Reducing polysaccharide degradation through inhibition of α-amylases and α-glucosidases is one of the strategies for glycemic control in the treatment of diabetes mellitus. Here we report the identification and characterization of a new β-defensin-like miniprotein magnificamide-2 (Mgf-2), one of the most potent inhibitors of mammalian α-amylases with subnanomolar binding affinity (Ki of recombinant Mgf-2 is 0.029 nM against human pancreatic α-amylase, 0.057 nM against human salivary α-amylase, and 0.011 nM against porcine pancreatic α-amylase). Using nuclear magnetic resonance (NMR) spectroscopy, ensemble protein-protein docking, and molecular dynamics (MD) simulations, the key structural determinants responsible for this ultra-tight inhibition were identified, which was subsequently confirmed by site-directed mutagenesis experiments. The β1-β2 loop containing the inhibitory motif 7YIYH10 plays a crucial role in the inhibitor-enzyme interaction by forming an extensive hydrophobic interface with the hydrophobic "rim" around the α-amylase active site. The data obtained enhance our understanding of the molecular mechanisms underlying high-affinity α-amylase inhibition and provide valuable insights into the design of novel proteinaceous α-amylase inhibitors for the treatment and prevention of postprandial hyperglycemia.

Psoriasis and allergic contact dermatitis (ACD) are the most common chronic inflammatory diseases, which are accompanied by epithelial alterations and a T cell-mediated immunopathology. In this study, we investigated the anti-ACD and anti-psoriasis effects of sea anemone Heteractis magnifica peptide HCRG21, a blocker of the TRPV1 channel, in 2,4-dinitrofluorobenzene (DNFB)- and imiquimod (IMQ)-induced mouse models, respectively. We found that topical application of 0.005–0.1% HCRG21 gels normalized hematological and immunological blood parameters in mice, significantly reduced the severity of ACD- and psoriasiform-like skin lesions, and increased the rate of tissue repair. The use of 0.005 and 0.05% HCRG21 gels decreased the production of IL-23-A and macrophage-derived chemokine (MDC) proteins in blood plasma, reduced the expression of Tnf, Il1β, Il6, Il23a, and Il17a genes, but increased the levels of the Il10 gene in scabs and/or blood of IMQ-treated mice. On the other hand, topical application of 0.05 and 0.1% HCRG21 reduced the expression of Il6 and Il23a in the DNFB-treated mice’s blood and it had no significant effects on TNF-α and IL-1β production. Thus, HCRG21 has the potential to be a treatment for psoriasis and dermatitis due to its potent anti-inflammatory properties. This effect is achieved by reducing pro-inflammatory cytokines associated with TRPV1 and normalizing immune cell levels in the bloodstream. This, in turn, leads to a decrease in clinical symptoms and an improvement in skin healing.

Voltage-gated sodium channels (NaV) are molecular targets for the development of drugs for the treatment of diseases such as epilepsy, neuropathic pain, long QT syndrome, etc., as well as for insecticides. Therefore, the search for novel selective NaV channel ligands is relevant. Using amplicon deep sequencing of tentacle cDNA libraries from sea anemones Heteractis magnifica, 36 transcripts related to RpIII neurotoxin, a NaV channel modulators, were revealed. The recombinant RpIII was moderately toxic for mice (LD50 0.030 ± 0.004 mg/kg) but did not demonstrate any activity towards NaV in human SH-SY5Y cells. The toxin inhibited inactivation of heterologously expressed mammalian, insect, and arachnid NaV channels with higher specificity to insect channels. Cockroach (Blattella germanica) sodium channel BgNaV1 (EC50 of 2.4 ± 0.2 nM) and yellow fever mosquito (Aedes aegypti) channel AaNaV1 (EC50 of 1.5 ± 0.3 nM) were the most sensitive to RpIII, while mammals NaV had EC50 values above 100 nM except mNaV1.6 (EC50 of 43.8 ± 3.6 nM). The low nanomolar RpIII affinity to insect AaNaV1 may be explained by the extensive intermolecular contacts found by docking study. According to the predicted data, the toxin lands on the ion channel between voltage-sensing domain IV and pore domain I, also known as toxin site 3, followed by stabilizing the channels in the open state what was measured at electrophysiological experiments.

