Inhibition of N-Type Calcium Channels by Fluorophenoxyanilide Derivatives

A set of fluorophenoxyanilides, designed to be simplified analogues of previously reported ω-conotoxin GVIA mimetics, were prepared and tested for N-type calcium channel inhibition in a SH-SY5Y neuroblastoma FLIPR assay. N-type or Ca_v2.2 channel is a validated target for the treatment of refractory chronic pain. Despite being significantly less complex than the originally designed mimetics, up to a seven-fold improvement in activity was observed.     

Subcutaneous ω-Conotoxins Alleviate Mechanical Pain in Rodent Models of Acute Peripheral Neuropathy

The peripheral effects of ω-conotoxins, selective blockers of N-type voltage-gated calcium channels (Ca_V2.2), have not been characterised across different clinically relevant pain models. This study examines the effects of locally administered ω-conotoxin MVIIA, GVIA, and CVIF on mechanical and thermal paw withdrawal threshold (PWT) in postsurgical pain (PSP), cisplatin-induced neuropathy (CisIPN), and oxaliplatin-induced neuropathy (OIPN) rodent models. Intraplantar injection of 300, 100 and 30 nM MVIIA significantly (p < 0.0001, p < 0.0001, and p < 0.05, respectively) alleviated mechanical allodynia of mice in PSP model compared to vehicle control group. Similarly, intraplantar injection of 300, 100, and 30 nM MVIIA (p < 0.0001, p < 0.01, and p < 0.05, respectively), and 300 nM and 100 nM GVIA (p < 0.0001 and p < 0.05, respectively) significantly increased mechanical thresholds of mice in OIPN model. The ED₅₀ of GVIA and MVIIA in OIPN was found to be 1.8 pmol/paw and 0.8 pmol/paw, respectively. However, none of the ω-conotoxins were effective in a mouse model of CisIPN. The intraplantar administration of 300 nM GVIA, MVIIA, and CVIF did not cause any locomotor side effects. The intraplantar administration of MVIIA can alleviate incision-induced mechanical allodynia, and GVIA and MVIIA effectively reduce OIPN associated mechanical pain, without locomotor side effects, in rodent models. In contrast, CVIF was inactive in these pain models, suggesting it is unable to block a subset of N-type voltage-gated calcium channels associated with nociceptors in the skin.     

A Conus regularis Conotoxin with a Novel Eight-Cysteine Framework Inhibits CaV2.2 Channels and Displays an Anti-Nociceptive Activity

A novel peptide, RsXXIVA, was isolated from the venom duct of Conus regularis, a worm-hunting species collected in the Sea of Cortez, México. Its primary structure was determined by mass spectrometry and confirmed by automated Edman degradation. This conotoxin contains 40 amino acids and exhibits a novel arrangement of eight cysteine residues (C-C-C-C-CC-CC). Surprisingly, two loops of the novel peptide are highly identical to the amino acids sequence of ω-MVIIA. The total length and disulfide pairing of both peptides are quite different, although the two most important residues for the described function of ω-MVIIA (Lys2 and Tyr13) are also present in the peptide reported here. Electrophysiological analysis using superior cervical ganglion (SCG) neurons indicates that RsXXIVA inhibits Ca_V2.2 channel current in a dose-dependent manner with an EC₅₀ of 2.8 μM, whose effect is partially reversed after washing. Furthermore, RsXXIVA was tested in hot-plate assays to measure the potential anti-nociceptive effect to an acute thermal stimulus, showing an analgesic effect in acute thermal pain at 30 and 45 min post-injection. Also, the toxin shows an anti-nociceptive effect in a formalin chronic pain test. However, the low affinity for Ca_V2.2 suggests that the primary target of the peptide could be different from that of ω-MVIIA.     

Nocapyrones: α- and γ-Pyrones from a Marine-Derived Nocardiopsis sp.

