| 28921982 |
Mortiamides A-D, Cyclic Heptapeptides from a Novel Mortierella sp. Obtained from Frobisher Bay |
10.1021/acs.jnatprod.7b00383. |
J Nat Prod |
Mortiamides A-D, Cyclic Heptapeptides from a Novel Mortierella sp. Obtained from Frobisher Bay
Abstract
- Four new cyclic heptapeptides, mortiamides A-D (1-4), were obtained from a novel Mortierella sp. isolate obtained from marine sediment collected from the intertidal zone of Frobisher Bay, Nunavut, Canada. The structures of the compounds were elucidated by NMR spectroscopy and tandem mass spectrometry. The absolute configurations of the amino acids were determined using Marfey's method. Localization of l and d amino acids within each compound was ascertained by retention time comparison of the partial hydrosylate products of each compound to synthesized dipeptide standards using LC-HRMS. Compounds 1-4 did not exhibit any significant antimicrobial or cytotoxic activity.
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| 28921993 |
Lactam-Stapled Cell-Penetrating Peptides: Cell Uptake and Membrane Binding Properties |
10.1021/acs.jmedchem.7b00813. |
J Med Chem |
Lactam-Stapled Cell-Penetrating Peptides: Cell Uptake and Membrane Binding Properties
Abstract
- Stapling of side chains to stabilize an α-helical structure has been generally associated with an increased uptake of CPPs. Here, we compare four amphiphilic stapled peptides with their linear counterparts in terms of their membrane binding and conformational features in order to correlate these with uptake efficiency and toxicological effects. The impact of lactam stapling was found to vary strongly with regard to the different aspects of peptide-membrane interactions. Nearly all stapled peptides caused less membrane perturbation (vesicle leakage, hemolysis, bacterial lysis) than their linear counterparts. In one case (MAP-1) where stapling enhanced α-helicity in aqueous and lipid environments, leakage was eliminated while cell uptake in HEK293 and HeLa cells remained high, which improved the overall characteristics. The other systems (DRIM, WWSP, KFGF) did not improve, however. The data suggest that cell uptake of amphipathic CPPs correlates with their adopted α-helix content in membranes rather than their helicity in solution.
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| 28933840 |
Divergent Synthesis of Revised Apratoxin E, 30-epi-Apratoxin E, and 30S/30R-Oxoapratoxin E |
10.1021/acs.joc.7b01598. |
J Org Chem |
Divergent Synthesis of Revised Apratoxin E, 30-epi-Apratoxin E, and 30S/30R-Oxoapratoxin E
Abstract
- In this report, originally proposed apratoxin E (30S-7), revised apratoxin E (30R-7), and (30S)/(30R)-oxoapratoxin E (30S)-38/(30R)-38 were efficiently prepared by two synthetic methods. The chiral lactone 10, recycled from the degradation of saponin glycosides, was utilized to prepare the key nonpeptide fragment 9. Our alternative convergent assembly strategy was applied to the divergent synthesis of revised apratoxin E and its three analogues. Moreover, ring-closing metathesis (RCM) was for the first time found to be an efficient strategy for the macrocyclization of apratoxins.
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| 28946884 |
Polycistronic gene expression in Aspergillus niger |
10.1186/s12934-017-0780-z. |
Microb Cell Fact |
Polycistronic gene expression in Aspergillus niger
Abstract
- Genome mining approaches predict dozens of biosynthetic gene clusters in each of the filamentous fungal genomes sequenced so far. However, the majority of these gene clusters still remain cryptic because they are not expressed in their natural host. Simultaneous expression of all genes belonging to a biosynthetic pathway in a heterologous host is one approach to activate biosynthetic gene clusters and to screen the metabolites produced for bioactivities. Polycistronic expression of all pathway genes under control of a single and tunable promoter would be the method of choice, as this does not only simplify cloning procedures, but also offers control on timing and strength of expression. However, polycistronic gene expression is a feature not commonly found in eukaryotic host systems, such as Aspergillus niger.
