737 results on '"Bridged Bicyclo Compounds metabolism"'
Search Results
2. Regiodivergent biosynthesis of bridged bicyclononanes.
- Author
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Ernst L, Lyu H, Liu P, Paetz C, Sayed HMB, Meents T, Ma H, Beerhues L, El-Awaad I, and Liu B
- Subjects
- Bridged Bicyclo Compounds metabolism, Bridged Bicyclo Compounds chemistry, Plant Proteins metabolism, Plant Proteins genetics, Molecular Docking Simulation, Phloroglucinol metabolism, Phloroglucinol analogs & derivatives, Phloroglucinol chemistry, Alkanes metabolism, Alkanes chemistry, Catalytic Domain, Terpenes metabolism, Terpenes chemistry, Models, Molecular, Hypericum metabolism, Hypericum genetics, Hypericum chemistry
- Abstract
Medicinal compounds from plants include bicyclo[3.3.1]nonane derivatives, the majority of which are polycyclic polyprenylated acylphloroglucinols (PPAPs). Prototype molecules are hyperforin, the antidepressant constituent of St. John's wort, and garcinol, a potential anticancer compound. Their complex structures have inspired innovative chemical syntheses, however, their biosynthesis in plants is still enigmatic. PPAPs are divided into two subclasses, named type A and B. Here we identify both types in Hypericum sampsonii plants and isolate two enzymes that regiodivergently convert a common precursor to pivotal type A and B products. Molecular modelling and substrate docking studies reveal inverted substrate binding modes in the two active site cavities. We identify amino acids that stabilize these alternative binding scenarios and use reciprocal mutagenesis to interconvert the enzymatic activities. Our studies elucidate the unique biochemistry that yields type A and B bicyclo[3.3.1]nonane cores in plants, thereby providing key building blocks for biotechnological efforts to sustainably produce these complex compounds for preclinical development., (© 2024. The Author(s).)
- Published
- 2024
- Full Text
- View/download PDF
3. A tuneable genetic switch for tight control of tac promoters in Escherichia coli boosts expression of synthetic injectisomes.
- Author
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Asensio-Calavia A, Ceballos-Munuera Á, Méndez-Pérez A, Álvarez B, and Fernández LÁ
- Subjects
- Promoter Regions, Genetic, Lac Repressors genetics, Lac Repressors metabolism, Escherichia coli genetics, Escherichia coli metabolism, Bridged Bicyclo Compounds metabolism, Thiadiazoles
- Abstract
Biosafety of engineered bacteria as living therapeutics requires a tight regulation to control the specific delivery of protein effectors, maintaining minimum leakiness in the uninduced (OFF) state and efficient expression in the induced (ON) state. Here, we report a three repressors (3R) genetic circuit that tightly regulates the expression of multiple tac promoters (Ptac) integrated in the chromosome of E. coli and drives the expression of a complex type III secretion system injectisome for therapeutic protein delivery. The 3R genetic switch is based on the tetracycline repressor (TetR), the non-inducible lambda repressor cI (ind-) and a mutant lac repressor (LacI
W220F ) with higher activity. The 3R switch was optimized with different protein translation and degradation signals that control the levels of LacIW220F . We demonstrate the ability of an optimized switch to fully repress the strong leakiness of the Ptac promoters in the OFF state while triggering their efficient activation in the ON state with anhydrotetracycline (aTc), an inducer suitable for in vivo use. The implementation of the optimized 3R switch in the engineered synthetic injector E. coli (SIEC) strain boosts expression of injectisomes upon aTc induction, while maintaining a silent OFF state that preserves normal growth in the absence of the inducer. Since Ptac is a commonly used promoter, the 3R switch may have multiple applications for tight control of protein expression in E. coli. In addition, the modularity of the 3R switch may enable its tuning for the control of Ptac promoters with different inducers., (© 2023 The Authors. Microbial Biotechnology published by Applied Microbiology International and John Wiley & Sons Ltd.)- Published
- 2024
- Full Text
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4. Bicyclic Picomolar OGA Inhibitors Enable Chemoproteomic Mapping of Its Endogenous Post-translational Modifications.
- Author
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González-Cuesta M, Sidhu P, Ashmus RA, Males A, Proceviat C, Madden Z, Rogalski JC, Busmann JA, Foster LJ, García Fernández JM, Davies GJ, Ortiz Mellet C, and Vocadlo DJ
- Subjects
- Amino Acid Sequence, Brain metabolism, Bridged Bicyclo Compounds metabolism, Catalytic Domain, Chromatography, High Pressure Liquid, Enzyme Inhibitors metabolism, Histone Acetyltransferases antagonists & inhibitors, Humans, Hyaluronoglucosaminidase antagonists & inhibitors, Mass Spectrometry, Peptides analysis, Peptides chemistry, Protein Processing, Post-Translational, Proteomics methods, Structure-Activity Relationship, Thiazolidines chemistry, Thiazolidines metabolism, beta-Hexosaminidase alpha Chain antagonists & inhibitors, beta-Hexosaminidase alpha Chain metabolism, Antigens, Neoplasm metabolism, Bridged Bicyclo Compounds chemistry, Enzyme Inhibitors chemistry, Histone Acetyltransferases metabolism, Hyaluronoglucosaminidase metabolism
- Abstract
Owing to its roles in human health and disease, the modification of nuclear, cytoplasmic, and mitochondrial proteins with O-linked N -acetylglucosamine residues (O-GlcNAc) has emerged as a topic of great interest. Despite the presence of O-GlcNAc on hundreds of proteins within cells, only two enzymes regulate this modification. One of these enzymes is O-GlcNAcase (OGA), a dimeric glycoside hydrolase that has a deep active site cleft in which diverse substrates are accommodated. Chemical tools to control OGA are emerging as essential resources for helping to decode the biochemical and cellular functions of the O-GlcNAc pathway. Here we describe rationally designed bicyclic thiazolidine inhibitors that exhibit superb selectivity and picomolar inhibition of human OGA. Structures of these inhibitors in complex with human OGA reveal the basis for their exceptional potency and show that they extend out of the enzyme active site cleft. Leveraging this structure, we create a high affinity chemoproteomic probe that enables simple one-step purification of endogenous OGA from brain and targeted proteomic mapping of its post-translational modifications. These data uncover a range of new modifications, including some that are less-known, such as O-ubiquitination and N-formylation. We expect that these inhibitors and chemoproteomics probes will prove useful as fundamental tools to decipher the mechanisms by which OGA is regulated and directed to its diverse cellular substrates. Moreover, the inhibitors and structures described here lay out a blueprint that will enable the creation of chemical probes and tools to interrogate OGA and other carbohydrate active enzymes.
- Published
- 2022
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5. Discovery of novel antibacterial agents: Recent developments in D-alanyl-D-alanine ligase inhibitors.
- Author
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Qin Y, Xu L, Teng Y, Wang Y, and Ma P
- Subjects
- Anti-Bacterial Agents metabolism, Anti-Bacterial Agents pharmacology, Bacteria drug effects, Bridged Bicyclo Compounds chemistry, Bridged Bicyclo Compounds metabolism, Bridged Bicyclo Compounds pharmacology, Enzyme Inhibitors metabolism, Heterocyclic Compounds, 1-Ring chemistry, Heterocyclic Compounds, 1-Ring metabolism, Heterocyclic Compounds, 1-Ring pharmacology, Peptide Synthases metabolism, Peptidoglycan biosynthesis, Structure-Activity Relationship, Anti-Bacterial Agents chemistry, Drug Discovery, Enzyme Inhibitors chemistry, Peptide Synthases chemistry
- Abstract
Bacterial infections can cause serious problems that threaten public health over a long period of time. Moreover, the continuous emergence of drug-resistant bacteria necessitates the development of novel antibacterial agents. D-alanyl-D-alanine ligase (Ddl) is an indispensable adenosine triphosphate-dependent bacterial enzyme involved in the biosynthesis of peptidoglycan precursor, which catalyzes the ligation of two D-alanine molecules into one D-alanyl-D-alanine dipeptide. This dipeptide is an essential component of the intracellular peptidoglycan precursor, uridine diphospho-N-acetylmuramic acid (UDP-MurNAc)-pentapeptide, that maintains the integrity of the bacterial cell wall by cross-linking the peptidoglycan chain, and is crucial for the survival of pathogens. Consequently, Ddl is expected to be a promising target for the development of antibacterial agents. In this review, we present a brief introduction regarding the structure and function of Ddl, as well as an overview of the various Ddl inhibitors currently being used as antibacterial agents, specifically highlighting their inhibitory activities, structure-activity relationships and mechanisms of action., (© 2021 John Wiley & Sons A/S.)
- Published
- 2021
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6. Comparison the dissipation behaviors and exposure risk of carbendazim and procymidone in greenhouse strawberries under different application method: Individual and joint applications.
- Author
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Di S, Wang Y, Xu H, Wang X, Yang G, Chen C, Yang X, and Qian Y
- Subjects
- Benzimidazoles analysis, Bridged Bicyclo Compounds analysis, Carbamates analysis, Chromatography, High Pressure Liquid, Fragaria metabolism, Greenhouse Effect, Half-Life, Humans, Pesticide Residues analysis, Risk Assessment, Spectrometry, Mass, Electrospray Ionization, Benzimidazoles metabolism, Bridged Bicyclo Compounds metabolism, Carbamates metabolism, Fragaria chemistry
- Abstract
The dissipation behaviors and exposure risks of individual and joint application of procymidone and carbendazim in greenhouse strawberries were studied. The initial concentrations were similar after individual or joint applications, while the dissipation half-lives and finial concentrations were significantly different. After joint application, the dissipation half-lives of procymidone and carbendazim were 12.9 and 16.0 days, respectively, which were about 1.8 times higher than those after individual application. Furthermore, the final residues under joint application condition were 1.8-3.5 times higher than those under individual application condition. The joint application decreased the dissipation rates of procymidone or carbendazim in strawberries, and increased the final residue concentrations. The dietary intake risks of procymidone and carbendazim (whether applied individually or jointly) were no higher than 0.12, which were acceptable for human health. This work would shed a light for the guidance of the joint application and risk assessment of the typical fungicides in strawberry., (Copyright © 2021 Elsevier Ltd. All rights reserved.)
- Published
- 2021
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7. Mirogabalin activates the descending noradrenergic system by binding to the α 2 δ-1 subunit of voltage-gated Ca 2+ channels to generate analgesic effects.
- Author
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Oyama M, Watanabe S, Iwai T, and Tanabe M
- Subjects
- Animals, Bridged Bicyclo Compounds metabolism, Calcium Channels genetics, Drug Administration Routes, Female, Male, Mice, Mice, Inbred Strains, Mice, Mutant Strains, Mutation, Protein Binding drug effects, Analgesics, Bridged Bicyclo Compounds administration & dosage, Bridged Bicyclo Compounds pharmacology, Calcium Channels metabolism, Neuralgia drug therapy, Norepinephrine metabolism
- Abstract
Gabapentinoids such as gabapentin and pregabalin, which bind specifically to the α
2 δ subunit of voltage-gated Ca2+ channels, are used for first-line treatment of neuropathic pain. Here, we examined the analgesic effect of mirogabalin besilate (referred to simply as mirogabalin), a novel gabapentinoid, focusing on its action on the spinal cord and the descending noradrenergic pain inhibitory system. When administered systemically (10 and 30 mg/kg, intraperitoneally (i.p.)) and locally (10 and 30 μg, intracerebroventricularly (i.c.v.) or intrathecally (i.t.)) to mice, mirogabalin was found to exert analgesic effects on thermal (plantar test) and mechanical (von Frey test) hypersensitivity developing after partial sciatic nerve ligation. Notably, its analgesic effects (30 mg/kg, i.p. and 30 μg, i.c.v.) disappeared in mice pretreated with yohimbine hydrochloride (3 μg, i.t.). Moreover, in mice harboring a mutation in the α2 δ-1 subunit resulting in substitution of arginine at position 217 with alanine to prevent gabapentinoid binding (R217A mutant mice), the analgesic effects of pregabalin and mirogabalin (30 μg, i.c.v., respectively) on mechanical hypersensitivity were almost completely suppressed. These results clearly demonstrate that mirogabalin also operates via the descending noradrenergic system, and that binding to the α2 δ-1 subunit supraspinally is essential for the pain relief effect of gabapentinoids., Competing Interests: Declaration of competing interest There is a financial relationship with Daiichi Sankyo Company Limited, Tokyo, Japan, that might lead to a conflict of interest., (Copyright © 2021 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)- Published
- 2021
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8. The Inactivation by Curcumin-Mediated Photosensitization of Botrytis cinerea Spores Isolated from Strawberry Fruits.
