BTB above preleptotene spermatocytes in transit in “clones” at the BTB, but also contribute to the assembly of “new” BTB below the transiting spermatocytes. Furthermore, hemidesmosomes at the Sertoli cell-basement membrane interface also contribute to the BTB restructuring events at stage VIII of the epithelial cycle. Additionally, the findings that a gap junction at the BTB provides a possible route for the passage of toxicants [e.g., bisphenol A (BPA)] and potential male contraceptives (e.g., adjudin) across the BTB also illustrate that these coexisting junctions, while helpful to maintain the immunological barrier integrity during the transit of spermatocytes, can be the “gateway” to making the BTB so vulnerable to toxicants and/or chemicals, causing male reproductive dysfunction.
Molecular docking is a reliable method with which to identify the binding conformations of substr... more Molecular docking is a reliable method with which to identify the binding conformations of substrates, inducers and inhibitors of cytochrome P450 (CYP) enzymes. We used the docking method to explore possible binding modes of an entry inhibitor (maraviroc) and nonnucleoside reverse transcriptase inhibitors (delavirdine, efavirenz and etravirine) to cytochrome P450 3A4 (CYP3A4). In addition, docking results were compared with the binding conformations of HIV protease drugs to infer the binding site residues and potential drug–drug interaction profiles for combination therapy in the treatment of AIDS. We observed that efavirenz and etravirine induce metabolism of co-administered drugs by binding to a unique position in the active site of CYP3A4. Dosage adjustment is required for delavirdine and maraviroc when combined with HIV protease drugs. The present results are in good agreement with experimental data from drug interaction profiles. The information provided in this paper will be helpful in furthering our understanding the functions of CYP3A4, and could aid in the design of new drugs that would be metabolized easily without having any drug–drug interaction profile.
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Papers by Jayakanthan M
Results: We performed molecular docking simulation with six different inhibitors (ligand), which includes DTNB, NSC695265, thionitrobenzoic acid, 2-nitro-5-thiocyanobenzoic acid, 2-nitro-5-sulfo-sulfonyl-benzoic acid and NSC517871 into the redox-active site [C37-G38-H39-C40] of the PDI enzyme and the activity was inferred by redox inhibitory models. All ligands showed favourable interactions and most of them seemed to bind to hydrophobic amino acids Ala34, Trp36, Cys37, Cys40, His39, Thr68 and Phe80. The redox inhibitory conformations were energetically and statistically favored and supported the evidence from wet laboratory experiments reported in the literature.
Conclusion: We demonstrated that in silico docking experiment can be effectively carried out to recognize the redox inhibitory models of PDI with inhibitor molecules. Interestingly we found that number of docked clusters with each ligand varies in the range of five to eight and conveys that the binding specificity of each inhibitor varies for PDI. We also identified that Cys37 of the enzyme plays an important role in hydrogen bonding with inhibitors. This residue can be considered to being an active site for anti-HIV drug design. Therefore, by inhibiting PDI, one can, not only prevent the viral entry but also circumvent the problem of viral resistance
Description: ZifBASE provides an extensive collection of various natural and engineered ZFP. It uses standard names and a genetic and structural classification scheme to present data retrieved from UniProtKB, GenBank, Protein Data Bank, ModBase, Protein Model Portal and the literature. It also incorporates specialized features of ZFP including finger sequences and positions, number of fingers,
physiochemical properties, classes, framework, PubMed itations with links to experimental structures (PDB, if available) and modeled structures of natural zinc finger proteins. ZifBASE provides information on zinc finger proteins (both natural and engineered ones), the number of finger units in each of the zinc finger proteins (with multiple fingers), the synergy between the adjacent fingers and their positions. Additionally, it gives the individual finger sequence and their target DNA site to which it binds for better and clear understanding on the interactions of adjacent fingers. The current version of ZifBASE contains 139 entries of which 89 are engineered ZFPs, containing 3-7F totaling to 296 fingers. There are 50 natural zinc finger protein entries ranging from 2-13F, totaling to 307 fingers. It has sequences and structures from literature, Protein Data Bank, ModBase and Protein Model Portal. The interface is cross linked to other public databases like UniprotKB, PDB, ModBase and Protein Model Portal and PubMed for making it more
informative.
Conclusion: A database is established to maintain the information of the sequence features, including the class, framework, number of fingers, residues, position, recognition site and physio-chemical properties (molecular weight, isoelectric point) of both natural and engineered zinc finger proteins and dissociation constant of few. ZifBASE can provide more effective and efficient way of accessing the zinc finger protein sequences and their target binding sites with the links to their three-dimensional structures. All the data and functions are available at the advanced web-based search interface http://web.iitd.ac.in/~sundar/
zifbase.
and CPT.
male reproductive dysfunction.
Results: We performed molecular docking simulation with six different inhibitors (ligand), which includes DTNB, NSC695265, thionitrobenzoic acid, 2-nitro-5-thiocyanobenzoic acid, 2-nitro-5-sulfo-sulfonyl-benzoic acid and NSC517871 into the redox-active site [C37-G38-H39-C40] of the PDI enzyme and the activity was inferred by redox inhibitory models. All ligands showed favourable interactions and most of them seemed to bind to hydrophobic amino acids Ala34, Trp36, Cys37, Cys40, His39, Thr68 and Phe80. The redox inhibitory conformations were energetically and statistically favored and supported the evidence from wet laboratory experiments reported in the literature.
Conclusion: We demonstrated that in silico docking experiment can be effectively carried out to recognize the redox inhibitory models of PDI with inhibitor molecules. Interestingly we found that number of docked clusters with each ligand varies in the range of five to eight and conveys that the binding specificity of each inhibitor varies for PDI. We also identified that Cys37 of the enzyme plays an important role in hydrogen bonding with inhibitors. This residue can be considered to being an active site for anti-HIV drug design. Therefore, by inhibiting PDI, one can, not only prevent the viral entry but also circumvent the problem of viral resistance
Description: ZifBASE provides an extensive collection of various natural and engineered ZFP. It uses standard names and a genetic and structural classification scheme to present data retrieved from UniProtKB, GenBank, Protein Data Bank, ModBase, Protein Model Portal and the literature. It also incorporates specialized features of ZFP including finger sequences and positions, number of fingers,
physiochemical properties, classes, framework, PubMed itations with links to experimental structures (PDB, if available) and modeled structures of natural zinc finger proteins. ZifBASE provides information on zinc finger proteins (both natural and engineered ones), the number of finger units in each of the zinc finger proteins (with multiple fingers), the synergy between the adjacent fingers and their positions. Additionally, it gives the individual finger sequence and their target DNA site to which it binds for better and clear understanding on the interactions of adjacent fingers. The current version of ZifBASE contains 139 entries of which 89 are engineered ZFPs, containing 3-7F totaling to 296 fingers. There are 50 natural zinc finger protein entries ranging from 2-13F, totaling to 307 fingers. It has sequences and structures from literature, Protein Data Bank, ModBase and Protein Model Portal. The interface is cross linked to other public databases like UniprotKB, PDB, ModBase and Protein Model Portal and PubMed for making it more
informative.
Conclusion: A database is established to maintain the information of the sequence features, including the class, framework, number of fingers, residues, position, recognition site and physio-chemical properties (molecular weight, isoelectric point) of both natural and engineered zinc finger proteins and dissociation constant of few. ZifBASE can provide more effective and efficient way of accessing the zinc finger protein sequences and their target binding sites with the links to their three-dimensional structures. All the data and functions are available at the advanced web-based search interface http://web.iitd.ac.in/~sundar/
zifbase.
and CPT.
male reproductive dysfunction.