In this study, we tested the suitability of the viral P2A peptide for co-expression of three genes in A. niger. Two genes descend from Fusarium oxysporum and are essential to produce the secondary metabolite enniatin (esyn1, ekivR). The third gene (luc) encodes the reporter luciferase which was included to study position effects. Expression of the polycistronic gene cassette was put under control of the Tet-On system to ensure tunable gene expression in A. niger. In total, three polycistronic expression cassettes which differed in the position of luc were constructed and targeted to the pyrG locus in A. niger. This allowed direct comparison of the luciferase activity based on the position of the luciferase gene. Doxycycline-mediated induction of the Tet-On expression cassettes resulted in the production of one long polycistronic mRNA as proven by Northern analyses, and ensured comparable production of enniatin in all three strains. Notably, gene position within the polycistronic expression cassette matters, as, luciferase activity was lowest at position one and had a comparable activity at positions two and three.
The P2A peptide can be used to express at least three genes polycistronically in A. niger. This approach can now be applied to heterologously express entire secondary metabolite gene clusters polycistronically or to co-express any genes of interest in equimolar amounts.
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| 28955446 |
Engineering of for the production of secondary metabolites |
10.1186/s40694-014-0004-9. |
Fungal Biol Biotechnol |
Engineering of for the production of secondary metabolites
Abstract
- Filamentous fungi can each produce dozens of secondary metabolites which are attractive as therapeutics, drugs, antimicrobials, flavour compounds and other high-value chemicals. Furthermore, they can be used as an expression system for eukaryotic proteins. Application of most fungal secondary metabolites is, however, so far hampered by the lack of suitable fermentation protocols for the producing strain and/or by low product titers. To overcome these limitations, we report here the engineering of the industrial fungus to produce high titers (up to 4,500 mg • l) of secondary metabolites belonging to the class of nonribosomal peptides.
For a proof-of-concept study, we heterologously expressed the 351 kDa nonribosomal peptide synthetase ESYN from in . ESYN catalyzes the formation of cyclic depsipeptides of the enniatin family, which exhibit antimicrobial, antiviral and anticancer activities. The encoding gene was put under control of a tunable bacterial-fungal hybrid promoter (Tet-on) which was switched on during early-exponential growth phase of cultures. The enniatins were isolated and purified by means of reverse phase chromatography and their identity and purity proven by tandem MS, NMR spectroscopy and X-ray crystallography. The initial yields of 1 mg • l of enniatin were increased about 950 fold by optimizing feeding conditions and the morphology of in liquid shake flask cultures. Further yield optimization (about 4.5 fold) was accomplished by cultivating in 5 l fed batch fermentations. Finally, an autonomous expression host was established, which was independent from feeding with the enniatin precursor d-2-hydroxyvaleric acid d-Hiv. This was achieved by constitutively expressing a fungal d-Hiv dehydrogenase in the -expressing strain, which used the intracellular α-ketovaleric acid pool to generate d-Hiv.
This is the first report demonstrating that is a potent and promising expression host for nonribosomal peptides with titers high enough to become industrially attractive. Application of the Tet-on system in allows precise control on the timing of product formation, thereby ensuring high yields and purity of the peptides produced.
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| 28960954 |
Structure-Activity and -Toxicity Relationships of the Antimicrobial Peptide Tachyplesin-1 |
10.1021/acsinfecdis.7b00123. |
ACS Infect Dis |
Structure-Activity and -Toxicity Relationships of the Antimicrobial Peptide Tachyplesin-1
Abstract
- Tachyplesin-1 (TP1; 1) is a cationic β-hairpin antimicrobial peptide with a membranolytic mechanism of action. While it possesses broad-spectrum, potent antimicrobial activity, 1 is highly hemolytic against mammalian erythrocytes, which precludes it from further development. In this study, we report a template-based approach to investigate the structure-function and structure-toxicity relationships of each amino acid of 1. We modulated charge and hydrophobicity by residue modification and truncation of the peptide. Antimicrobial activity was then assessed against six key bacterial pathogens and two fungi, with toxicity profiled against mammalian cells. The internal disulfide bridge Cys7-Cys12 of 1 was shown to play an important role in broad-spectrum antimicrobial activity against all pathogenic strains tested. Novel peptides based on the progenitor were then designed, including 5 (TP1[F4A]), 12 (TP1[I11A]), and 19 (TP1[C3A,C16A]). These had 26- to 64-fold improved activity/toxicity indices and show promise for further development. Structural studies of 5 (TP1[F4A]) and 12 (TP1[I11A]) identified a conserved β-hairpin secondary structure motif correlating with their very high stablility in mouse and human plasma. Membrane binding affinity determined by surface plasmon resonance confirmed their selectivity toward bacterial membranes, but the degree of membrane binding did not correlate with the degree of hemolysis, suggesting that other factors may drive toxicity.
