��-Conotoxin GVIA Mimetics that Bind and Inhibit Neuronal Cav2.2 Ion Channels

The neuronal voltage-gated N-type calcium channel (Ca_v2.2) is a validated target for the treatment of neuropathic pain. A small library of anthranilamide-derived ω-Conotoxin GVIA mimetics bearing the diphenylmethylpiperazine moiety were prepared and tested using three experimental measures of calcium channel blockade. These consisted of a ¹²⁵I-ω-conotoxin GVIA displacement assay, a fluorescence-based calcium response assay with SH-SY5Y neuroblastoma cells, and a whole-cell patch clamp electrophysiology assay with HEK293 cells stably expressing human Ca_v2.2 channels. A subset of compounds were active in all three assays. This is the first time that compounds designed to be mimics of ω-conotoxin GVIA and found to be active in the ¹²⁵I-ω-conotoxin GVIA displacement assay have also been shown to block functional ion channels in a dose-dependent manner.     

New Conotoxin SO-3 Targeting N-type Voltage-Sensitive Calcium Channels

Selective blockers of the N-type voltage-sensitive calcium (Ca_V) channels are useful in the management of severe chronic pain. Here, the structure and function characteristics of a novel N-type Ca_V channel blocker, SO-3, are reviewed. SO-3 is a 25- amino acid conopeptide originally derived from the venom of Conus striatus, and contains the same 4-loop, 6-cysteine framework (C-C-CC-C-C) as O-superfamily conotoxins. The synthetic SO-3 has high analgesic activity similar to ω-conotoxin MVIIA (MVIIA), a selective N-type Ca_V channel blocker approved in the USA and Europe for the alleviation of persistent pain states. In electrophysiological studies, SO-3 shows more selectivity towards the N-type Ca_V channels than MVIIA. The dissimilarity between SO-3 and MVIIA in the primary and tertiary structures is further discussed in an attempt to illustrate the difference in selectivity of SO-3 and MVIIA towards N-type Ca_V channels.     

ω-Conotoxins GVIA,MVIIA and CVID: SAR and Clinical Potential

Highly selective N-type voltage-gated calcium (Ca_V) channel inhibitors from cone snail venom (the ω-conotoxins) have emerged as a new class of therapeutics for the treatment of chronic and neuropathic pain. Earlier in 2005, Prialt (Elan) or synthetic ω-conotoxin MVIIA, was the first ω-conotoxin to be approved by Food and Drug Administration for human use. This review compares the action of three ω-conotoxins, GVIA, MVIIA and CVID, describing their structure-activity relationships and potential as leads for the design of improved N-type therapeutics that are more useful in the treatment of chronic pain.     

Development of Highly Selective Kv1.3-Blocking Peptides Based on the Sea Anemone Peptide ShK

ShK, from the sea anemone Stichodactyla helianthus, is a 35-residue disulfide-rich peptide that blocks the voltage-gated potassium channel Kv1.3 at ca. 10 pM and the related channel Kv1.1 at ca. 16 pM. We developed an analog of this peptide, ShK-186, which is currently in Phase 1b-2a clinical trials for the treatment of autoimmune diseases such as multiple sclerosis and rheumatoid arthritis. While ShK-186 displays a >100-fold improvement in selectivity for Kv1.3 over Kv1.1 compared with ShK, there is considerable interest in developing peptides with an even greater selectivity ratio. In this report, we describe several variants of ShK that incorporate p-phophono-phenylalanine at the N-terminus coupled with internal substitutions at Gln16 and Met21. In addition, we also explored the combinatorial effects of these internal substitutions with an alanine extension at the C-terminus. Their selectivity was determined by patch-clamp electrophysiology on Kv1.3 and Kv1.1 channels stably expressed in mouse fibroblasts. The peptides with an alanine extension blocked Kv1.3 at low pM concentrations and exhibited up to 2250-fold selectivity for Kv1.3 over Kv1.1. Analogs that incorporates p-phosphono-phenylalanine at the N-terminus blocked Kv1.3 with IC₅₀s in the low pM range and did not affect Kv1.1 at concentrations up to 100 nM, displaying a selectivity enhancement of >10,000-fold for Kv1.3 over Kv1.1. Other potentially important Kv channels such as Kv1.4 and Kv1.6 were only partially blocked at 100 nM concentrations of each of the ShK analogs.     