One new α-pyrone (nocapyrone R (1)), and three known γ-pyrones (nocapyrones B, H and L (2–4)) were isolated from the culture extract of a Nocardiopsis strain collected from marine sediment. Structures of these compounds were determined on the basis of spectroscopic data including NMR and MS. γ-Pyrones 2–4 were found to induce adiponectin production in murine ST-13 preadipocyte cells but the α-pyrone 1 had no activity. The absolute configuration of the anteiso-methyl branching in 4 was determined by HPLC comparison of a degraded product of 4 with standard samples as a 2:3 enantiomeric mixture of (R)- and (S)-isomers.     

Nano- and Microdelivery Systems for Marine Bioactive Lipids

There is an increasing body of evidence of the positive impact of several marine lipids on human health. These compounds, which include ω-3 polyunsaturated fatty acids, have been shown to improve blood lipid profiles and exert anti-inflammatory and cardioprotective effects. The high instability of these compounds to oxidative deterioration and their hydrophobicity have a drastic impact in their pharmacokinetics. Thus, the bioavailability of these compounds may be affected, resulting in their inability to reach the target sites at effective concentrations. In this regard; micro/nanoparticles can offer a wide range of solutions that can prevent the degradation of targeted molecules, increase their absorption, uptake and bioavailability. In this work we will present the options currently available concerning micro- and nanodelivery systems for marine lipids; with emphasis on micro/nanoparticles; such as micro/nanocapsules and emulsions. A wide range of bottom-up approaches using casein, chitosan, cyclodextrins, among others; will be discussed.     

Violapyrones H and I,New Cytotoxic Compounds Isolated from Streptomyces sp. Associated with the Marine Starfish Acanthaster planci

Two new α-pyrone derivatives, violapyrones H (1) and I (2), along with known violapyrones B (3) and C (4) were isolated from the fermentation broth of a marine actinomycete Streptomyces sp. The strain was derived from a crown-of-thorns starfish, Acanthaster planci, collected from Chuuk, Federated States of Micronesia. The structures of violapyrones were elucidated by the analysis of 1D and 2D NMR and HR-ESIMS data. Violapyrones (1–4) exhibited cytotoxicity against 10 human cancer cell lines with GI₅₀ values of 1.10–26.12 μg/mL when tested using sulforhodamine B (SRB) assay. This is the first report on the cytotoxicity of violapyrones against cancer cell lines and the absolute configuration of violapyrone C.     

New and Rare Carotenoids Isolated from Marine Bacteria and Their Antioxidant Activities

Marine bacteria have not been examined as extensively as land bacteria. We screened carotenoids from orange or red pigments-producing marine bacteria belonging to rare or novel species. The new acyclic carotenoids with a C₃₀ aglycone, diapolycopenedioc acid xylosylesters A–C and methyl 5-glucosyl-5,6-dihydro-apo-4,4′-lycopenoate, were isolated from the novel Gram-negative bacterium Rubritalea squalenifaciens, which belongs to phylum Verrucomicrobia, as well as the low-GC Gram-positive bacterium Planococcus maritimus strain iso-3 belonging to the class Bacilli, phylum Firmicutes, respectively. The rare monocyclic C₄₀ carotenoids, (3R)-saproxanthin and (3R,2′S)-myxol, were isolated from novel species of Gram-negative bacteria belonging to the family Flavobacteriaceae, phylum Bacteroidetes. In this review, we report the structures and antioxidant activities of these carotenoids, and consider relationships between bacterial phyla and carotenoid structures.     

Thalassospiramide G,a New γ-Amino-Acid-Bearing Peptide from the Marine Bacterium Thalassospira sp

In the chemical investigation of marine unicellular bacteria, a new peptide, thalassospiramide G (1), along with thalassospiramides A and D (2–3), was discovered from a large culture of Thalassospira sp. The structure of thalassospiramide G, bearing γ-amino acids, such as 4-amino-5-hydroxy-penta-2-enoic acid (AHPEA), 4-amino-3,5-dihydroxy-pentanoic acid (ADPA), and unique 2-amino-1-(1H-indol-3-yl)ethanone (AIEN), was determined via extensive spectroscopic analysis. The absolute configuration of thalassospiramide D (3), including 4-amino-3-hydroxy-5-phenylpentanoic acid (AHPPA), was rigorously determined by ¹H–¹H coupling constant analysis and chemical derivatization. Thalassospiramides A and D (2–3) inhibited nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated mouse macrophage RAW 264.7 cells, with IC₅₀ values of 16.4 and 4.8 μM, respectively.     