- Author
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Huang L, Yong KWL, Fernando WC, Carpinelli de Jesus M, De Voss JJ, Sultanbawa Y, and Fletcher MT
- Subjects
- Aldehydes metabolism, Aldehydes toxicity, Botrytis growth & development, Botrytis metabolism, Bridged Bicyclo Compounds metabolism, Bridged Bicyclo Compounds toxicity, Chromatography, Liquid, Food Microbiology, Food Preservation, Secondary Metabolism, Spectrometry, Mass, Electrospray Ionization, Spores, Fungal growth & development, Spores, Fungal metabolism, Tandem Mass Spectrometry, Botrytis drug effects, Curcumin pharmacology, Fragaria microbiology, Fruit microbiology, Fungicides, Industrial pharmacology, Photosensitizing Agents pharmacology, Spores, Fungal drug effects
- Abstract
Photosensitization is a novel environmentally friendly technology with promising applications in the food industry to extend food shelf life. In this study, the natural food dye curcumin, when combined with visible light (430 nm), was shown to be an effective photosensitizer against the common phytopathogenic fungi Botrytis cinerea (the cause of grey mould). Production of the associated phytotoxic metabolites botrydial and dihydrobotrydial was measured by our newly developed and validated HRAM UPLC-MS/MS method, and was also shown to be reduced by this treatment. With a light dose of 120 J/cm
2 , the reduction in spore viability was directly proportional to curcumin concentrations, and the overall concentration of both botrydial and dihydrobotrydial also decreased with increasing curcumin concentration above 200 µM. With curcumin concentrations above 600 µM, the percentage reduction in fungal spores was close to 100%. When the dye concentration was increased to 800 µM, the spores were completely inactive and neither botrydial nor dihydrobotrydial could be detected. These results suggest that curcumin-mediated photosensitization is a potentially effective method to control B. cinerea spoilage, and also to reduce the formation of these phytotoxic botryane secondary metabolites.- Published
- 2021
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9. Structural Characterization of Diazabicyclooctane β-Lactam "Enhancers" in Complex with Penicillin-Binding Proteins PBP2 and PBP3 of Pseudomonas aeruginosa.
- Author
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Rajavel M, Kumar V, Nguyen H, Wyatt J, Marshall SH, Papp-Wallace KM, Deshpande P, Bhavsar S, Yeole R, Bhagwat S, Patel M, Bonomo RA, and van den Akker F
- Subjects
- Anti-Bacterial Agents metabolism, Azabicyclo Compounds metabolism, Bridged Bicyclo Compounds metabolism, Crystallization, Cyclooctanes metabolism, Microbial Sensitivity Tests, Octanes metabolism, Penicillin-Binding Proteins antagonists & inhibitors, Piperidines metabolism, Protein Binding, Pseudomonas aeruginosa enzymology, Pseudomonas aeruginosa genetics, beta-Lactamase Inhibitors pharmacology, Anti-Bacterial Agents pharmacology, Azabicyclo Compounds pharmacology, Bridged Bicyclo Compounds pharmacology, Cyclooctanes pharmacology, Octanes pharmacology, Penicillin-Binding Proteins chemistry, Penicillin-Binding Proteins metabolism, Piperidines pharmacology, Pseudomonas aeruginosa chemistry, Pseudomonas aeruginosa drug effects
- Abstract
Multidrug-resistant (MDR) pathogens pose a significant public health threat. A major mechanism of resistance expressed by MDR pathogens is β-lactamase-mediated degradation of β-lactam antibiotics. The diazabicyclooctane (DBO) compounds zidebactam and WCK 5153, recognized as β-lactam "enhancers" due to inhibition of Pseudomonas aeruginosa penicillin-binding protein 2 (PBP2), are also class A and C β-lactamase inhibitors. To structurally probe their mode of PBP2 inhibition as well as investigate why P. aeruginosa PBP2 is less susceptible to inhibition by β-lactam antibiotics compared to the Escherichia coli PBP2, we determined the crystal structure of P. aeruginosa PBP2 in complex with WCK 5153. WCK 5153 forms an inhibitory covalent bond with the catalytic S327 of PBP2. The structure suggests a significant role for the diacylhydrazide moiety of WCK 5153 in interacting with the aspartate in the S-X-N/D PBP motif. Modeling of zidebactam in the active site of PBP2 reveals a similar binding mode. Both DBOs increase the melting temperature of PBP2, affirming their stabilizing interactions. To aid in the design of DBOs that can inhibit multiple PBPs, the ability of three DBOs to interact with P. aeruginosa PBP3 was explored crystallographically. Even though the DBOs show covalent binding to PBP3, they destabilized PBP3. Overall, the studies provide insights into zidebactam and WCK 5153 inhibition of PBP2 compared to their inhibition of PBP3 and the evolutionarily related KPC-2 β-lactamase. These molecular insights into the dual-target DBOs advance our knowledge regarding further DBO optimization efforts to develop novel potent β-lactamase-resistant, non-β-lactam PBP inhibitors. IMPORTANCE Antibiotic resistance is a significant clinical problem. Developing novel antibiotics that overcome known resistance mechanisms is highly desired. Diazabicyclooctane inhibitors such as zidebactam possess this potential as they readily inactivate penicillin-binding proteins, yet cannot be degraded by β-lactamases. In this study, we characterized the inhibition by diazabicyclooctanes of penicillin-binding proteins PBP2 and PBP3 from Pseudomonas aeruginosa using protein crystallography and biophysical analyses. These structures and analyses help define the antibiotic properties of these inhibitors, explain the decreased susceptibility of P. aeruginosa PBP2 to be inhibited by β-lactam antibiotics, and provide insights that could be used for further antibiotic development.
- Published
- 2021
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10. Reaction mechanism of the farnesyl pyrophosphate C-methyltransferase towards the biosynthesis of pre-sodorifen pyrophosphate by Serratia plymuthica 4Rx13.
- Author
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Lemfack MC, Brandt W, Krüger K, Gurowietz A, Djifack J, Jung JP, Hopf M, Noack H, Junker B, von Reuß S, and Piechulla B
- Subjects
- Amino Acid Motifs, Bacterial Proteins chemistry, Bacterial Proteins genetics, Binding Sites, Biocatalysis, Bridged Bicyclo Compounds chemistry, Cloning, Molecular, Cyclization, Escherichia coli genetics, Escherichia coli metabolism, Gene Expression, Genetic Vectors chemistry, Genetic Vectors metabolism, Methylation, Methyltransferases chemistry, Methyltransferases genetics, Molecular Docking Simulation, Mutagenesis, Site-Directed, Octanes chemistry, Polyisoprenyl Phosphates chemistry, Polyisoprenyl Phosphates metabolism, Protein Binding, Protein Conformation, alpha-Helical, Protein Conformation, beta-Strand, Protein Interaction Domains and Motifs, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Serratia chemistry, Serratia genetics, Sesquiterpenes chemistry, Sesquiterpenes metabolism, Substrate Specificity, Bacterial Proteins metabolism, Bridged Bicyclo Compounds metabolism, Methyltransferases metabolism, Octanes metabolism, Serratia enzymology
- Abstract
Classical terpenoid biosynthesis involves the cyclization of the linear prenyl pyrophosphate precursors geranyl-, farnesyl-, or geranylgeranyl pyrophosphate (GPP, FPP, GGPP) and their isomers, to produce a huge number of natural compounds. Recently, it was shown for the first time that the biosynthesis of the unique homo-sesquiterpene sodorifen by Serratia plymuthica 4Rx13 involves a methylated and cyclized intermediate as the substrate of the sodorifen synthase. To further support the proposed biosynthetic pathway, we now identified the cyclic prenyl pyrophosphate intermediate pre-sodorifen pyrophosphate (PSPP). Its absolute configuration (6R,7S,9S) was determined by comparison of calculated and experimental CD-spectra of its hydrolysis product and matches with those predicted by semi-empirical quantum calculations of the reaction mechanism. In silico modeling of the reaction mechanism of the FPP C-methyltransferase (FPPMT) revealed a S
N 2 mechanism for the methyl transfer followed by a cyclization cascade. The cyclization of FPP to PSPP is guided by a catalytic dyad of H191 and Y39 and involves an unprecedented cyclopropyl intermediate. W46, W306, F56, and L239 form the hydrophobic binding pocket and E42 and H45 complex a magnesium cation that interacts with the diphosphate moiety of FPP. Six additional amino acids turned out to be essential for product formation and the importance of these amino acids was subsequently confirmed by site-directed mutagenesis. Our results reveal the reaction mechanism involved in methyltransferase-catalyzed cyclization and demonstrate that this coupling of C-methylation and cyclization of FPP by the FPPMT represents an alternative route of terpene biosynthesis that could increase the terpenoid diversity and structural space.- Published
- 2021
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11. Dynamics in Secondary Metabolite Gene Clusters in Otherwise Highly Syntenic and Stable Genomes in the Fungal Genus Botrytis.
- Author
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Valero-Jiménez CA, Steentjes MBF, Slot JC, Shi-Kunne X, Scholten OE, and van Kan JAL
- Subjects
- Aldehydes metabolism, Botrytis metabolism, Bridged Bicyclo Compounds metabolism, Multigene Family, Polyketides metabolism, Secondary Metabolism genetics, Synteny, Allium microbiology, Botrytis genetics, Genome, Fungal, Host Specificity genetics, Phylogeny
- Abstract
Fungi of the genus Botrytis infect >1,400 plant species and cause losses in many crops. Besides the broad host range pathogen Botrytis cinerea, most other species are restricted to a single host. Long-read technology was used to sequence genomes of eight Botrytis species, mostly pathogenic on Allium species, and the related onion white rot fungus, Sclerotium cepivorum. Most assemblies contained <100 contigs, with the Botrytis aclada genome assembled in 16 gapless chromosomes. The core genome and pan-genome of 16 Botrytis species were defined and the secretome, effector, and secondary metabolite repertoires analyzed. Among those genes, none is shared among all Allium pathogens and absent from non-Allium pathogens. The genome of each of the Allium pathogens contains 8-39 predicted effector genes that are unique for that single species, none stood out as potential determinant for host specificity. Chromosome configurations of common ancestors of the genus Botrytis and family Sclerotiniaceae were reconstructed. The genomes of B. cinerea and B. aclada were highly syntenic with only 19 rearrangements between them. Genomes of Allium pathogens were compared with ten other Botrytis species (nonpathogenic on Allium) and with 25 Leotiomycetes for their repertoire of secondary metabolite gene clusters. The pattern was complex, with several clusters displaying patchy distribution. Two clusters involved in the synthesis of phytotoxic metabolites are at distinct genomic locations in different Botrytis species. We provide evidence that the clusters for botcinic acid production in B. cinerea and Botrytis sinoallii were acquired by horizontal transfer from taxa within the same genus., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)
- Published
- 2020
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12. Inorganic polyphosphate is required for sustained free mitochondrial calcium elevation, following calcium uptake.
- Author
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Solesio ME, Garcia Del Molino LC, Elustondo PA, Diao C, Chang JC, and Pavlov EV
- Subjects
- Adenosine Triphosphate pharmacology, Benzoates metabolism, Bridged Bicyclo Compounds metabolism, Calcium Signaling drug effects, Cell Membrane Permeability drug effects, Cycloheptanes metabolism, HEK293 Cells, Humans, Membrane Potential, Mitochondrial drug effects, Mitochondria drug effects, Models, Biological, Ruthenium Red metabolism, Sesquiterpenes metabolism, Calcium metabolism, Mitochondria metabolism, Polyphosphates pharmacology
- Abstract
Mitochondrial free calcium is critically linked to the regulation of cellular metabolism. Free ionic calcium concentration within these organelles is determined by the interplay between two processes: exchange across the mitochondrial inner membrane and calcium-buffering within the matrix. During stimulated calcium uptake, calcium is primarily buffered by orthophosphate, preventing calcium toxicity while allowing for well-regulated yet elevated calcium loads. However, if limited to orthophosphates only, this buffering system is expected to lead to the irreversible formation of insoluble precipitates, which are not observed in living cells, under physiological conditions. Here, we demonstrate that the regulation of free mitochondrial calcium requires the presence of free inorganic polyphosphate (polyP) within the organelle. We found that the overexpression of a mitochondrial-targeted enzyme hydrolyzing polyP leads to the loss of the cellular ability to maintain elevated calcium concentrations within the organelle, following stimulated cytoplasmic signal. We hypothesize that the presence of polyP prevents the formation of calcium-phosphate insoluble clusters, allowing for the maintenance of elevated free calcium levels, during stimulated calcium uptake., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2019 Elsevier Ltd. All rights reserved.)
- Published
- 2020
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13. Studies on the selectivity of proline hydroxylases reveal new substrates including bicycles.
- Author
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Smart TJ, Hamed RB, Claridge TDW, and Schofield CJ
- Subjects
- Biocatalysis, Bridged Bicyclo Compounds chemistry, Molecular Structure, Prolyl Hydroxylases chemistry, Substrate Specificity, Bridged Bicyclo Compounds metabolism, Prolyl Hydroxylases metabolism
- Abstract
Studies on the substrate selectivity of recombinant ferrous-iron- and 2-oxoglutarate-dependent proline hydroxylases (PHs) reveal that they can catalyse the production of dihydroxylated 5-, 6-, and 7-membered ring products, and can accept bicyclic substrates. Ring-substituted substrate analogues (such hydroxylated and fluorinated prolines) are accepted in some cases. The results highlight the considerable, as yet largely untapped, potential for amino acid hydroxylases and other 2OG oxygenases in biocatalysis., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2020
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14. Botrydial confers Botrytis cinerea the ability to antagonize soil and phyllospheric bacteria.