Design and Synthesis of Novel Xyloketal Derivatives and Their Protective Activities against H2O2-Induced HUVEC Injury

In this work, we designed and synthesized a series of amide derivatives (1–13), benzoxazine derivatives (16–28) and amino derivatives (29–30) from xyloketal B. All 28 new derivatives and seven known compounds (14, 15, 31–35) were evaluated for their protection against H₂O₂-induced HUVEC injury. 23 and 24 exhibited more potential protective activities than other derivatives; and the EC₅₀ values of them and the leading compound 31 (xyloketal B) were 5.10, 3.59 and 15.97 μM, respectively. Meanwhile, a comparative molecular similarity indices analysis (CoMSIA) was constructed to explain the structural activity relationship of these xyloketal derivatives. This 3D QSAR model from CoMSIA suggested that the derived model exhibited good predictive ability in the external test-set validation. Derivative 24 fit well with the COMSIA map, therefore it possessed the highest activity of all compounds. Compounds 23, 24 and 31 (xyloketal B) were further to examine in the JC-1 mitochondrial membrane potential (MMP) assay of HUVECs using flow cytometry (FCM). The result indicated that 23 and 24 significantly inhibited H₂O₂-induced decrease of the cell mitochondrial membrane potential (ΔΨm) at 25 μM. Collectively, the protective effects of xyloketals on H₂O₂-induced endothelial cells may be generated from oxidation action by restraining ROS and reducing the MMP.     

Action of Clathrodin and Analogues on Voltage-Gated Sodium Channels

Clathrodin is a marine alkaloid and believed to be a modulator of voltage-gated sodium (Na_V ) channels. Since there is an urgent need for small molecule Na_V channel ligands as novel therapeutics, clathrodin could represent an interesting lead compound. Therefore, clathrodin was reinvestigated for its potency and Na_V channel subtype selectivity. Clathrodin and its synthetic analogues were subjected to screening on a broad range of Na_V channel isoforms, both in voltage clamp and patch clamp conditions. Even though clathrodin was not found to exert any activity, some analogues were capable of modulating the Na_V channels, hereby validating the pyrrole-2-aminoimidazole alkaloid structure as a core structure for future small molecule-based Na_V channel modulators.     

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.     

Selective Blocking Effects of 4,9-Anhydrotetrodotoxin,Purified from a Crude Mixture of Tetrodotoxin Analogues,on NaV1.6 Channels and Its Chemical Aspects

Tetrodotoxin (TTX) is a potent neurotoxin found in a number of marine creatures including the pufferfish, where it is synthesized by bacteria and accumulated through the food chain. It is a potent and selective blocker of some types of voltage-gated Na⁺ channel (Na_V channel). 4,9-Anhydrotetrodotoxin (4,9-anhydroTTX) was purified from a crude mixture of TTX analogues (such as TTX, 4-epiTTX, 6-epiTTX, 11-oxoTTX and 11-deoxyTTX) by the use of liquid chromatography-fluorescence detection (LC-FLD) techniques. Recently, it has been reported that 4,9-anhydroTTX selectively blocks the activity of Na_V1.6 channels with a blocking efficacy 40–160 times higher than that for other TTX-sensitive Na_V1.x channel isoforms. However, little attention has been paid to the molecular properties of the α-subunit in Na_V1.6 channels and the characteristics of binding of 4,9-anhydroTTX. From a functional point of view, it is important to determine the relative expression of Na_V1.6 channels in a wide variety of tissues. The aim of this review is to discuss briefly current knowledge about the pharmacology of 4,9-anhydroTTX, and provide an analysis of the molecular structure of native Na_V1.6 channels. In addition, chemical aspects of 4,9-anhydroTTX are briefly covered.     

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.     

Bioactive Alkaloids from the Sea: A Review

In our ongoing search for bioactive substances from marine organisms, novel alkaloids have been isolated. Pinnatoxins and pinnamine, potent shellfish poisons, were purified from the Okinawan bivalve Pinna muricata. Pinnatoxins activate Ca²⁺ channels. Halichlorine was isolated from the marine sponge Halichondria okadai. This compound inhibits the induction of VCAM-1. Drugs that block VCAM-1 may be useful for treating coronary artery diseases, angina, and noncardiovascular inflammatory diseases. Pinnaic acids, which are cPLA₂ inhibitors, were also obtained from P. muricata. Interestingly, the structures of pinnaic acids are closely related to that of halichlorine. Norzoanthamine hydrochloride, isolated from the colonial zoanthid Zoanthus sp., suppresses decreases in bone weight and strength in ovariectomized mice, and could be a good candidate for an osteoporotic drug. Ircinamine, purified from the marine sponge Ircinia sp., has a reactive thioester. Aburatubolactams, inhibitors of superoxide anion generation, were isolated from Streptomyces sp. This article covers the bioactive marine alkaloids that have been recently isolated by this research group.     

