| 30160099 |
Unexpected Bacterial Origin of the Antibiotic Icosalide: Two-Tailed Depsipeptide Assembly in Multifarious Burkholderia Symbionts |
10.1021/acschembio.8b00600. |
ACS Chem Biol |
Unexpected Bacterial Origin of the Antibiotic Icosalide: Two-Tailed Depsipeptide Assembly in Multifarious Burkholderia Symbionts
Abstract
- Icosalide is an unusual two-tailed lipocyclopeptide antibiotic that was originally isolated from a fungal culture. Yet, its biosynthesis and ecological function have remained enigmatic. By genome mining and metabolic profiling of a bacterial endosymbiont ( Burkholderia gladioli) of the pest beetle Lagria villosa, we unveiled a bacterial origin of icosalide. Functional analysis of the biosynthetic gene locus revealed an unprecedented nonribosomal peptide synthetase (NRPS) that incorporates two β-hydroxy acids by means of two starter condensation domains in different modules. This unusual assembly line, which may inspire new synthetic biology approaches, is widespread among many symbiotic Burkholderia species from diverse habitats. Biological assays showed that icosalide is active against entomopathogenic bacteria, thus adding to the chemical armory protecting beetle offspring. By creating a null mutant, we found that icosalide is a swarming inhibitor, which may play a role in symbiotic interactions and bears the potential for therapeutic applications.
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| 30194442 |
Novel analgesic ω-conotoxins from the vermivorous cone snail Conus moncuri provide new insights into the evolution of conopeptides |
10.1038/s41598-018-31245-4. |
Sci Rep |
Novel analgesic ω-conotoxins from the vermivorous cone snail Conus moncuri provide new insights into the evolution of conopeptides
Abstract
- Cone snails are a diverse group of predatory marine invertebrates that deploy remarkably complex venoms to rapidly paralyse worm, mollusc or fish prey. ω-Conotoxins are neurotoxic peptides from cone snail venoms that inhibit Ca2.2 voltage-gated calcium channel, demonstrating potential for pain management via intrathecal (IT) administration. Here, we isolated and characterized two novel ω-conotoxins, MoVIA and MoVIB from Conus moncuri, the first to be identified in vermivorous (worm-hunting) cone snails. MoVIA and MoVIB potently inhibited human Ca2.2 in fluorimetric assays and rat Ca2.2 in patch clamp studies, and both potently displaced radiolabeled ω-conotoxin GVIA (I-GVIA) from human SH-SY5Y cells and fish brain membranes (IC 2-9 pM). Intriguingly, an arginine at position 13 in MoVIA and MoVIB replaced the functionally critical tyrosine found in piscivorous ω-conotoxins. To investigate its role, we synthesized MoVIB-[R13Y] and MVIIA-[Y13R]. Interestingly, MVIIA-[Y13R] completely lost Ca2.2 activity and MoVIB-[R13Y] had reduced activity, indicating that Arg at position 13 was preferred in these vermivorous ω-conotoxins whereas tyrosine 13 is preferred in piscivorous ω-conotoxins. MoVIB reversed pain behavior in a rat neuropathic pain model, confirming that vermivorous cone snails are a new source of analgesic ω-conotoxins. Given vermivorous cone snails are ancestral to piscivorous species, our findings support the repurposing of defensive venom peptides in the evolution of piscivorous Conidae.
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| 30230885 |
Frequency- and Amplitude-Dependent Microbial Population Dynamics during Cycles of Feast and Famine |
10.1103/PhysRevLett.121.098101. |
Phys Rev Lett |
Frequency- and Amplitude-Dependent Microbial Population Dynamics during Cycles of Feast and Famine
Abstract
- In nature microbial populations are subject to fluctuating nutrient levels. Nutrient fluctuations are important for evolutionary and ecological dynamics in microbial communities since they impact growth rates, population sizes, and biofilm formation. Here we use automated continuous-culture devices and high-throughput imaging to show that when populations of Escherichia coli are subjected to cycles of nutrient excess (feasts) and scarcity (famine) their abundance dynamics during famines depend on the frequency and amplitude of feasts. We show that frequency and amplitude dependent dynamics in planktonic populations arise from nutrient and history dependent rates of aggregation and dispersal. A phenomenological model recapitulates our experimental observations. Our results show that the statistical properties of environmental fluctuations have substantial impacts on spatial structure in bacterial populations driving large changes in abundance dynamics.