Assignment of the CD Cotton Effect to the Chiral Center in Pseurotins,and the Stereochemical Revision of Pseurotin A2

Pseurotins A₁ (1) and A₂ (2) were isolated from a culture broth of the fungal strain Aspergillus fumigatus WFZ-25 as stereoisomers of pseurotin A (3) in 2011. We also isolated 1 and 2 together with 3 from A. fumigatus OUPS-T106B-5 separated from the marine fish Mugil cephalus. In this study, we re-examined the stereochemistry of 1 and 2 using chemical transformation and the CD spectra, and found the relationship between the CD Cotton effect and the absolute configurations of 1 and 2, which led us to revise the stereostructure of pseurotin A₂.     

Characterization of a Novel Conus bandanus Conopeptide Belonging to the M-Superfamily Containing Bromotryptophan

A novel conotoxin (conopeptide) was biochemically characterized from the crude venom of the molluscivorous marine snail, Conus bandanus (Hwass in Bruguière, 1792), collected in the south-central coast of Vietnam. The peptide was identified by screening bromotryptophan from chromatographic fractions of the crude venom. Tandem mass spectrometry techniques were used to detect and localize different post-translational modifications (PTMs) present in the BnIIID conopeptide. The sequence was confirmed by Edman’s degradation and mass spectrometry revealing that the purified BnIIID conopeptide had 15 amino acid residues, with six cysteines at positions 1, 2, 7, 11, 13, and 14, and three PTMs: bromotryptophan, γ-carboxy glutamate, and amidated aspartic acid, at positions “4”, “5”, and “15”, respectively. The BnIIID peptide was synthesized for comparison with the native peptide. Homology comparison with conopeptides having the III-cysteine framework (–CCx₁x₂x₃x₄Cx₁x₂x₃Cx₁CC–) revealed that BnIIID belongs to the M-1 family of conotoxins. This is the first report of a member of the M-superfamily containing bromotryptophan as PTM.     

MytiLec,a Mussel R-Type Lectin,Interacts with Surface Glycan Gb3 on Burkitt’s Lymphoma Cells to Trigger Apoptosis through Multiple Pathways

MytiLec; a novel lectin isolated from the Mediterranean mussel (Mytilus galloprovincialis); shows strong binding affinity to globotriose (Gb3: Galα1-4Galβ1-4Glc). MytiLec revealed β-trefoil folding as also found in the ricin B-subunit type (R-type) lectin family, although the amino acid sequences were quite different. Classification of R-type lectin family members therefore needs to be based on conformation as well as on primary structure. MytiLec specifically killed Burkitt''s lymphoma Ramos cells, which express Gb3. Fluorescein-labeling assay revealed that MytiLec was incorporated inside the cells. MytiLec treatment of Ramos cells resulted in activation of both classical MAPK/ extracellular signal-regulated kinase and extracellular signal-regulated kinase (MEK-ERK) and stress-activated (p38 kinase and JNK) Mitogen-activated protein kinases (MAPK) pathways. In the cells, MytiLec treatment triggered expression of tumor necrosis factor (TNF)-α (a ligand of death receptor-dependent apoptosis) and activation of mitochondria-controlling caspase-9 (initiator caspase) and caspase-3 (activator caspase). Experiments using the specific MEK inhibitor U0126 showed that MytiLec-induced phosphorylation of the MEK-ERK pathway up-regulated expression of the cyclin-dependent kinase inhibitor p21, leading to cell cycle arrest and TNF-α production. Activation of caspase-3 by MytiLec appeared to be regulated by multiple different pathways. Our findings, taken together, indicate that the novel R-type lectin MytiLec initiates programmed cell death of Burkitt’s lymphoma cells through multiple pathways (MAPK cascade, death receptor signaling; caspase activation) based on interaction of the lectin with Gb3-containing glycosphingolipid-enriched microdomains on the cell surface.     