The Structural Basis and Functional Consequences of Interactions Between Tetrodotoxin and Voltage-Gated Sodium Channels

Tetrodotoxin (TTX) is a highly specific blocker of voltage-gated sodium channels. The dissociation constant of block varies with different channel isoforms. Until recently, channel resistance was thought to be primarily imparted by amino acid substitutions at a single position in domain I. Recent work reveals a novel site for tetrodotoxin resistance in the P-region of domain IV.     

Development of a Rapid Throughput Assay for Identification of hNav1.7 Antagonist Using Unique Efficacious Sodium Channel Agonist,Antillatoxin

Voltage-gated sodium channels (VGSCs) are responsible for the generation of the action potential. Among nine classified VGSC subtypes (Na_v1.1–Na_v1.9), Na_v1.7 is primarily expressed in the sensory neurons, contributing to the nociception transmission. Therefore Na_v1.7 becomes a promising target for analgesic drug development. In this study, we compared the influence of an array of VGSC agonists including veratridine, BmK NT1, brevetoxin-2, deltamethrin and antillatoxin (ATX) on membrane depolarization which was detected by Fluorescence Imaging Plate Reader (FLIPR) membrane potential (FMP) blue dye. In HEK-293 cells heterologously expressing hNa_v1.7 α-subunit, ATX produced a robust membrane depolarization with an EC₅₀ value of 7.8 ± 2.9 nM whereas veratridine, BmK NT1, and deltamethrin produced marginal response. Brevetoxin-2 was without effect on membrane potential change. The ATX response was completely inhibited by tetrodotoxin suggesting that the ATX response was solely derived from hNa_v1.7 activation, which was consistent with the results where ATX produced a negligible response in null HEK-293 cells. Six VGSC antagonists including lidocaine, lamotrigine, phenytoin, carbamazepine, riluzole, and 2-amino-6-trifluoromethylthiobenzothiazole all concentration-dependently inhibited ATX response with IC₅₀ values comparable to that reported from patch-clamp experiments. Considered together, we demonstrate that ATX is a unique efficacious hNa_v1.7 activator which offers a useful probe to develop a rapid throughput screening assay to identify hNa_v1.7 antagonists.     

Micrococcin P1 and P2 from Epibiotic Bacteria Associated with Isolates of Moorea producens from Kenya

Epibiotic bacteria associated with the filamentous marine cyanobacterium Moorea producens were explored as a novel source of antibiotics and to establish whether they can produce cyclodepsipeptides on their own. Here, we report the isolation of micrococcin P1 (1) (C₄₈H₄₉N₁₃O₉S₆; obs. m/z 1144.21930/572.60381) and micrococcin P2 (2) (C₄₈H₄₇N₁₃O₉S₆; obs. m/z 1142.20446/571.60370) from a strain of Bacillus marisflavi isolated from M. producens’ filaments. Interestingly, most bacteria isolated from M. producens’ filaments were found to be human pathogens. Stalked diatoms on the filaments suggested a possible terrestrial origin of some epibionts. CuSO₄·5H₂O assisted differential genomic DNA isolation and phylogenetic analysis showed that a Kenyan strain of M. producens differed from L. majuscula strain CCAP 1446/4 and L. majuscula clones. Organic extracts of the epibiotic bacteria Pseudoalteromonas carrageenovora and Ochrobactrum anthropi did not produce cyclodepsipeptides. Further characterization of 24 Firmicutes strains from M. producens identified extracts of B. marisflavi as most active. Our results showed that the genetic basis for synthesizing micrococcin P1 (1), discovered in Bacillus cereus ATCC 14579, is species/strain-dependent and this reinforces the need for molecular identification of M. producens species worldwide and their epibionts. These findings indicate that M. producens-associated bacteria are an overlooked source of antimicrobial compounds.     