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| 30248263 |
Targeted Delivery of Cyclotides via Conjugation to a Nanobody |
10.1021/acschembio.8b00653. |
ACS Chem Biol |
Targeted Delivery of Cyclotides via Conjugation to a Nanobody
Abstract
- Many naturally occurring peptides have poor proteolytic stability, which limits their therapeutic applications. Cyclotides are plant-derived cyclic peptides that resist proteolysis due to their highly constrained structure, comprising a head-to-tail cyclic backbone and three disulfide bonds that form a cystine-knotted core. This structure makes them useful as scaffolds onto which peptide sequences (epitopes) can be grafted. In this study, VHH7, an alpaca-derived nanobody that targets murine class II MHC molecules, was used for the targeted delivery of cyclotides to antigen-presenting cells (APCs). The cyclotides MCoTI-I, and MCoTI-I with a HA-tag (YPYDVPDYA) grafted into loop 6 (MCoTI-HA), were tested for immunogenic properties. To produce the requisite VHH7-peptide conjugates, a site-specific sortase A-catalyzed reaction in combination with a copper-free strain-promoted cycloaddition reaction was used. MCoTI-I alone did not display any obvious antibody response, thus showing the capacity of cyclotides as immunologically silent scaffolds. By contrast, MCoTI-I conjugated to VHH7 elicited antibodies against cyclic or linear MCoTI-I, thus suggesting a simple and robust approach for targeting cyclotides to APCs, and potentially to other cell types. A similar antibody response was observed when MCoTI-HA was conjugated to VHH7, but there was no reactivity toward a linear HA-tag itself, suggesting differences in conformational constraint between cyclotide-presented and linear epitopes. Studies of commercially available HA antibodies applied to MCoTI-HA confirmed that the conformation of peptide immunogens affects their reactivity. Thus, the production of antibodies that recognize constrained epitopes may benefit from engraftment onto scaffolds such as cyclotides. More broadly, this study validates that a prototypic cyclotide, a member of a peptide family that has proven to be useful as drug design scaffolds in many other studies, can efficiently reach a specific target in vivo.
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| 30252466 |
Single Amino Acid Substitution in α-Conotoxin TxID Reveals a Specific α3β4 Nicotinic Acetylcholine Receptor Antagonist |
10.1021/acs.jmedchem.8b00967. |
J Med Chem |
Single Amino Acid Substitution in α-Conotoxin TxID Reveals a Specific α3β4 Nicotinic Acetylcholine Receptor Antagonist
Abstract
- The α3β4 nicotinic acetylcholine receptor (nAChR) is an important target implicated in various disease states. α-Conotoxin TxID (1) is the most potent antagonist of α3β4 nAChR, but it also exhibits inhibition of α6/α3β4 nAChR. The results of alanine scanning of 1 suggested a vital role for Ser9 in the selectivity of the peptide. In this study, Ser9 was substituted with a series of 14 amino acids, including some non-natural amino acids, displaying different physicochemical characteristics to further improve the selectivity of 1 toward α3β4 nAChR. The pharmacological activities of the mutants were evaluated using an electrophysiological approach. The best selectivity was obtained with [S9K]TxID, 12, which inhibited α3β4 nAChR with an IC of 6.9 nM and had no effects on other nAChRs. Molecular modeling suggested a possible explanation for the high selectivity of 12 toward α3β4 nAChR, providing deeper insight into the interaction between α-conotoxins and nAChRs as well as potential treatments for nAChR-related diseases.
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| 30255960 |
A Lasso-Inspired Bicyclic Peptide: Synthesis, Structure and Properties |
10.1002/chem.201803899. |
Chemistry |
A Lasso-Inspired Bicyclic Peptide: Synthesis, Structure and Properties
Abstract
- The chemical synthesis of a bicycle inspired by the natural lasso peptide sungsanpin using a combination of solid-phase and in-solution chemistries is described. The bicyclic-derived topoisomer was designed by introducing a covalent linkage between the ring and the loop, which allowed the tying of these two parts of the peptide, rendering the bicyclic structure. Several structural techniques, such as MS fragmentation, ion-mobility and NMR spectroscopic analysis were used to characterize the bicycle. Ion-mobility spectroscopy studies revealed that it showed lasso-like behavior. Its 3D structure was predicted on the basis of the NMR restraints. In addition, the high proteolytic and thermal stability of the bicycle potentially make it a suitable scaffold for epitope grafting.