- Author
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Vignatti P, Gonzalez ME, Jofré EC, Bolívar-Anillo HJ, Moraga J, Viaud M, Collado IG, and Pieckenstain FL
- Subjects
- Aldehydes metabolism, Bacillus drug effects, Bacillus growth & development, Bacillus amyloliquefaciens drug effects, Bacillus amyloliquefaciens growth & development, Bacillus amyloliquefaciens physiology, Bacteria growth & development, Botrytis growth & development, Bridged Bicyclo Compounds metabolism, Lipopeptides metabolism, Peptides, Cyclic metabolism, Aldehydes pharmacology, Antibiosis, Bacillus physiology, Bacterial Physiological Phenomena, Botrytis physiology, Bridged Bicyclo Compounds pharmacology, Soil Microbiology
- Abstract
The role of the sesquiterpene botrydial in the interaction of the phytopathogenic fungus Botrytis cinerea and plant-associated bacteria was analyzed. From a collection of soil and phyllospheric bacteria, nine strains sensitive to growth-inhibition by B. cinerea were identified. B. cinerea mutants unable to produce botrydial caused no bacterial inhibition, thus demonstrating the inhibitory role of botrydial. A taxonomic analysis showed that these bacteria corresponded to different Bacillus species (six strains), Pseudomonas yamanorum (two strains) and Erwinia aphidicola (one strain). Inoculation of WT and botrydial non-producing mutants of B. cinerea along with Bacillusamyloliquefaciens strain MEP
2 18 in soil demonstrated that both microorganisms exert reciprocal inhibitory effects; the inhibition caused by B. cinerea being dependent on botrydial production. Moreover, botrydial production was modulated by the presence of B. amyloliquefaciens MEP2 18 in confrontation assays in vitro. Purified botrydial in turn, inhibited growth of Bacillus strains in vitro and cyclic lipopeptide (surfactin) production by B. amyloliquefaciens MEP2 18. As a whole, results demonstrate that botrydial confers B. cinerea the ability to inhibit potential biocontrol bacteria of the genus Bacillus. We propose that resistance to botrydial could be used as an additional criterion for the selection of biocontrol agents of plant diseases caused by B. cinerea., (Copyright © 2019 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)- Published
- 2020
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15. Sevoflurane-induced inflammation development: involvement of cholinergic anti-inflammatory pathway.
- Author
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Yin J, Zhao X, Wang L, Xie X, Geng H, Zhan X, and Teng J
- Subjects
- Acetylcholinesterase metabolism, Anesthetics, Inhalation pharmacology, Animals, Benzamides metabolism, Brain drug effects, Bridged Bicyclo Compounds metabolism, Cognition physiology, Cognitive Dysfunction metabolism, Hippocampus drug effects, Inflammation immunology, Male, Methyl Ethers pharmacology, Rats, Rats, Sprague-Dawley, Signal Transduction drug effects, alpha7 Nicotinic Acetylcholine Receptor metabolism, Inflammation physiopathology, Neuroimmunomodulation drug effects, Sevoflurane pharmacology
- Abstract
Chronic inflammation plays an important role in the mechanisms underpinning the development of anesthesia-induced cognitive dysfunction. However, less is known about how anesthesia causes inflammation. One possibility is that the inflammation is related to alteration of the activity of the alpha 7 nicotinic acetylcholine receptor cholinergic anti-inflammatory pathway. This study analyzed the effect of sevoflurane administration on the cognitive function by using a novel object recognition test and Y-maze test, and on acetylcholinesterase activity and expression in hippocampal tissue by using an acetylcholinesterase assay kit and quantitative real-time PCR. This study also evaluated the effect of alpha 7 nicotinic acetylcholine receptor agonist PNU-282987 and antagonist methyllycaconitine on cognitive function and the level of hippocampal tumor necrosis factor-α in aged rats exposed to sevoflurane anesthesia. We found that 3% sevoflurane significantly impaired cognitive function and increased acetylcholinesterase activity by upregulating its expression in hippocampal tissue. Sevoflurane-induced impairment of cognitive function was significantly rescued by PNU-282987 but aggravated by methyllycaconitine. In addition to impairment of cognitive function, sevoflurane also significantly increased tumor necrosis factor-α level in plasma and hippocampal tissue. Similarly, this sevoflurane-induced change of tumor necrosis factor-α level in rats was antagonized by PNU-282987 but amplified by methyllycaconitine. In conclusion, our data show that the development of inflammation in sevoflurane-induced cognitive decline is associated with the downregulation of alpha 7 nicotinic acetylcholine receptor cholinergic anti-inflammatory pathway in aged rats.
- Published
- 2019
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16. Type II flavoprotein monooxygenase PsFMO_A from the bacterium Pimelobacter sp. Bb-B catalyzes enantioselective Baeyer-Villiger oxidations with a relaxed cofactor specificity.
- Author
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Löwe J, Blifernez-Klassen O, Baier T, Wobbe L, Kruse O, and Gröger H
- Subjects
- Bridged Bicyclo Compounds metabolism, Camphor metabolism, Escherichia coli genetics, Flavoproteins, Oxidation-Reduction, Stereoisomerism, Actinobacteria enzymology, Bacterial Proteins genetics, Bacterial Proteins metabolism, Mixed Function Oxygenases genetics, Mixed Function Oxygenases metabolism
- Abstract
Microbial consortia, which degrade branched, long-chain hydrocarbons, can be regarded as a promising source of novel enzymes for the stereo- and regio-selective oxyfunctionalization of hydrocarbons. The hydrocarbon-degrading bacterium Pimelobacter sp. Bb-B was isolated from the consortium associated with the colonial hydrocarbon-excreting microalga Botryococcus braunii. Three new type II flavoprotein monooxygenases (type II FMOs) from this bacterium have been made available in recombinant form through cloning and overexpression in an E. coli host organism. These enzymes (PsFMO_A-C) were characterized in terms of their capability of catalyzing Baeyer-Villiger oxidations with distinct substrates. The highest activity was detected when utilizing camphor and bicyclo[3.2.0]hept-2-en-6-one as substrate in combination with PsFMO_A as the most promising enzyme. Furthermore, synthetic biotransformations with 5 mM of the substrate bicyclo[3.2.0]hept-2-en-6-one, formate and formate dehydrogenase for in situ-cofactor recycling were conducted with this enzyme, leading to a substrate consumption of 85% after 66 h and excellent enantioselectivity of 99% ee for the (1R,5S)-enantiomer. Additionally, an alternative in situ-cofactor recycling based on the use of microalgae for in situ-production of formate from carbon dioxide, water and light together with a formate dehydrogenase was combined successfully with the enzyme PsFMO_A, leading to a substrate consumption of 94% and an enantioselectivity of >99% ee for the so-called "normal lactone"-enantiomer with the absolute configuration 1R,5S., (Copyright © 2019 Elsevier B.V. All rights reserved.)
- Published
- 2019
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17. Synthesis of All Stereoisomers of RK460 and Evaluation of Their Activity and Selectivity as Abscisic Acid Receptor Antagonists.
- Author
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Mikame Y, Yoshida K, Hashizume D, Hirai G, Nagasawa K, Osada H, and Sodeoka M
- Subjects
- Arabidopsis metabolism, Arabidopsis Proteins metabolism, Bridged Bicyclo Compounds chemical synthesis, Bridged Bicyclo Compounds chemistry, Intracellular Signaling Peptides and Proteins metabolism, Membrane Transport Proteins metabolism, Plant Growth Regulators chemical synthesis, Plant Growth Regulators chemistry, Receptors, Cell Surface metabolism, Stereoisomerism, Abscisic Acid metabolism, Arabidopsis drug effects, Arabidopsis Proteins antagonists & inhibitors, Bridged Bicyclo Compounds metabolism, Intracellular Signaling Peptides and Proteins antagonists & inhibitors, Plant Growth Regulators metabolism, Receptors, Cell Surface antagonists & inhibitors
- Abstract
The PYR/PYL/RCAR protein families have recently emerged as receptors of the phytohormone abscisic acid (ABA, 1), which regulates plant responses to environmental stress. These families have multiple members with different physiological actions, and so selective agonists or antagonists are needed both as tools to elucidate functional differences and as lead compounds for agrochemicals. We previously identified RK460 (rac-3 a) as a PYR1-selective antagonist, and showed that it possesses five stereocenters on a 6,5-cis-bicyclo skeleton. Here, we synthesized all the stereoisomers of RK460 and evaluated their activity towards a panel of receptors. Relative stereochemistry as well as absolute stereochemistry was important for selective action., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2019
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18. Direct pathway cloning of the sodorifen biosynthetic gene cluster and recombinant generation of its product in E. coli.
- Author
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Duell ER, D'Agostino PM, Shapiro N, Woyke T, Fuchs TM, and Gulder TAM
- Subjects
- Bacterial Proteins metabolism, Biosynthetic Pathways, Cloning, Molecular, Computational Biology, Escherichia coli genetics, Genome, Bacterial, Pyrophosphatases metabolism, Bridged Bicyclo Compounds metabolism, Escherichia coli metabolism, Multigene Family, Octanes metabolism, Serratia genetics
- Abstract
Background: Serratia plymuthica WS3236 was selected for whole genome sequencing based on preliminary genetic and chemical screening indicating the presence of multiple natural product pathways. This led to the identification of a putative sodorifen biosynthetic gene cluster (BGC). The natural product sodorifen is a volatile organic compound (VOC) with an unusual polymethylated hydrocarbon bicyclic structure (C
16 H26 ) produced by selected strains of S. plymuthica. The BGC encoding sodorifen consists of four genes, two of which (sodA, sodB) are homologs of genes encoding enzymes of the non-mevalonate pathway and are thought to enhance the amounts of available farnesyl pyrophosphate (FPP), the precursor of sodorifen. Proceeding from FPP, only two enzymes are necessary to produce sodorifen: an S-adenosyl methionine dependent methyltransferase (SodC) with additional cyclisation activity and a terpene-cyclase (SodD). Previous analysis of S. plymuthica found sodorifen production titers are generally low and vary significantly among different producer strains. This precludes studies on the still elusive biological function of this structurally and biosynthetically fascinating bacterial terpene., Results: Sequencing and mining of the S. plymuthica WS3236 genome revealed the presence of 38 BGCs according to antiSMASH analysis, including a putative sodorifen BGC. Further genome mining for sodorifen and sodorifen-like BGCs throughout bacteria was performed using SodC and SodD as queries and identified a total of 28 sod-like gene clusters. Using direct pathway cloning (DiPaC) we intercepted the 4.6 kb candidate sodorifen BGC from S. plymuthica WS3236 (sodA-D) and transformed it into Escherichia coli BL21. Heterologous expression under the control of the tetracycline inducible PtetO promoter firmly linked this BGC to sodorifen production. By utilizing this newly established expression system, we increased the production yields by approximately 26-fold when compared to the native producer. In addition, sodorifen was easily isolated in high purity by simple head-space sampling., Conclusions: Genome mining of all available genomes within the NCBI and JGI IMG databases led to the identification of a wealth of sod-like pathways which may be responsible for producing a range of structurally unknown sodorifen analogs. Introduction of the S. plymuthica WS3236 sodorifen BGC into the fast-growing heterologous expression host E. coli with a very low VOC background led to a significant increase in both sodorifen product yield and purity compared to the native producer. By providing a reliable, high-level production system, this study sets the stage for future investigations of the biological role and function of sodorifen and for functionally unlocking the bioinformatically identified putative sod-like pathways.- Published
- 2019
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19. β-Pinene moderates Cr(VI) phytotoxicity by quenching reactive oxygen species and altering antioxidant machinery in maize.
- Author
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Mahajan P, Singh HP, Kaur S, Batish DR, and Kohli RK
- Subjects
- Antioxidants, Bicyclic Monoterpenes, Hydrogen Peroxide pharmacology, Oxidative Stress drug effects, Reactive Oxygen Species, Superoxides, Zea mays drug effects, Bridged Bicyclo Compounds metabolism, Chromium toxicity, Monoterpenes metabolism, Zea mays physiology
- Abstract
We examined the possible role of monoterpene β-pinene in providing protection against Cr(VI) toxicity in maize (Zea mays). Treatment with β-pinene (10 μM) significantly alleviated Cr(VI) accumulation and recuperated Cr(VI) caused decline in root and coleoptile growth in maize. β-Pinene addition caused a decline in Cr(VI)-induced accumulation of superoxide anion, hydroxyl ion, hydrogen peroxide and confirmed by in-situ detection of ROS using histochemical localization. It suggested that the β-pinene quenches/neutralizes enhanced ROS generated under Cr(VI) exposure. β-Pinene also reduced Cr(VI)-induced electrolyte leakage, thereby suggesting its role in membrane stabilization. Further, β-pinene regulated the activity of scavenging enzymes, thereby suggesting a role in modulating Cr(VI)-induced oxidative damage. In conclusion, our results suggest that the addition of β-pinene has a protective role against Cr(VI) stress and provides resistance to maize against Cr(VI) toxicity.
- Published
- 2019
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20. Interspecies interaction of Serratia plymuthica 4Rx13 and Bacillus subtilis B2g alters the emission of sodorifen.