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| 29030449 |
Plantaricyclin A, a Novel Circular Bacteriocin Produced by Lactobacillus plantarum NI326: Purification, Characterization, and Heterologous Production |
10.1128/AEM.01801-17. |
Appl Environ Microbiol |
Plantaricyclin A, a Novel Circular Bacteriocin Produced by Lactobacillus plantarum NI326: Purification, Characterization, and Heterologous Production
Abstract
- Bacteriocins from lactic acid bacteria (LAB) are of increasing interest in recent years due to their potential as natural preservatives against food and beverage spoilage microorganisms. In a screening study for LAB, we isolated from olives a strain, NI326, with activity against the beverage-spoilage bacterium Genome sequencing of NI326 enabled the identification of a gene cluster (designated ) encoding a putative circular bacteriocin and proteins involved in its modification, transport, and immunity. This novel bacteriocin, named plantaricyclin A (PlcA), was grouped into the circular bacteriocin subgroup II due to its high degree of similarity with other gassericin A-like bacteriocins. Purification of PlcA from the supernatant of NI326 resulted in an active peptide with a molecular mass of 5,570 Da, corresponding to that predicted from the (processed) PlcA amino acid sequence. The gene cluster was cloned and expressed in NZ9000, resulting in the production of an active 5,570-Da bacteriocin in the supernatant. PlcA is believed to be produced as a 91-amino-acid precursor with a 33-amino-acid leader peptide, which is predicted to be removed, followed by joining of the N and C termini via a covalent linkage to form the mature 58-amino-acid circular bacteriocin PlcA. We report the characterization of a circular bacteriocin produced by The inhibition displayed against highlights its potential use as a preservative in food and beverages. In this work, we describe the purification and characterization of an antimicrobial peptide, termed plantaricyclin A (PlcA), produced by a strain isolated from olives. This peptide has a circular structure, and all genes involved in its production, circularization, and secretion were identified. PlcA shows antimicrobial activity against different strains, including , a common spoilage bacterium, which causes substantial economic losses in the beverage industry every year. In this study, we describe a circular antimicrobial peptide, PlcA, for a strain.
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| 29033832 |
Cyclotides Isolated from an Ipecac Root Extract Antagonize the Corticotropin Releasing Factor Type 1 Receptor |
10.3389/fphar.2017.00616. |
Front Pharmacol |
Cyclotides Isolated from an Ipecac Root Extract Antagonize the Corticotropin Releasing Factor Type 1 Receptor
Abstract
- Cyclotides are plant derived, cystine-knot stabilized peptides characterized by their natural abundance, sequence variability and structural plasticity. They are abundantly expressed in Rubiaceae, Psychotrieae in particular. Previously the cyclotide kalata B7 was identified to modulate the human oxytocin and vasopressin G protein-coupled receptors (GPCRs), providing molecular validation of the plants' uterotonic properties and further establishing cyclotides as valuable source for GPCR ligand design. In this study we screened a cyclotide extract derived from the root powder of the South American medicinal plant ipecac () for its GPCR modulating activity of the corticotropin-releasing factor type 1 receptor (CRFR). We identified and characterized seven novel cyclotides. One cyclotide, caripe 8, isolated from the most active fraction, was further analyzed and found to antagonize the CRFR. A nanomolar concentration of this cyclotide (260 nM) reduced CRF potency by ∼4.5-fold. In contrast, caripe 8 did not inhibit forskolin-, or vasopressin-stimulated cAMP responses at the vasopressin V receptor, suggesting a CRFR-specific mode-of-action. These results in conjunction with our previous findings establish cyclotides as modulators of both classes A and B GPCRs. Given the diversity of cyclotides, our data point to other cyclotide-GPCR interactions as potentially important sources of drug-like molecules.