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| 30267875 |
Identifying novel conopepetides from the venom ducts of Conus litteratus through integrating transcriptomics and proteomics |
10.1016/j.jprot.2018.09.015. |
J Proteomics |
Identifying novel conopepetides from the venom ducts of Conus litteratus through integrating transcriptomics and proteomics
Abstract
- The venom ducts of marine cone snails secrete highly complex mixtures of cysteine-rich active peptides, which are generally known as conotoxins or conopeptides and provide a potential fertile resource for pharmacological neuroscience research and the discovery of new drugs. Previous studies have devoted substantial effort to the identification of novel conopeptides, and the 109 cone snail species have yielded 7000 known conopeptides to date. Here, we used de novo deep transcriptome sequencing analyses combined with traditional Sanger sequencing and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) to identify 30 distinct conopeptide precursors. Twenty of these were previously reported and the other 10 were novel conopeptide precursors. The study provides the first identification of the Con-ikot-ikot, NSF-bt05, O3 and I1 gene superfamilies in C. litteratus. A new putative superfamily was identified. In addition, the following cysteine frameworks were first identified in this study: CC-C-C-C-C-C-C-C-C-C-C-C-CC-C-C-C-C-C and C-C-C-C-C-CC-C. Several isomerases involved in post-translational modification of conopeptides were identified as well. The discovery of new conopeptides in C. litteratus will enhance our understanding of the conopeptide diversity in this particular clade of cone snails. We also found the existence of intraspecific variations in vermivorous species. Finally, the analysis strategy offers a relatively reliable workflow for screening for peptide drug candidates. SIGNIFICANCE: These novel conopeptides provide a potential resource for the development of new channel-targeting drugs. The intraspecific variation in C. litteratus enhance our understanding of the conopeptide diversity in this particular clade of cone snails. The identified three cysteine residues, which might participate in the formation of disulfide bonds, provide a clue to get the connectivity of cysteine frameworks. Finally, the analysis strategy offers a relatively reliable workflow for screening for peptide drug candidates.
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| 30301313 |
Anti-Angiogenesis Effects Induced by Octaminomycins A and B against HUVECs |
10.4014/jmb.1806.06046. |
J Microbiol Biotechnol |
Anti-Angiogenesis Effects Induced by Octaminomycins A and B against HUVECs
Abstract
- In the course of studies to discover natural products with anti-angiogenic properties, two cyclic octapeptides, octaminomycins A () and B (), were isolated from the cultures of sp. RK85-270. Octaminomycins suppressed the vascular endothelial growth factor (VEGF)-induced proliferation, adhesion, tube formation, migration, and invasion of HUVECs. Anti-angiogenic activity was futher confirmed in vivo by the chicken chorioallantoic membrane assay. We also identified that and inhibited the phosphorylation of VEGF receptor 2, AKT, and ERK1/2 and the expression and activities of MMP-2 and MMP-9. These results suggest that and may serve as potential scaffolds for the development of therapeutic agents to angiogenesis-dependent diseases.
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| 30310586 |
Prediction of disulfide dihedral angles using chemical shifts |
10.1039/c8sc01423j. |
Chem Sci |
Prediction of disulfide dihedral angles using chemical shifts
Abstract
- Cystine residues result from the formation of disulfide bonds between pairs of cysteine residues. This cross linking of the backbone is essential for the structure and activity of peptides and proteins. The conformation of a cystine side chain can be described using five dihedral angles, 1, 2, 3, 2', and 1', with cystines favouring certain combinations of these angles. 2D NMR spectroscopy is ideally suited for structure determination of disulfide-rich peptides, because of their small size and constrained nature. However, only limited information of the cystine side chain conformation can be determined by NMR spectroscopy, leading to ambiguity in the deduced 3D structures. Resolving accurate structures is important as disulfide-rich peptides have proven to be promising drug candidates in a number of fields, either as bioactive leads or scaffolds. Using a database of NMR chemical shifts combined with crystallographic structures, we have developed a method called DISH that uses support vector machines to predict the dihedral angles of cysteine side chains. It is able to successfully predict 2 angles with 91% accuracy, and has improved performance over existing prediction methods for 1 angles, with 87% accuracy. For 81% of cysteine residues, DISH successfully predicted both the 1 and 2 angles. By revisiting published solution structures of peptides determined using NMR spectroscopy, we assessed the impact of additional cystine dihedral restraints on the quality of 3D models. DISH improved the resolution and accuracy, highlighting the potential for improving the understanding of structure-activity relationships and rational development of peptide drugs.