- Author
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Kai M and Piechulla B
- Subjects
- Environmental Microbiology, Gas Chromatography-Mass Spectrometry, Rhizosphere, Solid Phase Microextraction, Bacillus subtilis physiology, Bridged Bicyclo Compounds metabolism, Microbial Interactions, Octanes metabolism, Serratia physiology, Volatile Organic Compounds metabolism
- Abstract
Sodorifen is the major volatile of Serratia plymuthica 4Rx13. It is assumed to be a long-distance communication signal. However, so far the emission patterns of sodorifen had been studied using mono-cultures of S. plymuthica 4Rx13 neglecting that in natura bacteria live in communities. Here, we show that the structured co-cultivation of S. plymuthica 4Rx13 and Bacillus subtilis B2g in a low-diversity model community grown under nutrient-rich conditions led to quantitative changes in sodorifen emission compared to self-paired mono-cultivations. Co-culturing revealed a decreased emission of sodorifen (50%) during exponential growth phase, whereas in the late stationary stage of growth, the amount of headspace sodorifen was increased compared to self-paired mono-cultivation (217% at 500 h of cultivation). Six other compounds that are most probably related to sodorifen or are isomers showed similar emission patterns. These data indicated that S. plymuthica 4Rx13 enhances its communication signal sodorifen as a consequence of interaction with B. subtilis B2g.
- Published
- 2018
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21. Cyanobacterial production of plant essential oils.
- Author
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Formighieri C and Melis A
- Subjects
- Abies enzymology, Abies genetics, Acyclic Monoterpenes, Bicyclic Monoterpenes, Bridged Bicyclo Compounds metabolism, Cyclohexane Monoterpenes, Gene Expression, Intramolecular Lyases genetics, Intramolecular Lyases metabolism, Lavandula enzymology, Lavandula genetics, Solanum lycopersicum enzymology, Solanum lycopersicum genetics, Metabolic Engineering, Photosynthesis, Picea enzymology, Picea genetics, Pinus enzymology, Pinus genetics, Plant Proteins genetics, Recombinant Fusion Proteins, Synechocystis genetics, Transgenes, Monoterpenes metabolism, Oils, Volatile metabolism, Plant Oils metabolism, Plant Proteins metabolism, Synechocystis metabolism
- Abstract
Main Conclusion: Synechocystis (a cyanobacterium) was employed as an alternative host for the production of plant essential oil constituents. β-Phellandrene synthase (PHLS) genes from different plants, when expressed in Synechocystis, enabled synthesis of variable monoterpene hydrocarbon blends, converting Synechocystis into a cell factory that photosynthesized and released useful products. Monoterpene synthases are secondary metabolism enzymes that catalyze the generation of essential oil constituents in terrestrial plants. Essential oils, including monoterpene hydrocarbons, are of interest for their commercial application and value. Therefore, heterologous expression of monoterpene synthases for high-capacity essential oil production in photosynthetic microorganism transformants is of current interest. In the present work, the cyanobacterium Synechocystsis PCC 6803 was employed as an alternative host for the production of plant essential oil constituents. As a case study, β-phellandrene synthase (PHLS) genes from different plants were heterologously expressed in Synechocystis. Genomic integration of individual PHLS-encoding sequences endowed Synechocystis with constitutive monoterpene hydrocarbons generation, occurring concomitant with photosynthesis and cell growth. Specifically, the β-phellandrene synthase from Lavandula angustifolia (lavender), Solanum lycopersicum (tomato), Pinus banksiana (pine), Picea sitchensis (Sitka spruce) and Abies grandis (grand fir) were active in Synechocystis transformants but, instead of a single product, they generated a blend of terpene hydrocarbons comprising β-phellandrene, α-phellandrene, β-myrcene, β-pinene, and δ-carene with variable percentage ratios ranging from < 10 to > 90% in different product combinations and proportions. Our results suggested that PHLS enzyme conformation and function depends on the cytosolic environment in which they reside, with the biochemical properties of the latter causing catalytic deviations from the products naturally observed in the corresponding gene-encoding plants, giving rise to the terpene hydrocarbon blends described in this work. These findings may have commercial application in the generation of designer essential oil blends and will further assist the development of heterologous cyanobacterial platforms for the generation of desired monoterpene hydrocarbon products.
- Published
- 2018
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22. α7 Nicotinic acetylcholine receptor contributes to the alleviation of lung ischemia-reperfusion injury by transient receptor potential vanilloid type 1 stimulation.
- Author
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Li X, Xu Y, Cheng Y, and Wang R
- Subjects
- Aconitine analogs & derivatives, Aconitine pharmacology, Animals, Apoptosis drug effects, Benzamides metabolism, Bridged Bicyclo Compounds metabolism, Capsaicin analogs & derivatives, Capsaicin therapeutic use, Disease Models, Animal, Down-Regulation, Humans, Lung drug effects, Lung metabolism, Lung pathology, Lung Injury pathology, Male, Mice, Mice, Knockout, Reperfusion Injury etiology, Reperfusion Injury pathology, TRPV Cation Channels agonists, TRPV Cation Channels antagonists & inhibitors, TRPV Cation Channels genetics, Treatment Outcome, alpha7 Nicotinic Acetylcholine Receptor agonists, alpha7 Nicotinic Acetylcholine Receptor antagonists & inhibitors, Capsaicin pharmacology, Lung Injury drug therapy, Reperfusion Injury drug therapy, TRPV Cation Channels metabolism, alpha7 Nicotinic Acetylcholine Receptor metabolism
- Abstract
Background: Activation of transient receptor potential vanilloid type 1 (TRPV1) decreases lung ischemia-reperfusion injury (LIRI) in rabbits and rats. Stimulation of α7 nicotinic acetylcholine receptors (α7nAChRs) protects against lung injury. Here we examined whether α7nAChRs contribute to TRPV1-mediated protection against LIRI., Methods: Wild-type (WT) and TRPV1-knockout (KO) mice were subjected to 1-h lung ischemia by clamping left hilum, followed by 2-h reperfusion. WT or KO mice were pretreated with vehicle, TRPV1 agonist capsaicin, TRPV1 antagonist capsazepine, α7nAChR antagonist methyllycaconitine, or α7nAChR agonist PNU-282987. Arterial blood and lung tissues were obtained for blood gas, lung wet-to-dry weight ratio, interleukin (IL)1β, IL6, tumor necrosis factor-α (TNF-α), apoptosis-related proteins (caspases, Bax, Fas), and pathologic scoring., Results: Capsaicin pretreatment reduced wet-to-dry ratio, pathologic score, alveolar-arterial oxygen gradient (A-aDO
2 ), and IL1β, IL6, and TNFα levels in WT mice, with no effects in KO mice. This reduction was reversed by TRPV1 blockade. Furthermore, α7nAChR blockade before capsaicin exacerbated LIRI as evidenced by enhanced alveolar-arterial oxygen gradient, pathologic score, and IL1β, IL6, and TNFα levels, while α7nAChR agonist pretreatment under TRPV1 blockade showed opposite changes. Capsaicin also decreased cleaved caspase-3, caspase-3/9, and Bax protein expression, effects abolished by TRPV1 blockade. Similarly, α7nAChR blockade diminished capsaicin-induced downregulation of apoptotic proteins, and α7nAChR activation decreased expression levels even under TRPV1 blockade., Conclusions: TRPV1 activation alleviates LIRI, partially dependent on α7nAChR activity. The α7nAChR stimulation with or without existence of TRPV1 alleviates LIRI. Thus, α7nAChR is involved in the pathway of TRPV1-mediated protection against LIRI and the specific mechanism remains to be revealed., (Copyright © 2018 Elsevier Inc. All rights reserved.)- Published
- 2018
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23. Sodorifen Biosynthesis in the Rhizobacterium Serratia plymuthica Involves Methylation and Cyclization of MEP-Derived Farnesyl Pyrophosphate by a SAM-Dependent C-Methyltransferase.
- Author
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von Reuss S, Domik D, Lemfack MC, Magnus N, Kai M, Weise T, and Piechulla B
- Subjects
- Bridged Bicyclo Compounds chemistry, Cyclization, Erythritol chemistry, Erythritol metabolism, Methylation, Molecular Structure, Octanes chemistry, Polyisoprenyl Phosphates chemistry, S-Adenosylmethionine chemistry, Serratia enzymology, Sesquiterpenes chemistry, Sugar Phosphates chemistry, Bridged Bicyclo Compounds metabolism, Erythritol analogs & derivatives, Methyltransferases metabolism, Octanes metabolism, Polyisoprenyl Phosphates metabolism, S-Adenosylmethionine metabolism, Serratia metabolism, Sesquiterpenes metabolism, Sugar Phosphates metabolism
- Abstract
The rhizobacterium Serratia plymuthica 4Rx13 releases a unique polymethylated hydrocarbon (C
16 H26 ) with a bicyclo[3.2.1]octadiene skeleton called sodorifen. Sodorifen production depends on a gene cluster carrying a C-methyltransferase and a terpene cyclase along with two enzymes of the 2- C-methyl-d-erythritol 4-phosphate (MEP) pathway of isoprenoid biosynthesis. Comparative analysis of wild-type and mutant volatile organic compound profiles revealed a C-methyltransferase-dependent C16 alcohol called pre-sodorifen, the production of which is upregulated in the terpene cyclase mutant. The monocyclic structure of this putative intermediate in sodorifen biosynthesis was identified by NMR spectroscopy. In vitro assays with the heterologously expressed S. plymuthica C-methyltransferase and terpene cyclase demonstrated that these enzymes act sequentially to convert farnesyl pyrophosphate (FPP) into sodorifen via a pre-sodorifen pyrophosphate intermediate, indicating that the S-adenosyl methionine (SAM)-dependent C-methyltransferase from S. plymuthica exhibits unprecedented cyclase activity. In vivo incorporation experiments with13 C-labeled succinate, l-alanine, and l-methionine confirmed a MEP pathway to FPP via the canonical glyceraldehyde-3-phosphate and pyruvate, as well as its SAM-dependent methylation in pre-sodorifen and sodorifen biosynthesis.13 C{1 H} NMR spectroscopy facilitated the localization of13 C labels and provided detailed insights into the biosynthetic pathway from FPP via pre-sodorifen pyrophosphate to sodorifen.- Published
- 2018
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24. Impact of allosteric modulation: Exploring the binding kinetics of glutamate and other orthosteric ligands of the metabotropic glutamate receptor 2.
- Author
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Doornbos MLJ, Vermond SC, Lavreysen H, Tresadern G, IJzerman AP, and Heitman LH
- Subjects
- Allosteric Regulation drug effects, Allosteric Regulation physiology, Amino Acids metabolism, Amino Acids pharmacology, Animals, Binding Sites drug effects, Binding Sites physiology, Bridged Bicyclo Compounds metabolism, Bridged Bicyclo Compounds pharmacology, CHO Cells, Cricetinae, Cricetulus, Dose-Response Relationship, Drug, Excitatory Amino Acid Agonists pharmacology, Excitatory Amino Acid Antagonists pharmacology, Glutamic Acid pharmacology, Ligands, Receptors, Metabotropic Glutamate agonists, Receptors, Metabotropic Glutamate antagonists & inhibitors, Xanthenes metabolism, Xanthenes pharmacology, Excitatory Amino Acid Agonists metabolism, Excitatory Amino Acid Antagonists metabolism, Glutamic Acid metabolism, Receptors, Metabotropic Glutamate metabolism
- Abstract
While many orthosteric ligands have been developed for the mGlu
2 receptor, little is known about their target binding kinetics and how these relate to those of the endogenous agonist glutamate. Here, the kinetic rate constants, i.e. kon and koff , of glutamate were determined for the first time followed by those of the synthetic agonist LY354740 and antagonist LY341495. To increase the understanding of the binding mechanism and impact of allosteric modulation thereon, kinetic experiments were repeated in the presence of allosteric modulators. Functional assays were performed to further study the interplay between the orthosteric and allosteric binding sites, including an impedance-based morphology assay. We found that dissociation rate constants of orthosteric mGlu2 ligands were all within a small 6-fold range, whereas association rate constants were ranging over more than three orders of magnitude and correlated to both affinity and potency. The latter showed that target engagement of orthosteric mGlu2 ligands is kon -driven in vitro. Moreover, only the off-rates of the two agonists were decreased by a positive allosteric modulator (PAM), thereby increasing their affinity. Interestingly, a PAM increased the duration of a glutamate-induced cellular response. A negative allosteric modulator (NAM) increased both on- and off-rate of glutamate without changing its affinity, while it did not affect these parameters for LY354740, indicating probe-dependency. In conclusion, we found that affinity- or potency-based orthosteric ligand optimization primarily results in ligands with high kon values. Moreover, positive allosteric modulators alter the binding kinetics of orthosteric agonists mainly by decreasing koff , which we were able to correlate to a lengthened cellular response. Together, this study shows the importance of studying binding kinetics in early drug discovery, as this may provide important insights towards improved efficacy in vivo., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2018
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25. Phytotoxic Activity of Metabolites Isolated from Rutstroemia sp.n. , the Causal Agent of Bleach Blonde Syndrome on Cheatgrass ( Bromus tectorum ).