Overexpression and Characterization of a Novel Thermostable β-Agarase YM01-3, from Marine Bacterium Catenovulum agarivorans YM01T

Genome sequencing of Catenovulum agarivorans YM01^T reveals 15 open-reading frames (ORFs) encoding various agarases. In this study, extracellular proteins of YM01^T were precipitated by ammonium sulfate and separated by one-dimensional gel electrophoresis. The results of in-gel agarase activity assay and mass spectrometry analysis revealed that the protein, YM01-3, was an agarase with the most evident agarolytic activity. Agarase YM01-3, encoded by the YM01-3 gene, consisted of 420 amino acids with a calculated molecular mass of 46.9 kDa and contained a glycoside hydrolase family 16 β-agarase module followed by a RICIN superfamily in the C-terminal region. The YM01-3 gene was cloned and expressed in Escherichia coli. The recombinant agarase, YM01-3, showed optimum activity at pH 6.0 and 60 °C and had a K_m of 3.78 mg mL⁻¹ for agarose and a V_max of 1.14 × 10⁴ U mg⁻¹. YM01-3 hydrolyzed the β-1,4-glycosidic linkages of agarose, yielding neoagarotetraose and neoagarohexaose as the main products. Notably, YM01-3 was stable below 50 °C and retained 13% activity after incubation at 80 °C for 1 h, characteristics much different from other agarases. The present study highlights a thermostable agarase with great potential application value in industrial production.     

A Collaborative Evaluation of LC-MS/MS Based Methods for BMAA Analysis: Soluble Bound BMAA Found to Be an Important Fraction

Exposure to β-N-methylamino-l-alanine (BMAA) might be linked to the incidence of amyotrophic lateral sclerosis, Alzheimer’s disease and Parkinson’s disease. Analytical chemistry plays a crucial role in determining human BMAA exposure and the associated health risk, but the performance of various analytical methods currently employed is rarely compared. A CYANOCOST initiated workshop was organized aimed at training scientists in BMAA analysis, creating mutual understanding and paving the way towards interlaboratory comparison exercises. During this workshop, we tested different methods (extraction followed by derivatization and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis, or directly followed by LC-MS/MS analysis) for trueness and intermediate precision. We adapted three workup methods for the underivatized analysis of animal, brain and cyanobacterial samples. Based on recovery of the internal standard D₃BMAA, the underivatized methods were accurate (mean recovery 80%) and precise (mean relative standard deviation 10%), except for the cyanobacterium Leptolyngbya. However, total BMAA concentrations in the positive controls (cycad seeds) showed higher variation (relative standard deviation 21%–32%), implying that D₃BMAA was not a good indicator for the release of BMAA from bound forms. Significant losses occurred during workup for the derivatized method, resulting in low recovery (<10%). Most BMAA was found in a trichloroacetic acid soluble, bound form and we recommend including this fraction during analysis.     

Apo-9′-Fucoxanthinone,Isolated from Sargassum muticum,Inhibits CpG-Induced Inflammatory Response by Attenuating the Mitogen-Activated Protein Kinase Pathway

Sargassum muticum (S. muticum) is a brown edible alga and widely distributed in Korea. This report was designed to evaluate the anti-inflammatory properties of apo-9′-fucoxanthinone (APO-9′) isolated from S. muticum on pro-inflammatory cytokine production. S. muticum extract (SME) exhibited significant inhibitory effects on pro-inflammatory cytokine production in bone marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs). APO-9′ pre-treatment in the CpG DNA-stimulated BMDMs and BMDCs showed a strong dose-dependent inhibitory effect on interleukin (IL)-12 p40, IL-6 and tumor necrosis factor (TNF)-α production with IC₅₀ values ranging from 5.31 to 13.79. It exhibited a strong inhibitory effect on the phosphorylation of ERK1/2 and on activator protein (AP)-1 reporter activity. APO-9′ pre-treatment exhibited significant inhibition of CpG DNA-induced production of inducible nitric oxide synthase. Taken together, these data suggest that SME and APO-9′ have a significant anti-inflammatory property and warrant further studies concerning the potentials of SME and APO-9′ for medicinal use.