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| 30345754 |
Synthesis, Racemic X-ray Crystallographic, and Permeability Studies of Bioactive Orbitides from Jatropha Species |
10.1021/acs.jnatprod.8b00447. |
J Nat Prod |
Synthesis, Racemic X-ray Crystallographic, and Permeability Studies of Bioactive Orbitides from Jatropha Species
Abstract
- Orbitides are small cyclic peptides with a diverse range of therapeutic bioactivities. They are produced by many plant species, including those of the Jatropha genus. Here, the objective was to provide new structural information on orbitides to complement the growing knowledge base on orbitide sequences and activities by focusing on three Jatropha orbitides: ribifolin (1), pohlianin C (7), and jatrophidin (12). To determine three-dimensional structures, racemic crystallography, an emerging structural technique that enables rapid crystallization of biomolecules by combining equal amounts of the two enantiomers, was used. The high-resolution structure of ribifolin (0.99 Å) was elucidated from its racemate and showed it was identical to the structure crystallized from its l-enantiomer only (1.35 Å). Racemic crystallography was also used to elucidate high-resolution structures of pohlianin C (1.20 Å) and jatrophidin (1.03 Å), for which there was difficulty forming crystals without using racemic mixtures. The structures were used to interpret membrane permeability data in PAMPA and a Caco-2 cell assay, showing they had poor permeability. Overall, the results show racemic crystallography can be used to obtain high-resolution structures of orbitides and is useful when enantiopure samples are difficult to crystallize or solution structures from NMR are of low resolution.
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| 30361948 |
Effects of lysine-to-arginine substitution on antimicrobial activity of cationic stapled heptapeptides |
10.1007/s12272-018-1084-5. |
Arch Pharm Res |
Effects of lysine-to-arginine substitution on antimicrobial activity of cationic stapled heptapeptides
Abstract
- We previously reported a series of amphipathic helices of stapled heptapeptides as membrane-lytic antimicrobial peptides. These peptides possess three lysine residues as the sole cationic amino acid residues in their hydrophilic face of the helix. Lysine-to-arginine substitution is often shown to increase antimicrobial activity of many natural AMPs due to the more favorable interactions of guanidinium moiety of arginine with membranes. In an effort to further improve the pharmacological properties of our novel AMP series, we here examined the impact of lysine-to-arginine substitution on their structures and antimicrobial and hemolytic activities. Our results indicate that the lysine-to-arginine substitution does not always guarantee enhancement in the antimicrobial potency of AMPs. Instead, we observed varied potency and selectivity depending on the number of substitutions and the positions substituted. Our results imply that, in the given helical scaffold stabilized by a hydrocarbon staple, antimicrobial potency and selectivity are influenced by a complex effect of various structural and chemical changes accompanied by lysine-to-arginine substitution rather than solely by the type of cationic residue. These data show potential for use in our scaffold-assisted development of short, selective, and metabolically stable AMPs.
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| 30387611 |
Discovery and Characterization of Cyclotides from Rinorea Species |
10.1021/acs.jnatprod.8b00572. |
J Nat Prod |
Discovery and Characterization of Cyclotides from Rinorea Species
Abstract
- Cyclotides are macrocyclic cystine-knotted peptides most commonly found in the Violaceae plant family. Although Rinorea is the second-largest genera within the Violaceae family, few studies have examined whether or not they contain cyclotides. To further our understanding of cyclotide diversity and evolution, we examined the cyclotide content of two Rinorea species found in Southeast Asia: R. virgata and R. bengalensis. Seven cyclotides were isolated from R. virgata (named Rivi1-7), and a known cyclotide (cT10) was found in R. bengalensis. Loops 2, 5, and 6 of Rivi1-4 contained sequences not previously seen in corresponding loops of known cyclotides, thereby expanding our understanding of the diversity of cyclotides. In addition, the sequence of loop 2 of Rivi3 and Rivi4 were identical to some related noncyclic "acyclotides" from the Poaceae plant family. As only acyclotides, but not cyclotides, have been reported in monocotyledons thus far, our findings support an evolutionary link between monocotyledon-derived ancestral cyclotide precursors and dicotyledon-derived cyclotides. Furthermore, Rivi2 and Rivi3 had comparable cytotoxic activities to the most cytotoxic cyclotide known to date: cycloviolacin O2 from Viola odorata; yet, unlike cycloviolacin O2, they did not show hemolytic activity. Therefore, these cyclotides represent novel scaffolds for use in future anticancer drug design.