Mechanisms for coping with submergence and waterlogging in rice

Rice (Oryza sativa L.), unlike other cereals, can grow well in paddy fields and is highly tolerant of excess water stress, from either submergence (in which part or all of the plant is under water) or waterlogging (in which excess water in soil limits gas diffusion). Rice handles submergence stress by internal aeration and growth controls. A quiescence strategy based on Submergence-1A (SUB1A) or an escape strategy based on SNORKEL1 (SK1) and SNORKEL2 (SK2) is used for the growth controls. On the other hand, rice handles waterlogging stress by forming lysigenous aerenchyma and a barrier to radial O₂ loss (ROL) in roots in order to supply O₂ to the root tip. In this article, we summarize recent advances in understanding the mechanisms of responding to excess water stresses (i.e., submergence and waterlogging) in rice and other gramineous plants.     

Extraction of Fucoxanthin from Raw Macroalgae excluding Drying and Cell Wall Disruption by Liquefied Dimethyl Ether

Macroalgae are one of potential sources for carotenoids, such as fucoxanthin, which are consumed by humans and animals. This carotenoid has been applied in both the pharmaceutical and food industries. In this study, extraction of fucoxanthin from wet brown seaweed Undaria pinnatifida (water content was 93.2%) was carried out with a simple method using liquefied dimethyl ether (DME) as an extractant in semi-continuous flow-type system. The extraction temperature and absolute pressure were 25 °C and 0.59 MPa, respectively. The liquefied DME was passed through the extractor that filled by U. pinnatifida at different time intervals. The time of experiment was only 43 min. The amount of fucoxanthin could approach to 390 μg/g dry of wet U. pinnatifida when the amount of DME used was 286 g. Compared with ethanol Soxhlet and supercritical CO₂ extraction, which includes drying and cell disruption, the result was quite high. Thus, DME extraction process appears to be a good method for fucoxanthin recovery from U. pinnatifida with improved yields.     

Heat stress effects around flowering on kernel set of temperate and tropical maize hybrids

Final kernel number in the uppermost ear of temperate maize (Zea mays L.) hybrids is smaller than the potential represented by the number of florets differentiated in this ear, and than the number of silks exposed from it (i.e., kernel set <1). This trend increases when stressful conditions affect plant growth immediately before (GS₁) or during (GS₂) silking, but the magnitude of change has not been documented for heat stress effects and hybrids of tropical background. In this work we evaluated mentioned traits in field experiments (Exp₁ and Exp₂), including (i) two temperature regimes, control and heated during daytime hours (ca. 33-40 °C at ear level), (ii) two 15-d periods during GS₁ and GS₂, and (iii) three hybrids (Te: temperate; Tr: tropical; TeTr: Te × Tr). We also measured crop anthesis and silking dynamics, silk exposure of individual plants, and the anthesis-silking interval (ASI). Three sources of kernel loss were identified: decreased floret differentiation, pollination failure, and kernel abortion. Heating affected all surveyed traits, but negative effects on flowering dynamics were larger (i) for anthesis than for silking with the concomitant decrease in ASI, and (ii) for GS₁ than for GS₂. Heat also caused a decrease in the number of (i) florets only when performed during GS₁ (−15.5% in Exp₁ and −9.1% in Exp₂), and only among Te and TeTr hybrids, (ii) exposed silks of all GS × Hybrid combinations, and (iii) harvestable kernels (mean of −51.8% in GS₁ and −74.5% in GS₂). Kernel abortion explained 95% of the variation in final kernel numbers (P < 0.001), and negative heat effects were larger on this loss (38.6%) than on other losses (≤11.3%). The tropical genetic background conferred an enhanced capacity for enduring most negative effects of heating.     