- Author
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Masi M, Meyer S, Górecki M, Pescitelli G, Clement S, Cimmino A, and Evidente A
- Subjects
- Bridged Bicyclo Compounds chemistry, Bridged Bicyclo Compounds metabolism, Quinones chemistry, Quinones metabolism, Ascomycota chemistry, Ascomycota isolation & purification, Ascomycota metabolism, Bromus microbiology, Plant Diseases microbiology
- Abstract
A fungal pathogen soon to be described as Rutstroemia capillus-albis (Rutstroemiaceae, Helotiales, Leotiomycetes) has been identified as the causal agent of 'bleach blonde syndrome' on the invasive annual grass weed Bromus tectorum (cheatgrass) in western North America. This apparently common but previously undescribed disease causes premature senescence and sterility, but does not affect seed germination or seedling emergence and growth. This study investigated whether the new species produces phytotoxins that could be implicated in pathogenesis. The compounds 9- O -methylfusarubin, 9- O -methylbostrycoidin, 5- O -methylnectriafurone, trans -methyl- p -coumarate and terpestacin were isolated from the solid culture of this fungus. The undescribed absolute stereochemistry at C-3 of 9- O -methylfusarubin and at C-1' of 5- O -methylnectriafurone were assigned by applying electronic and vibrational circular dichroism (ECD and VCD) combined with computational methods and the advanced Mosher's method, respectively. The first three listed compounds are naphtoquinone pigments, while terpestacin is a sesterterpene, and trans -methyl- p -coumarate could be the product of an unusual fungal phenylpropanoid biosynthesis pathway. In a juvenile plant immersion bioassay, both 9- O -methylfusarubin and terpestacin proved to be highly toxic at 10
-4 M, causing wilting and plant death within 10 days. This finding suggests that these two compounds could play a role in pathogenesis on B. tectorum .- Published
- 2018
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26. The efficiency of 18 F labelling of a prostate specific membrane antigen ligand via strain-promoted azide-alkyne reaction: reaction speed versus hydrophilicity.
- Author
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Wang M, McNitt CD, Wang H, Ma X, Scarry SM, Wu Z, Popik VV, and Li Z
- Subjects
- Animals, Bridged Bicyclo Compounds chemical synthesis, Bridged Bicyclo Compounds chemistry, Bridged Bicyclo Compounds pharmacology, Cyclooctanes chemical synthesis, Cyclooctanes chemistry, Cyclooctanes pharmacology, Dipeptides metabolism, Fluorine Radioisotopes, Heterocyclic Compounds, 1-Ring metabolism, Humans, Hydrocarbons, Fluorinated chemical synthesis, Hydrocarbons, Fluorinated chemistry, Hydrocarbons, Fluorinated pharmacology, Hydrophobic and Hydrophilic Interactions, Kinetics, Ligands, Male, Mice, Positron-Emission Tomography, Prostate-Specific Antigen, Prostatic Neoplasms diagnostic imaging, Protein Binding, Radiopharmaceuticals chemical synthesis, Radiopharmaceuticals chemistry, Radiopharmaceuticals pharmacology, Xenograft Model Antitumor Assays, Alkynes chemistry, Antigens, Surface metabolism, Azides chemistry, Bridged Bicyclo Compounds metabolism, Cyclooctanes metabolism, Glutamate Carboxypeptidase II metabolism, Hydrocarbons, Fluorinated metabolism, Radiopharmaceuticals metabolism
- Abstract
Here we report the 18F labeling of a prostate specific membrane antigen (PSMA) ligand via a strain promoted oxa-dibenzocyclooctyne (ODIBO)- or bicyclo[6.1.0]nonyne (BCN)-azide reaction. Although ODIBO reacts with azide 20 fold faster than BCN, in vivo PET imaging suggests that 18F-BCN-azide-PSMA demonstrated much higher tumor uptake and a much higher tumor to background contrast.
- Published
- 2018
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27. Total Biosynthesis of Antiangiogenic Agent (-)-Terpestacin by Artificial Reconstitution of the Biosynthetic Machinery in Aspergillus oryzae.
- Author
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Narita K, Minami A, Ozaki T, Liu C, Kodama M, and Oikawa H
- Subjects
- Alkyl and Aryl Transferases genetics, Alkyl and Aryl Transferases metabolism, Angiogenesis Inhibitors chemistry, Aspergillus oryzae enzymology, Aspergillus oryzae genetics, Bridged Bicyclo Compounds chemistry, Bridged Bicyclo Compounds metabolism, Catalysis, Cytochrome P-450 Enzyme System genetics, Cytochrome P-450 Enzyme System metabolism, Genes, Fungal, Magnetic Resonance Spectroscopy methods, Mass Spectrometry methods, Molecular Structure, Oxidoreductases genetics, Oxidoreductases metabolism, Stereoisomerism, Angiogenesis Inhibitors biosynthesis, Aspergillus oryzae metabolism
- Abstract
The total biosynthesis of (-)-terpestacin was achieved by heterologous expression of four biosynthetic enzyme genes ( tpcA- D) in Aspergillus oryzae. After construction of preterpestacin I by the action of bifunctional terpene synthase (TpcA), two cytochrome P450s (TpcBC) activate inert C-H bond to install three hydroxyl groups on the A-ring in stereo- and regioselective manners. Subsequently, a flavin-dependent oxidase (TpcD) catalyzes oxidation of the vicinal diol moiety to give a α-diketone, which undergoes an enolization to furnish terpestacin. The successful synthesis of structurally elaborated terpestacin showed that a reconstitution approach that harnesses several biosynthetic enzyme genes in A. oryzae could be a promising alternative to the current chemical synthesis of natural terpenoids.
- Published
- 2018
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28. Phosphorylation-induced conformational changes of photoactivated rhodopsin probed by fluorescent labeling at Cys 140 and Cys 316 .
- Author
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Rodríguez S, Silva ML, Benaím G, and Bubis J
- Subjects
- Animals, Biofilms, Bridged Bicyclo Compounds chemistry, Bridged Bicyclo Compounds metabolism, Cattle, Cysteine chemistry, Fluorescence, Molecular Conformation, Phosphorylation physiology, Protein Conformation, Cysteine metabolism, Rhodopsin chemistry, Rhodopsin metabolism
- Abstract
In order to monitor conformational changes following photoactivation and phosphorylation of bovine rhodopsin, the two reactive sulfhydryl groups at Cys
140 and Cys316 were specifically labeled with the monobromobimane (mBBr) fluorophore. Although alterations in conformation after light exposure of rhodopsin were not detected by fluorescence excitation scans (300-450 nm) of the mBBr-labeled protein, the fluorescence signal was reduced ∼ 90% in samples containing photoactivated phosphorhodopsin. Predominant labeling at either Cys140 or Cys316 in light-activated and phosphorylated rhodopsin merely generated a decrease of ∼38% and 28%, respectively, in the fluorescence excitation intensity. Thus, neither mBBr-modified Cys140 nor mBBr-modified Cys316 were involved single-handedly in the remarkable fall seen on the signal following phosphorylation of the protein; rather, the incorporation of phosphate groups on the mBBr-labeled light-activated rhodopsin appeared to affect its fluorescence signal in a cooperative or synergistic manner. These findings demonstrated that the phosphorylation of specific hydroxyl groups at the carboxyl terminal tail of rhodopsin causes definite conformational changes in the three-dimensional fold of the protein. Apparently, amino acid residues that are buried in the interior of the inactive protein become accessible following illumination and phosphorylation of rhodopsin, quenching in turn the fluorescence excitation signal of mBBr-modified rhodopsin., (Copyright © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)- Published
- 2018
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29. Harnessing biosynthesis and quantitative NMR for late stage functionalization of lead molecules: Application to the M1 positive allosteric modulator (PAM) program.
- Author
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Brodney MA, Sharma R, Lazzaro JT, Walker GS, and Scott Obach R
- Subjects
- Bridged Bicyclo Compounds chemistry, Cyclohexanes chemistry, Dose-Response Relationship, Drug, Heterocyclic Compounds chemistry, Magnetic Resonance Spectroscopy, Microsomes, Liver chemistry, Microsomes, Liver metabolism, Molecular Structure, Structure-Activity Relationship, Bridged Bicyclo Compounds metabolism, Cyclohexanes metabolism, Heterocyclic Compounds metabolism
- Abstract
A facile method for late stage diversification of lead molecules for the M1 PAM program using biosynthesis is described. Liver microsomes from several species are screened to identify a high turnover system. Subsequent incubations using less than 1 mg of substrate generate nanomole quantities of drug metabolites that are purified, characterized by microcryoprobe NMR spectroscopy, and quantified to known concentrations to enable rapid biology testing. The late-stage diversification of lead compounds provides rapid SAR feedback to the medicinal chemistry design cycle., (Copyright © 2018 Elsevier Ltd. All rights reserved.)
- Published
- 2018
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30. Binding Characteristics and Analgesic Effects of Mirogabalin, a Novel Ligand for the α 2 δ Subunit of Voltage-Gated Calcium Channels.
- Author
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Domon Y, Arakawa N, Inoue T, Matsuda F, Takahashi M, Yamamura N, Kai K, and Kitano Y
- Subjects
- Analgesics adverse effects, Analgesics therapeutic use, Animals, Bridged Bicyclo Compounds adverse effects, Bridged Bicyclo Compounds therapeutic use, Diabetes Mellitus, Experimental drug therapy, HEK293 Cells, Humans, Ligands, Locomotion drug effects, Male, Protein Binding, Rats, Safety, Analgesics metabolism, Analgesics pharmacology, Bridged Bicyclo Compounds metabolism, Bridged Bicyclo Compounds pharmacology, NAV1.2 Voltage-Gated Sodium Channel metabolism
- Abstract
Mirogabalin ([(1 R ,5 S ,6 S )-6-(aminomethyl)-3-ethylbicyclo[3.2.0]hept-3-en-6-yl]acetic acid), a novel ligand for the α
2 δ subunit of voltage-gated calcium channels, is being developed to treat pain associated with diabetic peripheral neuropathy and postherpetic neuralgia. In the present study, we investigated the in vitro binding characteristics and in vivo analgesic effects of mirogabalin compared with those of pregabalin, a standard α2 δ ligand. Mirogabalin showed potent and selective binding affinities for the α2 δ subunits, while having no effects on 186 off-target proteins. Similar to pregabalin, mirogabalin did not show clear subtype selectivity ( α2 δ -1 vs. α2 δ -2) or species differences (human vs. rat). However, in contrast to pregabalin, mirogabalin showed greater binding affinities for human α2 δ -1, human α2 δ -2, rat α2 δ -1, and rat α2 δ -2 subunits; further, it had a slower dissociation rate for the α2 δ -1 subunit than the α2 δ -2 subunit. Additionally, in experimental neuropathic pain models, partial sciatic nerve ligation rats and streptozotocin-induced diabetic rats, mirogabalin showed more potent and longer lasting analgesic effects. In safety pharmacological evaluations, mirogabalin and pregabalin inhibited rota-rod performance and locomotor activity in rats; however, the safety indices of mirogabalin were superior to those of pregabalin. In conclusion, mirogabalin shows potent and selective binding affinities for the human and rat α2 δ subunits, and slower dissociation rates for the α2 δ -1 subunit than the α2 δ -2 subunit. It shows potent and long-lasting analgesic effects in rat models of neuropathic pain, and wider safety margins for side effects of the central nervous system. These properties of mirogabalin can be associated with its unique binding characteristics., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)- Published
- 2018
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31. Phenotypic Effects and Inhibition of Botrydial Biosynthesis Induced by Different Plant-Based Elicitors in Botrytis cinerea.
- Author
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Liñeiro E, Macias-Sánchez AJ, Espinazo M, Cantoral JM, Moraga J, Collado IG, and Fernández-Acero FJ
- Subjects
- Botrytis genetics, Fungal Proteins genetics, Fungal Proteins metabolism, Gene Expression Regulation, Fungal, Host-Pathogen Interactions, Aldehydes metabolism, Botrytis metabolism, Bridged Bicyclo Compounds metabolism, Solanum lycopersicum microbiology, Mycotoxins metabolism, Plant Diseases microbiology
- Abstract
Botrytis cinerea is considered a model organism for the study of plant-pathogen interaction showing great genetic diversity and a high degree of morphological variability depending on environmental conditions. The use of new compounds and plant-based elicitors may trigger the expression of different B. cinerea genes, providing new sources of virulence factors. This work is focused on elucidating the phenotypic effect in B. cinerea of different carbon sources such as glucose, cellulose and tomato cell walls (TCW). Production of botrydial and dihydrobotrydial toxins was evaluated using thin-layer chromatography (TLC), proton nuclear magnetic resonance spectroscopy (
1 H-NMR) and mass spectrometry (UPLC-HRESIMS). Expression of the toxin biosynthesis gene BcBOT2 was followed using RT-qPCR. Results show an inhibition of the toxin biosynthesis pathway when TCW are present as a sole carbon source, suggesting that the toxin is only produced when rich molecules, like glucose, are available for fungal metabolism. That suggests a connection between gene expression of virulence factors and environmental conditions, where the silent genes can be induced by different culture conditions.- Published
- 2018
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32. Spectroscopic profiling and computational study of the binding of tschimgine: A natural monoterpene derivative, with calf thymus DNA.