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| 30417166 |
A family of small, cyclic peptides buried in preproalbumin since the Eocene epoch |
10.1002/pld3.42. |
Plant Direct |
A family of small, cyclic peptides buried in preproalbumin since the Eocene epoch
Abstract
- Orbitides are cyclic ribosomally-synthesized and post-translationally modified peptides (RiPPs) from plants; they consist of standard amino acids arranged in an unbroken chain of peptide bonds. These cyclic peptides are stable and range in size and topologies making them potential scaffolds for peptide drugs; some display valuable biological activities. Recently two orbitides whose sequences were buried in those of seed storage albumin precursors were said to represent the first observable step in the evolution of larger and hydrophilic bicyclic peptides. Here, guided by transcriptome data, we investigated peptide extracts of 40 species specifically for the more hydrophobic orbitides and confirmed 44 peptides by tandem mass spectrometry, as well as obtaining solution structures for four of them by NMR. Acquiring transcriptomes from the phylogenetically important Corymboideae family confirmed the precursor genes for the peptides (called or ) are confined to the Asteroideae, a subfamily of the huge plant family Asteraceae. To be confined to the Asteroideae indicates these peptides arose during the Eocene epoch around 45 Mya. Unlike other orbitides, all PawL-derived Peptides contain an Asp residue, needed for processing by asparaginyl endopeptidase. This study has revealed what is likely to be a very large new family of orbitides, uniquely buried alongside albumin and processed by asparaginyl endopeptidase.
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| 30472978 |
Development, Characterization, and Pharmacokinetic Evaluation of a CRV431 Loaded Self-Microemulsifying Drug Delivery System |
10.18433/jpps30245. |
J Pharm Pharm Sci |
Development, Characterization, and Pharmacokinetic Evaluation of a CRV431 Loaded Self-Microemulsifying Drug Delivery System
Abstract
- The objective of this study was to develop a self-microemulsifying drug delivery system (SMEDDS) formulation for the oral delivery of CRV431, a non-immunosuppressive analogue of cyclosporine A. Relative to cyclosporine A, CRV431 is poorly soluble in lipid solvents and thusly presents a challenge for the development of a formulation of sufficient oral bioavailability for clinical use.
The solubility of CRV431, a cyclosporine derivative, was determined in a range of commonly used surfactants, oils and co-solvents. A pseudo-ternary phase diagram was constructed from the most soluble excipients and prototype formulations, SERIES 1 and SERIES 2 were developed. The pharmacokinetics, following single oral doses of 1 and 3 mg/kg of CRV431 SMEDDS, was studied in healthy human volunteers using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS).
The maximum drug load for the SERIES 1 formulations was less than 40 mg/ml. Manipulation of the excipient ratios allowed for the development of SERIES 2 formulations, which had higher drug loading capacity and stability for CRV431 compared to SERIES 1. Further improvements allowed for the development of an optimized SMEDDS formulation containing up to 90 mg/ml CRV431 and which generated a microemulsion mean particle size of 25 nm when dispersed into aqueous media. The pharmacokinetics of the optimized CRV431 SMEDDS displayed excellent total body exposure and dose-proportional effects in humans, and high drug levels in the liver of rats.
The developed SMEDDS formulation should allow for effective clinical development of CRV431, targeted to the treatment of liver diseases including hepatitis B (HBV), fibrosis, and hepatocellular carcinoma.
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| 30602509 |
Structure-Activity Relationships of Wollamide Cyclic Hexapeptides with Activity against Drug-Resistant and Intracellular |
10.1128/AAC.01773-18. |
Antimicrob Agents Chemother |
Structure-Activity Relationships of Wollamide Cyclic Hexapeptides with Activity against Drug-Resistant and Intracellular
Abstract
- Wollamides are cyclic hexapeptides, recently isolated from an Australian soil isolate, that exhibit promising antimycobacterial activity against Bacille Calmette Guérin without displaying cytotoxicity against a panel of mammalian cells. Here, we report the synthesis and antimycobacterial activity of 36 new synthetic wollamides, collated with all known synthetic and natural wollamides, to reveal structure characteristics responsible for growth-inhibitory activity against (H37Rv, H37Ra, CDC1551, HN878, and HN353). The most potent antimycobacterial wollamides were those where residue VI d-Orn (wollamide B) was replaced by d-Arg (wollamide B1) or d-Lys (wollamide B2), with all activity being lost when residue VI was replaced by Gly, l-Arg, or l-Lys (wollamide B3). Substitution of other amino acid residues mainly reduced or ablated antimycobacterial activity. Significantly, whereas wollamide B2 was the most potent in restricting , wollamide B1 restricted intracellular burden in infected macrophages. Wollamide B1 synergized with pretomanid (PA-824) in inhibiting growth but did not antagonize prominent first- and second-line tuberculosis antibiotics. Furthermore, wollamide B1 exerted bactericidal activity against nonreplicating and impaired growth of multidrug- and extensively drug-resistant clinical isolates. pharmacokinetic profiles for wollamide B1 in rats and mice encourage further optimization of the wollamide pharmacophore for bioavailability. Collectively, these observations highlight the potential of the wollamide antimycobacterial pharmacophore.
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