Relationship between P and N concentrations in maize and wheat leaves

Simple plant-based diagnostic tools can be used to determine crop P status. Our objectives were to establish the relationships between P and N concentrations of the uppermost collared leaf (P_L and N_L) of spring wheat (Triticum aestivum L.) and maize (Zea mays L.) during the growing season and, in particular, to determine the critical leaf P concentrations required to diagnose P deficiencies. Various N applications were evaluated over six site-years for wheat and eight site-years for maize (2004-2006) with adequate soil P for growth. Phosphorus and N concentrations of the uppermost collared leaf were determined weekly and the relationships between leaf N and P concentrations were established using only the sampling dates from the stem elongation stage for wheat and from the V8 stage of development for maize. Leaf P concentration generally decreased with decreasing N fertilization. Relationships between P_L and N_L concentrations (mg g⁻¹ DM) using all site-years and sampling dates were described by significant linear-plateau functions in both maize (P_L = 0.82 + 0.089 N_L if N_L ≤ 32.1 and P_L = 3.7 if N_L > 32.1; R² = 0.41; P < 0.001) and wheat (P_L = 0.02 + 0.106 N_L if N_L ≤ 33.2 and P_L = 3.5 if N_L > 33.2; R² = 0.42; P < 0.001). Variation among sampling dates in the relationships were noted. By restricting the sampling dates [413-496 growing degree days (5 °C basis) in wheat (i.e., stem elongation) and 1494-1579 crop heat units in maize (i.e., silking), relationships for wheat (P_L = 0.29 + 0.073 N_L, R² = 0.66; P < 0.001) and maize (P_L = 1.04 + 0.084 N_L, R² = 0.66; P < 0.001) were improved. In maize, expressing P and N concentrations on a leaf area basis (P_LA and N_LA) at silking further improved the relationship (P_LA = 0.002 + 0.101 N_LA, R² = 0.80; P < 0.001). Predictive models of critical P concentration as a function of N concentration in the uppermost collared leaf of wheat and maize were established which could be used for diagnostic purposes.     

Biological Properties of Fucoxanthin in Oil Recovered from Two Brown Seaweeds Using Supercritical CO2 Extraction

The bioactive materials in brown seaweeds hold great interest for developing new drugs and healthy foods. The oil content in brown seaweeds (Saccharina japonica and Sargassum horneri) was extracted by using environmentally friendly supercritical CO₂ (SC-CO₂) with ethanol as a co-solvent in a semi-batch flow extraction process and compared the results with a conventional extraction process using hexane, ethanol, and acetone mixed with methanol (1:1, v/v). The SC-CO₂ method was used at a temperature of 45 °C and pressure of 250 bar. The flow rate of CO₂ (27 g/min) was constant for the entire extraction period of 2 h. The obtained oil from the brown seaweeds was analyzed to determine their valuable compounds such as fatty acids, phenolic compounds, fucoxanthin and biological properties including antioxidant, antimicrobial, and antihypertension effects. The amounts of fucoxanthin extracted from the SC-CO₂ oils of S. japonica and S. horneri were 0.41 ± 0.05 and 0.77 ± 0.07 mg/g, respectively. High antihypertensive activity was detected when using mixed acetone and methanol, whereas the phenolic content and antioxidant property were higher in the oil extracted by SC-CO₂. The acetone–methanol mix extracts exhibited better antimicrobial activities than those obtained by other means. Thus, the SC-CO₂ extraction process appears to be a good method for obtaining valuable compounds from both brown seaweeds, and showed stronger biological activity than that obtained by the conventional extraction process.     

Construction and Characterization of an Escherichia coli Mutant Producing Kdo2-Lipid A

3-deoxy-d-manno-oct-2-ulosonic acid (Kdo)₂-lipid A is the conserved structure domain of lipopolysaccharide found in most Gram-negative bacteria, and it is believed to stimulate the innate immune system through the TLR4/MD2 complex. Therefore, Kdo₂-lipid A is an important stimulator for studying the mechanism of the innate immune system and for developing bacterial vaccine adjuvants. Kdo₂-lipid A has not been chemically synthesized to date and could only be isolated from an Escherichia coli mutant strain, WBB06. WBB06 cells grow slowly and have to grow in the presence of tetracycline. In this study, a novel E. coli mutant strain, WJW00, that could synthesize Kdo₂-lipid A was constructed by deleting the rfaD gene from the genome of E. coli W3110. The rfaD gene encodes ADP-l-glycero-d-manno-heptose-6-epimerase RfaD. Based on the analysis by SDS-PAGE, thin layer chromatography (TLC) and electrospray ionization mass spectrometry (ESI/MS), WJW00 could produce similar levels of Kdo₂-lipid A to WBB06. WJW00 cells grow much better than WBB06 cells and do not need to add any antibiotics during growth. Compared with the wild-type strain, W3110, WJW00 showed increased hydrophobicity, higher cell permeability, greater autoaggregation and decreased biofilm-forming ability. Therefore, WJW00 could be a more suitable strain than WBB06 for producing Kdo₂-lipid A and a good base strain for developing lipid A adjuvants.