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| 29048160 |
Destruxin A Induces and Binds HSPs in Bombyx mori Bm12 Cells |
10.1021/acs.jafc.7b03734. |
J Agric Food Chem |
Destruxin A Induces and Binds HSPs in Bombyx mori Bm12 Cells
Abstract
- Destruxin A (DA) is a cyclodepsipeptidic mycotoxin isolated from the entomopathogenic fungus, Metarhizium anisopliae. It has insecticidal activity against host insect's innate immunity system, but the molecular mechanism is not yet elucidated. In our previous experiment, four HSPs (heat shock proteins, BmHSP70-3, BmHSP75, BmHSP83, and BmHSCP) were characterized from the specific protein electrophoretic bands of Bombyx mori Bm12 cell line treated with DA in the test of drug affinity responsive target stability (DARTS), which implied that these HSPs might be kinds of DA-affinity proteins, or DA induces them up-regulated expression. Therefore, in current research, the interactions of DA and HSPs were explored through analysis of bio-layer interferometry (BLI) employing FortBio OcteteQK. The expression levels of HSPs genes were surveyed by quantitative real-time polymerase chain reaction (qPCR). The results indicated that DA had no interactions with BmHSP70-3, BmHSP75, and BmHSP83, but had affinity to BmHSCP with a K value of 88.1 μM, in BLI analysis. However, the expression levels of all HSPs genes were significantly up-regulated after the Bm12 cells were treated by DA. In conclusion, DA can induce the four HSPs expression in Bm12 cells, but DA only binds to BmHSCP. Our research provides new insights on understanding of the action mechanisms of destruxins.
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| 29051530 |
Characterization of a dual function macrocyclase enables design and use of efficient macrocyclization substrates |
10.1038/s41467-017-00862-4. |
Nat Commun |
Characterization of a dual function macrocyclase enables design and use of efficient macrocyclization substrates
Abstract
- Peptide macrocycles are promising therapeutic molecules because they are protease resistant, structurally rigid, membrane permeable, and capable of modulating protein-protein interactions. Here, we report the characterization of the dual function macrocyclase-peptidase enzyme involved in the biosynthesis of the highly toxic amanitin toxin family of macrocycles. The enzyme first removes 10 residues from the N-terminus of a 35-residue substrate. Conformational trapping of the 25 amino-acid peptide forces the enzyme to release this intermediate rather than proceed to macrocyclization. The enzyme rebinds the 25 amino-acid peptide in a different conformation and catalyzes macrocyclization of the N-terminal eight residues. Structures of the enzyme bound to both substrates and biophysical analysis characterize the different binding modes rationalizing the mechanism. Using these insights simpler substrates with only five C-terminal residues were designed, allowing the enzyme to be more effectively exploited in biotechnology.
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| 29075625 |
Chemical Proteomics for Target Discovery of Head-to-Tail Cyclized Mini-Proteins |
10.3389/fchem.2017.00073. |
Front Chem |
Chemical Proteomics for Target Discovery of Head-to-Tail Cyclized Mini-Proteins
Abstract
- Target deconvolution is one of the most challenging tasks in drug discovery, but a key step in drug development. In contrast to small molecules, there is a lack of validated and robust methodologies for target elucidation of peptides. In particular, it is difficult to apply these methods to cyclic and cysteine-stabilized peptides since they exhibit reduced amenability to chemical modification and affinity capture; however, such ribosomally synthesized and post-translationally modified peptide natural products are rich sources of promising drug candidates. For example, plant-derived circular peptides called cyclotides have recently attracted much attention due to their immunosuppressive effects and oral activity in the treatment of multiple sclerosis in mice, but their molecular target has hitherto not been reported. In this study, a chemical proteomics approach using photo-affinity crosslinking was developed to determine a target for the circular peptide [T20K]kalata B1. Using this prototypic nature-derived peptide enabled the identification of a possible functional modulation of 14-3-3 proteins. This biochemical interaction was validated via competition pull down assays as well as a cellular reporter assay indicating an effect on 14-3-3-dependent transcriptional activity. As proof of concept, the presented approach may be applicable for target elucidation of various cyclic peptides and mini-proteins, in particular cyclotides, which represent a promising class of molecules in drug discovery and development.
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| 29075946 |
Antimicrobial activity and stability of stapled helices of polybia-MP1 |
10.1007/s12272-017-0963-5. |
Arch Pharm Res |
Antimicrobial activity and stability of stapled helices of polybia-MP1
Abstract
- Polybia-MP1 is a well-known natural antimicrobial peptide isolated from the venom of the social wasp Polybia paulista. A recent study showed that this peptide displays a broad antibacterial spectrum as well as low toxicity to human red blood cells and normal fibroblasts. However, its moderate antimicrobial activity and high susceptibility to protease have been a major hurdle for clinical use. This study examined the possibility of developing biologically more potent, yet metabolically more stable, analogues of MP1 using an emerging technology termed "all-hydrocarbon stapling." The stapled analogues of MP1 showed more than a threefold increase in helicity as well as an approximately 70-fold enhancement in proteolytic stability. These stapled analogues also exhibited a significant increase in inhibition against some Gram-positive bacteria while displaying a modest enhancement in hemolytic activity. Overall, the current study demonstrated that the all-hydrocarbon stapling system is a highly useful tool for the development of biologically more potent and metabolically more stable analogues of natural antimicrobial peptides.