- Author
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Khajeh MA, Dehghan G, Dastmalchi S, Shaghaghi M, and Iranshahi M
- Subjects
- Animals, Binding, Competitive, Bisbenzimidazole chemistry, Bisbenzimidazole metabolism, Bridged Bicyclo Compounds chemistry, Cattle, Circular Dichroism, Hydrogen-Ion Concentration, Hydroxybenzoates chemistry, Methylene Blue chemistry, Methylene Blue metabolism, Monoterpenes chemistry, Osmolar Concentration, Spectrometry, Fluorescence, Spectrophotometry, Ultraviolet, Thermodynamics, Viscosity, Bridged Bicyclo Compounds metabolism, DNA metabolism, Hydroxybenzoates metabolism, Molecular Docking Simulation, Monoterpenes metabolism
- Abstract
DNA is a major target for a number of anticancer substances. Interaction studies between small molecules and DNA are essential for rational drug designing to influence main biological processes and also introducing new probes for the assay of DNA. Tschimgine (TMG) is a monoterpene derivative with anticancer properties. In the present study we tried to elucidate the interaction of TMG with calf thymus DNA (CT-DNA) using different spectroscopic methods. UV-visible absorption spectrophotometry, fluorescence and circular dichroism (CD) spectroscopies as well as molecular docking study revealed formation of complex between TMG and CT-DNA. Binding constant (K
b ) between TMG and DNA was 2.27×104 M-1 , that is comparable to groove binding agents. The fluorescence spectroscopic data revealed that the quenching mechanism of fluorescence of TMG by CT-DNA is static quenching. Thermodynamic parameters (ΔH<0 and ΔS<0) at different temperatures indicated that van der Waals forces and hydrogen bonds were involved in the binding process of TMG with CT-DNA. Competitive binding assay with methylene blue (MB) and Hoechst 33258 using fluorescence spectroscopy displayed that TMG possibly binds to the minor groove of CT-DNA. These observations were further confirmed by CD spectral analysis, viscosity measurements and molecular docking., (Copyright © 2017 Elsevier B.V. All rights reserved.)- Published
- 2018
- Full Text
- View/download PDF
33. Novel Bacterial Topoisomerase Inhibitors Exploit Asp83 and the Intrinsic Flexibility of the DNA Gyrase Binding Site.
- Author
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Franco-Ulloa S, La Sala G, Miscione GP, and De Vivo M
- Subjects
- Amino Acid Motifs, Anti-Bacterial Agents metabolism, Aspartic Acid metabolism, Binding Sites, Bridged Bicyclo Compounds metabolism, DNA Gyrase genetics, DNA Gyrase metabolism, Escherichia coli chemistry, Escherichia coli enzymology, Gene Expression, Hydrogen Bonding, Molecular Docking Simulation, Molecular Dynamics Simulation, Protein Binding, Protein Conformation, alpha-Helical, Protein Conformation, beta-Strand, Protein Interaction Domains and Motifs, Protein Multimerization, Protein Subunits antagonists & inhibitors, Protein Subunits genetics, Protein Subunits metabolism, Staphylococcus aureus enzymology, Topoisomerase Inhibitors metabolism, Anti-Bacterial Agents chemistry, Aspartic Acid chemistry, Bridged Bicyclo Compounds chemistry, DNA Gyrase chemistry, Protein Subunits chemistry, Staphylococcus aureus chemistry, Topoisomerase Inhibitors chemistry
- Abstract
DNA gyrases are enzymes that control the topology of DNA in bacteria cells. This is a vital function for bacteria. For this reason, DNA gyrases are targeted by widely used antibiotics such as quinolones. Recently, structural and biochemical investigations identified a new class of DNA gyrase inhibitors called NBTIs (i.e., novel bacterial topoisomerase inhibitors). NBTIs are particularly promising because they are active against multi-drug resistant bacteria, an alarming clinical issue. Structural data recently demonstrated that these NBTIs bind tightly to a newly identified pocket at the dimer interface of the DNA-protein complex. In the present study, we used molecular dynamics (MD) simulations and docking calculations to shed new light on the binding of NBTIs to this site. Interestingly, our MD simulations demonstrate the intrinsic flexibility of this binding site, which allows the pocket to adapt its conformation and form optimal interactions with the ligand. In particular, we examined two ligands, AM8085 and AM8191, which induced a repositioning of a key aspartate (Asp83B), whose side chain can rotate within the binding site. The conformational rearrangement of Asp83B allows the formation of a newly identified H-bond interaction with an NH on the bound NBTI, which seems important for the binding of NBTIs having such functionality. We validated these findings through docking calculations using an extended set of cognate oxabicyclooctane-linked NBTIs derivatives (~150, in total), screened against multiple target conformations. The newly identified H-bond interaction significantly improves the docking enrichment. These insights could be helpful for future virtual screening campaigns against DNA gyrase., Competing Interests: The authors declare no conflicts of interest.
- Published
- 2018
- Full Text
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34. A2A Adenosine Receptor Antagonists as Therapeutic Candidates: Are They Still an Interesting Challenge?
- Author
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Cacciari B, Spalluto G, and Federico S
- Subjects
- Adenosine A2 Receptor Antagonists chemistry, Bridged Bicyclo Compounds chemistry, Bridged Bicyclo Compounds metabolism, Bridged Bicyclo Compounds therapeutic use, Caffeine chemistry, Caffeine metabolism, Caffeine therapeutic use, Dopamine Agents chemistry, Dopamine Agents metabolism, Dopamine Agents therapeutic use, Humans, Neoplasms drug therapy, Neoplasms pathology, Parkinson Disease pathology, Receptor, Adenosine A2A chemistry, Xanthines chemistry, Xanthines metabolism, Xanthines therapeutic use, Adenosine A2 Receptor Antagonists therapeutic use, Parkinson Disease drug therapy, Receptor, Adenosine A2A metabolism
- Abstract
In the past decades, many efforts were undertaken to develop ligands for the adenosine receptors, with the purpose to individuate agonists and antagonists affinity and selectivity for each subtypes, namely A1, A2A, A2B, and A3. These intense studies allowed a deeper knowledge of the nature and, moreover, of the pathophysiological roles of all the adenosine receptor subtypes. In particular, the involvement of the A2A adenosine receptor subtype in some physiological mechanisms in the brain, that could be related to important diseases such as the Parkinson's disease, encouraged the research in this field. Particular attention was given to the antagonists endowed with high affinity and selectivity since they could have a real employment in the treatment of Parkinson's disease, and some compounds, such as istradefylline, preladenant and tozadenant, are already studied in clinical trials. Actually, the role of A2A antagonists in Parkinson's disease is becoming contradictory due to contrasting results in the last studies, but, at the same time, new possible employments are emerging for this class of antagonists in cancer pathologies as much interesting to legitimate further efforts in the research of A2A ligands., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)
- Published
- 2018
- Full Text
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35. Structural Transformation Detection Contributes to Screening of Behaviorally Active Compounds: Dynamic Binding Process Analysis of DhelOBP21 from Dastarcus helophoroides.
- Author
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Yang RN, Li DZ, Yu G, Yi SC, Zhang Y, Kong DX, and Wang MQ
- Subjects
- Animals, Behavior, Animal drug effects, Bicyclic Monoterpenes, Binding Sites, Bridged Bicyclo Compounds chemistry, Bridged Bicyclo Compounds metabolism, Bridged Bicyclo Compounds pharmacology, Circular Dichroism, Insect Proteins chemistry, Insect Proteins genetics, Molecular Dynamics Simulation, Monoterpenes chemistry, Monoterpenes metabolism, Monoterpenes pharmacology, Polycyclic Sesquiterpenes, RNA Interference, RNA, Double-Stranded metabolism, Receptors, Odorant chemistry, Receptors, Odorant genetics, Recombinant Proteins biosynthesis, Recombinant Proteins chemistry, Recombinant Proteins isolation & purification, Sesquiterpenes chemistry, Sesquiterpenes metabolism, Sesquiterpenes pharmacology, Spectrometry, Fluorescence, Coleoptera metabolism, Insect Proteins metabolism, Receptors, Odorant metabolism
- Abstract
In light of reverse chemical ecology, the fluorescence competitive binding assays of functional odorant binding proteins (OBPs) is a recent advanced approach for screening behaviorally active compounds of insects. Previous research on Dastareus helophoroides identified a minus-C OBP, DhelOBP21, which preferably binds to several ligands. In this study, only (+)-β-pinene proved attractive to unmated adult beetles. To obtain a more in-depth explanation of the lack of behavioral activity of other ligands we selected compounds with high (camphor) and low (β-caryophyllene) binding affinities. The structural transformation of OBPs was investigated using well-established approaches for studying binding processes, such as fluorescent quenching assays, circular dichroism, and molecular dynamics. The dynamic binding process revealed that the flexibility of DhelOBP21 seems conducive to binding specific ligands, as opposed to broad substrate binding. The compound (+)-β-pinene and DhelOBP21 formed a stable complex through a secondary structural transformation of DhelOBP21, in which its amino-terminus transformed from random coil to an α-helix to cover the binding pocket. On the other hand, camphor could not efficiently induce a stable structural transformation, and its high binding affinities were due to strong hydrogen-bonding, compromising the structure of the protein. The other compound, β-caryophyllene, only collided with DhelOBP21 and could not be positioned in the binding pocket. Studying structural transformation of these proteins through examining the dynamic binding process rather than using approaches that just measure binding affinities such as fluorescence competitive binding assays can provide a more efficient and reliable approach for screening behaviorally active compounds.
- Published
- 2017
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36. Design and synthesis of a novel series of (1'S,2R,4'S)-3H-4'-azaspiro[benzo[4,5]imidazo[2,1-b]oxazole-2,2'-bicyclo[2.2.2]octanes] with high affinity for the α7 neuronal nicotinic receptor.
- Author
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Cook J, Zusi FC, Hill MD, Fang H, Pearce B, Park H, Gallagher L, McDonald IM, Bristow L, Macor JE, and Olson RE
- Subjects
- Bridged Bicyclo Compounds chemical synthesis, Bridged Bicyclo Compounds chemistry, Crystallography, X-Ray, Humans, Hydrogen Bonding, Molecular Conformation, Nicotinic Agonists chemistry, Nicotinic Agonists metabolism, Octanes chemical synthesis, Octanes chemistry, Protein Binding, Receptors, Serotonin, 5-HT3 chemistry, Receptors, Serotonin, 5-HT3 metabolism, Stereoisomerism, alpha7 Nicotinic Acetylcholine Receptor chemistry, Bridged Bicyclo Compounds metabolism, Drug Design, Octanes metabolism, alpha7 Nicotinic Acetylcholine Receptor metabolism
- Abstract
We describe an efficient and convergent synthesis of a series of (1'S,2R,4'S)-3H-4'-azaspiro[benzo[4,5]imidazo[2,1-b]oxazole-2,2'-bicyclo[2.2.2]octanes] displaying potency for the α7 nicotinic acetylcholine receptor (nAChR) and good selectivity vs. the related 5-HT
3A receptor., (Copyright © 2017 Elsevier Ltd. All rights reserved.)- Published
- 2017
- Full Text
- View/download PDF
37. Dissipation of the Herbicide Benzobicyclon Hydrolysate in a Model California Rice Field Soil.
- Author
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Williams KL, Gladfelder JJ, Quigley LL, Ball DB, and Tjeerdema RS
- Subjects
- Bacteria metabolism, Biodegradation, Environmental, Bridged Bicyclo Compounds metabolism, California, Herbicides metabolism, Kinetics, Molecular Structure, Soil Microbiology, Soil Pollutants chemistry, Soil Pollutants metabolism, Sulfones metabolism, Bridged Bicyclo Compounds chemistry, Herbicides chemistry, Oryza growth & development, Soil chemistry, Sulfones chemistry
- Abstract
The herbicide benzobicyclon (BZB; 3-(2-chloro-4-(methylsulfonyl)benzoyl)-2-phenylthiobicyclo[3.2.1]oct-2-en-4-one) has recently been approved for use on California rice fields by the United States Environmental Protection Agency (U.S. EPA). Hydrolysis of BZB rapidly forms the active compound, benzobicyclon hydrolysate (BH), whose fate is currently not well understood. A model California rice soil was used to determine BH soil dissipation. The pK
a and aqueous solubility were also determined, as experimental values are not currently available. Sorption data indicate BH does not bind tightly, or irreversibly, with this soil. Flooding resulted in decreased BH loss, indicating anaerobic microbes are less likely to transform BH compared to aerobic microorganisms. Temperature increased dissipation, while autoclaving decreased BH loss. Overall, dissipation was slow regardless of treatment. Further investigation is needed to elucidate the exact routes of loss in soil, though BH is expected to dissipate slowly in flooded rice field soil.- Published