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| 29125222 |
Total Synthesis and Antibacterial Study of Cyclohexapeptides Desotamide B, Wollamide B and Their Analogs |
10.1002/cbdv.201700414. |
Chem Biodivers |
Total Synthesis and Antibacterial Study of Cyclohexapeptides Desotamide B, Wollamide B and Their Analogs
Abstract
- As natural-product-derived antibiotics, desotamides A - D and wollamides exhibit growth inhibitory activity against Gram-posivite bacteria (IC 0.6 - 7 μm) and are noncytotoxic to mammalian cells (IC > 30 μm). Herein we firstly report the total synthesis of above two cyclohexapeptides as well as a series of structural variants through solid phase peptide synthesis, of which 3 displayed a 2-fold increase of antibacterial activity when compared with the original peptide 1. This strategy may offer good improvements for the synthesis of other cyclic peptides.
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| 29133552 |
CD101, a Novel Echinocandin, Possesses Potent Antibiofilm Activity against Early and Mature Candida albicans Biofilms |
10.1128/AAC.01750-17. |
Antimicrob Agents Chemother |
CD101, a Novel Echinocandin, Possesses Potent Antibiofilm Activity against Early and Mature Candida albicans Biofilms
Abstract
- Currently available echinocandins are generally effective against biofilms, but the recent emergence of resistance has underscored the importance of developing new antifungal agents that are effective against biofilms. CD101 is a long-acting novel echinocandin with distinctive pharmacokinetic properties and improved stability and safety relative to other drugs in the same class. CD101 is currently being evaluated as a once-weekly intravenous (i.v.) infusion for the treatment of candidemia and invasive candidiasis. In this study, we determined (i) the effect of CD101 against early and mature phase biofilms formed by and (ii) the temporal effect of CD101 on the formation of biofilms using time-lapse microscopy (TLM). Early- or mature-phase biofilms were formed on silicone elastomer discs and were exposed to the test compounds for 24 h and quantified by measuring their metabolic activity. Separate batches were observed under a confocal microscope or used to capture TLM images from 0 to 16 h. Measurements of their metabolic activity showed that CD101 (0.25 or 1 μg/ml) significantly prevented adhesion-phase cells from developing into mature biofilms ( = 0.0062 or 0.0064, respectively) and eradicated preformed mature biofilms ( = 0.04 or 0.01, respectively) compared to those of untreated controls. Confocal microscopy showed significant reductions in biofilm thicknesses for both early and mature phases ( < 0.05). TLM showed that CD101 stopped the growth of adhesion- and early-phase biofilms within minutes. CD101-treated hyphae failed to grow into mature biofilms. These results suggest that CD101 may be effective in the prevention and treatment of biofilm-associated nosocomial infections.
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| 29169187 |
Biologically Active Orbitides from the Euphorbiaceae Family |
10.1055/s-0043-122604. |
Planta Med |
Biologically Active Orbitides from the Euphorbiaceae Family
Abstract
- A comprehensive overview of natural orbitides isolated from Euphorbiaceae species and their most relevant biological activities are presented. Euphorbiaceae is a large and diverse family, which comprises about 300 genera, and is known as an important source of medicines and toxins. Several classes of secondary metabolites have been described for this taxon, however, orbitides have been broadly reported in and genera. Additionally, the latex is documented as the main source of orbitides in this family. Based on their structural and functional diversity, orbitides present a large variety of biological activities described as cytotoxicity, antimalarial, antibacterial, antifungal, enzymatic inhibition, and immunosuppressive, although the mechanism of action still needs to be further investigated. In recent years, the discovery of bioactive cyclic peptides from different sources has grown exponentially, making them promising molecules in the search for new drug leads. This review also highlights the attempts made by many researchers to organize the orbitides nomenclature and amino acid numbering, as well the important progress recently achieved in the biosynthetic study area.
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