- 2017
- Full Text
- View/download PDF
38. Enzymatic Synthesis of 7',5'-Bicyclo-DNA Oligonucleotides.
- Author
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Diafa S, Evéquoz D, Leumann CJ, and Hollenstein M
- Subjects
- Bridged Bicyclo Compounds chemistry, DNA chemistry, DNA-Directed DNA Polymerase chemistry, Molecular Conformation, Oligonucleotides chemistry, Bridged Bicyclo Compounds metabolism, DNA biosynthesis, DNA-Directed DNA Polymerase metabolism, Oligonucleotides biosynthesis
- Abstract
The selection of artificial genetic polymers with tailor-made properties for their application in synthetic biology requires the exploration of new nucleosidic scaffolds that can be used in selection experiments. Herein, we describe the synthesis of a bicyclo-DNA triphosphate (i.e., 7',5'-bc-TTP) and show its potential to serve for the generation of new xenonucleic acids (XNAs) based on this scaffold. 7',5'-bc-TTP is a good substrate for Therminator DNA polymerase, and up to seven modified units can be incorporated into a growing DNA chain. In addition, this scaffold sustains XNA-dependent DNA synthesis and potentially also XNA-dependent XNA synthesis. However, DNA-dependent XNA synthesis on longer templates is hampered by competitive misincorporation of deoxyadenosine triphosphate (dATP) caused by the slow rate of incorporation of 7',5'-bc-TTP., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2017
- Full Text
- View/download PDF
39. Isoprenoids Production from Lipid-Extracted Microalgal Biomass Residues Using Engineered E. coli.
- Author
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Wang S and Yang J
- Subjects
- Acetic Acid isolation & purification, Bicyclic Monoterpenes, Biofuels, Biomass, Bridged Bicyclo Compounds isolation & purification, Butadienes isolation & purification, Cellulose metabolism, Escherichia coli genetics, Fermentation, Furaldehyde analogs & derivatives, Furaldehyde isolation & purification, Glucose metabolism, Hemiterpenes isolation & purification, Hydrolysis, Kinetics, Monoterpenes isolation & purification, Pentanes isolation & purification, Sulfuric Acids chemistry, Bridged Bicyclo Compounds metabolism, Escherichia coli metabolism, Genetic Engineering methods, Hemiterpenes biosynthesis, Microalgae chemistry, Monoterpenes metabolism
- Abstract
Microalgae are recognized as a third generation feedstock for biofuel production due to their rapid growth rates and lignin-free characteristics. In this study, a lipid extracted microalgal biomass residues was used as the raw material to produce isoprene, α-pinene and β-pinene with an engineered E. coli strain. We adopted an optimal sulfuric acid hydrolysis method (1:7 ratio of solid to acid solution, 32% ( w/v ) concentration of sulfuric acid solution at 90 °C for 90 min) to efficiently convert holocellulose into glucose efficiently (6.37 g/L). Futhermore, we explored a novel detoxification strategy (phosphoric acid/calcium hydroxide) to remove inhibitors and notably acetic acid, furfural and 5-hydroxymethylfurfural (5-HMF) were reduced by 5.32%, different number given later 99.19% and 98.22%, respectively. Finally, the fermentation concentrations of isoprene (223.23 mg/L), α-pinene (382.21 μg/L) and β-pinene (17.4 mg/L) were achieved using the detoxified hydrolysate as the carbon source, equivalent to approximately 86.02%, 90.16% and 88.32% of those produced by the engineered E. coli strain fermented on pure glucose, respectively., Competing Interests: The authors declare no conflict of interest.
- Published
- 2017
- Full Text
- View/download PDF
40. Tissue-Specific Emission of (E)-α-Bergamotene Helps Resolve the Dilemma When Pollinators Are Also Herbivores.
- Author
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Zhou W, Kügler A, McGale E, Haverkamp A, Knaden M, Guo H, Beran F, Yon F, Li R, Lackus N, Köllner TG, Bing J, Schuman MC, Hansson BS, Kessler D, Baldwin IT, and Xu S
- Subjects
- Alkyl and Aryl Transferases metabolism, Animals, Flowers chemistry, Flowers cytology, Flowers physiology, Organ Specificity, Plant Leaves chemistry, Plant Leaves cytology, Nicotiana cytology, Bridged Bicyclo Compounds metabolism, Herbivory, Manduca physiology, Plant Leaves physiology, Pollination, Nicotiana chemistry, Nicotiana physiology
- Abstract
More than 87% of flowering plant species are animal-pollinated [1] and produce floral scents and other signals to attract pollinators. These floral cues may however also attract antagonistic visitors, including herbivores [2]. The dilemma is exacerbated when adult insects pollinate the same plant that their larvae consume. It remains largely unclear how plants maximize their fitness under these circumstances. Here we show that in the night-flowering wild tobacco Nicotiana attenuata, the emission of a sesquiterpene, (E)-α-bergamotene, in flowers increases adult Manduca sexta moth-mediated pollination success, while the same compound in leaves is known to mediate indirect defense against M. sexta larvae [3, 4]. Forward and reverse genetic analyses demonstrated that both herbivory-induced and floral (E)-α-bergamotene are regulated by the expression of a monoterpene-synthase-derived sesquiterpene synthase (NaTPS38). The expression pattern of NaTPS38 also accounts for variation in (E)-α-bergamotene emission among natural accessions. These results highlight that differential expression of a single gene that results in tissue-specific emission of one compound contributes to resolving the dilemma for plants when their pollinators are also herbivores. Furthermore, this study provides genetic evidence that pollinators and herbivores interactively shape the evolution of floral signals and plant defense., (Copyright © 2017 Elsevier Ltd. All rights reserved.)
- Published
- 2017
- Full Text
- View/download PDF
41. Fungal volatile compounds induce production of the secondary metabolite Sodorifen in Serratia plymuthica PRI-2C.
- Author
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Schmidt R, Jager V, Zühlke D, Wolff C, Bernhardt J, Cankar K, Beekwilder J, Ijcken WV, Sleutels F, Boer W, Riedel K, and Garbeva P
- Subjects
- Bacterial Proteins genetics, Energy Metabolism drug effects, Fungal Proteins pharmacology, Gene Expression Profiling, Gene Expression Regulation, Bacterial drug effects, Metabolome drug effects, Secondary Metabolism drug effects, Sequence Analysis, DNA methods, Serratia drug effects, Serratia genetics, Serratia metabolism, Bridged Bicyclo Compounds metabolism, Fusarium chemistry, Octanes metabolism, Serratia growth & development, Volatile Organic Compounds pharmacology
- Abstract
The ability of bacteria and fungi to communicate with each other is a remarkable aspect of the microbial world. It is recognized that volatile organic compounds (VOCs) act as communication signals, however the molecular responses by bacteria to fungal VOCs remain unknown. Here we perform transcriptomics and proteomics analyses of Serratia plymuthica PRI-2C exposed to VOCs emitted by the fungal pathogen Fusarium culmorum. We find that the bacterium responds to fungal VOCs with changes in gene and protein expression related to motility, signal transduction, energy metabolism, cell envelope biogenesis, and secondary metabolite production. Metabolomic analysis of the bacterium exposed to the fungal VOCs, gene cluster comparison, and heterologous co-expression of a terpene synthase and a methyltransferase revealed the production of the unusual terpene sodorifen in response to fungal VOCs. These results strongly suggest that VOCs are not only a metabolic waste but important compounds in the long-distance communication between fungi and bacteria.
- Published
- 2017
- Full Text
- View/download PDF
42. Translational Pharmacology of the Metabotropic Glutamate 2 Receptor-Preferring Agonist LY2812223 in the Animal and Human Brain.
- Author
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Felder CC, Schober DA, Tu Y, Quets A, Xiao H, Watt M, Siuda E, Nisenbaum E, Xiang C, Heinz B, Prieto L, McKinzie DL, and Monn JA
- Subjects
- Animals, Brain metabolism, Bridged Bicyclo Compounds metabolism, Dose-Response Relationship, Drug, Excitatory Amino Acid Agonists metabolism, Humans, Mice, Mice, Knockout, Protein Binding drug effects, Protein Binding physiology, Rats, Rats, Sprague-Dawley, Receptors, Metabotropic Glutamate metabolism, Translational Research, Biomedical, Triazoles metabolism, Brain drug effects, Bridged Bicyclo Compounds pharmacology, Drug Partial Agonism, Excitatory Amino Acid Agonists pharmacology, Receptors, Metabotropic Glutamate agonists, Triazoles pharmacology
- Abstract
LY2812223 [(1 R ,2 S ,4 R ,5 R ,6 R )-2-amino-4-(1 H -1,2,4-triazol-3-ylsulfanyl)bicyclo[3.1.0]hexane-2,6-dicarboxylic acid] was identified via structure-activity studies arising from the potent metabotropic glutamate mGlu2/3 receptor agonist LY354740 [(+)-2-aminobicyclo[3.1.0] hexane-2,6-dicarboxylic acid] as an mGlu2-preferring agonist. This pharmacology was determined using stably transfected cells containing either the human mGlu2 or mGlu3 receptor. We extended the pharmacological evaluation of LY2812223 to native brain tissues derived from relevant species used for preclinical drug development as well as human postmortem brain tissue. This analysis was conducted to ensure pharmacological translation from animals to human subjects in subsequent clinical studies. A guanosine 5'- O -(3-[
35 S]thio)triphosphate (GTP γ S) functional binding assay, a method for measuring Gi -coupled signaling that is inherent to the group 2 mGlu receptors, was used to evaluate LY2812223 pharmacology of native mGlu receptors in mouse, rat, nonhuman primate, and human cortical brain tissue samples. In native tissue membranes, LY2812223 unexpectedly acted as a partial agonist across all species tested. Activity of LY2812223 was lost in cortical membranes collected from mGlu2 knockout mice, but not those from mGlu3 knockout mice, providing additional support for mGlu2-preferring activity. Other signal transduction assays were used for comparison with the GTP binding assay (cAMP, calcium mobilization, and dynamic mass redistribution). In ectopic cell line-based assays, LY2812223 displayed near maximal agonist responses at the mGlu2 receptor across all assay formats, while it showed no functional agonist activity at the mGlu3 receptor except in the cAMP assay. In native brain slices or membranes that express both mGlu2 and mGlu3 receptors, LY2812223 displayed unexpected partial agonist activity, which may suggest a functional interplay between these receptor subtypes in the brain., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)- Published
- 2017
- Full Text
- View/download PDF
43. Reprint of Pharmacological and molecular characterization of the positive allosteric modulators of metabotropic glutamate receptor 2.
- Author
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Lundström L, Bissantz C, Beck J, Dellenbach M, Woltering TJ, Wichmann J, and Gatti S
- Subjects
- Allosteric Regulation drug effects, Allosteric Regulation physiology, Animals, Binding Sites physiology, Bridged Bicyclo Compounds chemistry, Bridged Bicyclo Compounds metabolism, Bridged Bicyclo Compounds pharmacology, Excitatory Amino Acid Agonists chemistry, Glutamic Acid metabolism, Glutamic Acid pharmacology, Humans, Protein Structure, Secondary, Excitatory Amino Acid Agonists metabolism, Excitatory Amino Acid Agonists pharmacology, Receptors, Metabotropic Glutamate agonists, Receptors, Metabotropic Glutamate physiology
- Abstract
The metabotropic glutamate receptor 2 (mGlu
2 ) plays an important role in the presynaptic control of glutamate release and several mGlu2 positive allosteric modulators (PAMs) have been under assessment for their potential as antipsychotics. The binding mode of mGlu2 PAMs is better characterized in functional terms while few data are available on the relationship between allosteric and orthosteric binding sites. Pharmacological studies characterizing binding and effects of two different chemical series of mGlu2 PAMs are therefore carried out here using the radiolabeled mGlu2 agonist3 [H]-LY354740 and mGlu2 PAM3 [H]-2,2,2-TEMPS. A multidimensional approach to the PAM mechanism of action shows that mGlu2 PAMs increase the affinity of3 [H]-LY354740 for the orthosteric site of mGlu2 as well as the number of3 [H]-LY354740 binding sites.3 [H]-2,2,2-TEMPS binding is also enhanced by the presence of LY354740. New residues in the allosteric rat mGlu2 binding pocket are identified to be crucial for the PAMs ligand binding, among these Tyr3.40 and Asn5.46 . Also of remark, in the described experimental conditions S731A (Ser5.42 ) residue is important only for the mGlu2 PAM LY487379 and not for the compound PAM-1: an example of the structural differences among these mGlu2 PAMs. This study provides a summary of the information generated in the past decade on mGlu2 PAMs adding a detailed molecular investigation of PAM binding mode. Differences among mGlu2 PAM compounds are discussed as well as the mGlu2 regions interacting with mGlu2 PAM and NAM agents and residues driving mGlu2 PAM selectivity. This article is part of the Special Issue entitled 'Metabotropic Glutamate Receptors, 5 years on'., (Copyright © 2016 Elsevier Ltd. All rights reserved.)- Published
- 2017
- Full Text
- View/download PDF
44. Efficient Liver Targeting by Polyvalent Display of a Compact Ligand for the Asialoglycoprotein Receptor.
- Author
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Sanhueza CA, Baksh MM, Thuma B, Roy MD, Dutta S, Préville C, Chrunyk BA, Beaumont K, Dullea R, Ammirati M, Liu S, Gebhard D, Finley JE, Salatto CT, King-Ahmad A, Stock I, Atkinson K, Reidich B, Lin W, Kumar R, Tu M, Menhaji-Klotz E, Price DA, Liras S, Finn MG, and Mascitti V
- Subjects
- Bridged Bicyclo Compounds metabolism, Crystallography, X-Ray, Drug Carriers chemistry, Humans, Ketones metabolism, Liver cytology, Models, Molecular, Molecular Structure, Polymers metabolism, Asialoglycoprotein Receptor metabolism, Bridged Bicyclo Compounds chemistry, Hepatocytes metabolism, Ketones chemistry, Liver metabolism, Polymers chemistry
- Abstract
A compact and stable bicyclic bridged ketal was developed as a ligand for the asialoglycoprotein receptor (ASGPR). This compound showed excellent ligand efficiency, and the molecular details of binding were revealed by the first X-ray crystal structures of ligand-bound ASGPR. This analogue was used to make potent di- and trivalent binders of ASGPR. Extensive characterization of the function of these compounds showed rapid ASGPR-dependent cellular uptake in vitro and high levels of liver/plasma selectivity in vivo. Assessment of the biodistribution in rodents of a prototypical Alexa647-labeled trivalent conjugate showed selective hepatocyte targeting with no detectable distribution in nonparenchymal cells. This molecule also exhibited increased ASGPR-directed hepatocellular uptake and prolonged retention compared to a similar GalNAc derived trimer conjugate. Selective release in the liver of a passively permeable small-molecule cargo was achieved by retro-Diels-Alder cleavage of an oxanorbornadiene linkage, presumably upon encountering intracellular thiol. Therefore, the multicomponent construct described here represents a highly efficient delivery vehicle to hepatocytes.
- Published
- 2017
- Full Text
- View/download PDF
45. Biotransformation of α- and β-pinene into flavor compounds.
- Author
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Vespermann KA, Paulino BN, Barcelos MC, Pessôa MG, Pastore GM, and Molina G
- Subjects
- Bicyclic Monoterpenes, Biotransformation, Food Additives chemistry, Limonene, Odorants, Oils, Volatile chemistry, Aspergillus metabolism, Bacteria metabolism, Bridged Bicyclo Compounds metabolism, Cyclohexenes metabolism, Monoterpenes metabolism, Penicillium metabolism, Terpenes metabolism
- Abstract
Products that bear the label "natural" have gained more attention in the marketplace. In this approach, the production of aroma compounds through biotransformation or bioconversion has been receiving more incentives in economic and research fields. Among the substrates used in these processes, terpenes can be highlighted for their versatility and low cost; some examples are limonene, α-pinene, and β-pinene. This work focused on the biotransformation of the two bicyclic monoterpenes, α-pinene and β-pinene; the use of different biocatalysts; the products obtained; and the conditions employed in the process.
- Published
- 2017
- Full Text
- View/download PDF
46. Biological properties of Hertia cheirifolia L. flower extracts and effect of the nopol on α-glucosidase.
- Author
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Majouli K, Mahjoub MA, Rahim F, Hamdi A, Wadood A, Besbes Hlila M, and Kenani A
- Subjects
- Bridged Bicyclo Compounds chemistry, Bridged Bicyclo Compounds metabolism, Glycoside Hydrolase Inhibitors chemistry, Glycoside Hydrolase Inhibitors metabolism, Molecular Docking Simulation, Plant Extracts chemistry, Plant Extracts metabolism, Protein Conformation, alpha-Glucosidases chemistry, Asteraceae chemistry, Bridged Bicyclo Compounds pharmacology, Flowers chemistry, Glycoside Hydrolase Inhibitors pharmacology, Plant Extracts pharmacology, alpha-Glucosidases metabolism
- Abstract
In screening for antioxidant and α-glucosidase inhibitors from the extracts of Hertia cheirifolia L. flowers, the petroleum ether extract showed interesting antioxidant activity and inhibitory effect on the activity of α-glucosidase. The fractionation of this extract resulted in the isolation of a compound which is characterized by NMR and ESI-MS as a nopol. The nopol exhibited potent α-glucosidase inhibitory potential with IC
50 value of 220μM. The kinetic evaluation indicated that it acts as a non-competitive inhibitor. A molecular docking study proved that the nopol presented a strong affinity with amino acid residues of α-glucosidase., (Copyright © 2016 Elsevier B.V. All rights reserved.)- Published
- 2017
- Full Text
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47. The botrydial biosynthetic gene cluster of Botrytis cinerea displays a bipartite genomic structure and is positively regulated by the putative Zn(II) 2 Cys 6 transcription factor BcBot6.
- Author
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Porquier A, Morgant G, Moraga J, Dalmais B, Luyten I, Simon A, Pradier JM, Amselem J, Collado IG, and Viaud M
- Subjects
- AT Rich Sequence, Botrytis metabolism, Botrytis pathogenicity, DNA Transposable Elements, DNA, Fungal, Fungal Proteins genetics, Nuclear Proteins genetics, Nuclear Proteins metabolism, Virulence, Aldehydes metabolism, Botrytis genetics, Bridged Bicyclo Compounds metabolism, Fungal Proteins metabolism, Gene Expression Regulation, Fungal, Multigene Family, Transcription Factors metabolism
- Abstract
Botrydial (BOT) is a non-host specific phytotoxin produced by the polyphagous phytopathogenic fungus Botrytis cinerea. The genomic region of the BOT biosynthetic gene cluster was investigated and revealed two additional genes named Bcbot6 and Bcbot7. Analysis revealed that the G+C/A+T-equilibrated regions that contain the Bcbot genes alternate with A+T-rich regions made of relics of transposable elements that have undergone repeat-induced point mutations (RIP). Furthermore, BcBot6, a Zn(II)
2 Cys6 putative transcription factor was identified as a nuclear protein and the major positive regulator of BOT biosynthesis. In addition, the phenotype of the ΔBcbot6 mutant indicated that BcBot6 and therefore BOT are dispensable for the development, pathogenicity and response to abiotic stresses in the B. cinerea strain B05.10. Finally, our data revealed that B. pseudocinerea, that is also polyphagous and lives in sympatry with B. cinerea, lacks the ability to produce BOT. Identification of BcBot6 as the major regulator of BOT synthesis is the first step towards a comprehensive understanding of the complete regulation network of BOT synthesis and of its ecological role in the B. cinerea life cycle., (Copyright © 2016 Elsevier Inc. All rights reserved.)- Published
- 2016
- Full Text
- View/download PDF
48. Selective activation of α7 nicotinic acetylcholine receptors augments hippocampal oscillations.
- Author
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Stoiljkovic M, Kelley C, Nagy D, Leventhal L, and Hajós M
- Subjects
- Animals, Benzamides metabolism, Benzamides pharmacology, Bridged Bicyclo Compounds metabolism, Bridged Bicyclo Compounds pharmacology, Hippocampus drug effects, Male, Mice, Mice, Knockout, Nicotinic Agonists pharmacology, Protein Binding physiology, Quinuclidines metabolism, Quinuclidines pharmacology, Theta Rhythm drug effects, Thiophenes metabolism, Thiophenes pharmacology, Hippocampus physiology, Nicotinic Agonists metabolism, Theta Rhythm physiology, alpha7 Nicotinic Acetylcholine Receptor agonists, alpha7 Nicotinic Acetylcholine Receptor metabolism
- Abstract
Neural α7 nicotinic acetylcholine receptors (α7 nAChRs) emerged as a potential pharmacologic target for treating cognitive deficits in schizophrenia and Alzheimer's disease. Experiments modeling these dysfunctions, as well as clinical evidence, demonstrate the relatively consistent procognitive effects of α7 nAChR agonists. One preclinical observation supporting the procognitive role of α7 nAChRs is their ability to modulate neuronal network oscillations closely associated with learning and memory, especially hippocampal oscillations. Due to the high degree of structural similarity between α7 nACh and 5-HT receptors, the majority of α7 nAChR agonists to date also act as 5-HT3 antagonists. To address this confounding property and determine the relevance of α7 nAChR agonist binding to 5-HT3 receptors in modulating hippocampal activity, we tested two well-described α7 nAChR agonists, PNU-282987 and FRM-17874, in mice lacking α7 nAChRs (α7 knock-out, α7KO) using the brainstem simulation-elicited hippocampal theta oscillation assay. Under urethane anesthesia both agonists at equivalent doses demonstrated efficacy in wild-type (WT) mice, significantly enhancing theta power and theta phase-gamma amplitude coupling as compared to saline treated control mice. These effects are comparable to those seen with drugs clinically used to treat Alzheimer's disease. Although α7KO mice showed no alterations in elicited hippocampal oscillations, both α7 nAChR agonists failed to enhance theta power or theta phase - gamma amplitude coupling in these mice. Our findings demonstrate that selective activation of α7 nAChRs can modulate hippocampal oscillation, and these receptors are the primary targets of the tested agonists, PNU-282987 and FRM-17874 and likely underlies their observed procognitive activity., (Copyright © 2016 Elsevier Ltd. All rights reserved.)
- Published
- 2016
- Full Text
- View/download PDF
49. Trichothecenes and aspinolides produced by Trichoderma arundinaceum regulate expression of Botrytis cinerea genes involved in virulence and growth.
- Author
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Malmierca MG, Izquierdo-Bueno I, McCormick SP, Cardoza RE, Alexander NJ, Barua J, Lindo L, Casquero PA, Collado IG, Monte E, and Gutiérrez S
- Subjects
- Botrytis metabolism, Botrytis pathogenicity, Fungal Proteins metabolism, Gene Expression Regulation, Fungal, Trichoderma chemistry, Trichoderma genetics, Virulence, Aldehydes metabolism, Botrytis genetics, Botrytis growth & development, Bridged Bicyclo Compounds metabolism, Fungal Proteins genetics, Plant Diseases microbiology, Trichoderma metabolism, Trichothecenes metabolism
- Abstract
Trichoderma arundinaceum (Ta37) and Botrytis cinerea (B05.10) produce the sesquiterpenoids harzianum A (HA) and botrydial (BOT), respectively. TaΔTri5, an HA non-producer mutant, produces high levels of the polyketide compounds aspinolides (Asp) B and C. We analyzed the role of HA and Asp in the B. cinerea-T. arundinaceum interaction, including changes in BOT production as well as transcriptomic changes of BcBOT genes involved in BOT biosynthesis, and also of genes associated with virulence and ergosterol biosynthesis. We found that exogenously added HA up-regulated the expression of the BcBOT and all the virulence genes analyzed when B. cinerea was grown alone. However, a decrease in the amount of BOT and a down-regulation of BcBOT gene expression was observed in the interaction zone of B05.10-Ta37 dual cultures, compared to TaΔTri5. Thus, the confrontation with T. arundinaceum results in an up-regulation of most of the B. cinerea genes involved in virulence yet the presence of T. arundinaceum secondary metabolites, HA and AspC, act separately and together to down-regulate the B. cinerea genes analyzed. The present work emphasizes the existence of a chemical cross-regulation between B. cinerea and T. arundinaceum and contributes to understanding how a biocontrol fungus and its prey interact with each other., (© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.)
- Published
- 2016
- Full Text
- View/download PDF
50. Genetic and Molecular Basis of Botrydial Biosynthesis: Connecting Cytochrome P450-Encoding Genes to Biosynthetic Intermediates.
- Author
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Moraga J, Dalmais B, Izquierdo-Bueno I, Aleu J, Hanson JR, Hernández-Galán R, Viaud M, and Collado IG
- Subjects
- Carbon-13 Magnetic Resonance Spectroscopy, Gas Chromatography-Mass Spectrometry, Genes, Fungal, Polyketides metabolism, Proton Magnetic Resonance Spectroscopy, Aldehydes metabolism, Botrytis genetics, Bridged Bicyclo Compounds metabolism, Cytochrome P-450 Enzyme System genetics
- Abstract
Over two hundred species of plants can be infected by the phytopathogenic fungus Botrytis cinerea under a range of different environmental conditions. In response to these, the fungus produces unique terpenoid and polyketide metabolites. Parts of the plants may be killed by the phytotoxin botrydial, enabling the fungus to feed on the dead cells. In this paper, we describe the genetic and molecular basis of botrydial biosynthesis and the function of the five genes of the genome of B. cinerea that together constitute the botrydial biosynthetic gene cluster. Genes BcBOT3 and BcBOT4, encoding two cytochrome P450 monooxygenases, were inactivated by homologous recombination and were shown to catalyze regio- and stereospecific hydroxylations at the carbons C-10 and C-4, respectively, of the presilphiperfolan-8β-ol skeleton. The null mutants, bcbot3Δ and bcbot4Δ, accumulated key intermediates in the botrydial biosynthesis enabling the complete genetic and molecular basis of the botrydial biosynthetic pathway to be established. Furthermore, the bcbot4Δ mutant overproduced a significant number of polyketides, which included, in addition to known botcinins, botrylactones and cinbotolide A, two new botrylactones and two new cinbotolides, cinbotolides B and C.
- Published
- 2016
- Full Text
- View/download PDF
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