The gene structure and amino acid sequences of the precursor proteins of the sea anemone Heteractis magnifica neurotoxin RpII, a modulator of voltage-gated sodium channels, have been determined. A technology has been developed for the recombinant production of RpII in the Escherichia coli BL21(DE3) expression system based on the pET32b vector containing the thioredoxin gene, which opens up the possibility of obtaining a pure neurotoxin for further structural and/or pharmacological studies. The neurotoxin exhibits a neurotropic effect when administered intravenously to mice in doses greater than 1 mg kg−1, but is not lethal in doses up to 6 mg kg−1 and, in addition, RpII in the nm range does significantly increase the metabolism of human malignant glioblastoma U87-MG.

NMDA receptors (NMDARs) belong to the ionotropic glutamate receptor (iGluR) family and are important for synaptic plasticity, learning and memory. Their biophysical and pharmacological characterization has been carried out in a few model organisms, such as nematode, fly, rat and human. We turned our attention to crustaceans, which have also been used in neurophysiological experiments, but whose NMDARs have not been investigated. We compiled NMDAR sequences from NCBI and Crustybase and generated an iGluR phylogeny.

Neurotransmitter ligands electrically excite neurons by activating ionotropic glutamate receptor (iGluR) ion channels. Knowledge of the iGluR amino acid residues that dominate ligand-induced activation would enable the prediction of function from sequence. We therefore explored the molecular determinants of activity in rat N-methyl-D-aspartate (NMDA)-type iGluRs (NMDA receptors), complex heteromeric iGluRs comprising two glycine-binding GluN1 and two glutamate-binding GluN2 subunits, using amino acid sequence analysis, mutagenesis, and electrophysiology. We find that a broadly conserved aspartate residue controls both ligand potency and channel activity, to the extent that certain substitutions at this position bypass the need for ligand binding in GluN1 subunits, generating NMDA receptors activated solely by glutamate. Furthermore, we identify a homomeric iGluR from the placozoan Trichoplax adhaerens that has utilized native mutations of this crucial residue to evolve into a leak channel that is inhibited by neurotransmitter binding, pointing to a dominant role of this residue throughout the iGluR superfamily.

Diabetes mellitus is one of the most serious diseases of our century. The drugs used are limited or have serious side effects. The search for new sources of compounds for effective treatment is relevant. Magnificamide, a peptide inhibitor of mammalian α-amylases, isolated from the venom of sea anemone Heteractis magnifica, can be used for the control of postprandial hyperglycemia in diabetes mellitus. Using the RACE approach, seven isoforms of magnificamide were detected in H. magnifica tentacles. The exon–intron structure of magnificamide genes was first established, and intron retention in the mature peptide-encoding region was revealed. Additionally, an α-amylase inhibitory domain was discovered in the mucins of some sea anemones. According to phylogenetics, sea anemones diverge into two groups depending on the presence of β-defensin-like α-amylase inhibitors and/or mucin-inhibitory domains. It is assumed that the intron retention phenomenon leads to additional diversity in the isoforms of inhibitors and allows for its neofunctionalization in sea anemone tentacles. Bioprospecting of sea anemones of the order Actiniaria for β-defensin-like α-amylase inhibitors revealed a diversity of inhibitory sequences that represents a starting point for the design of effective glucose-lowering drugs.

Purinergic P2X7 receptors (P2X7) have now been proven to play an important role and represent an important therapeutic target in many pathological conditions including neurodegeneration. Here, we investigated the impact of peptides on purinergic signaling in Neuro-2a cells through the P2X7 subtype in in vitro models. We have found that a number of recombinant peptides, analogs of sea anemone Kunitz-type peptides, are able to influence the action of high concentrations of ATP and thereby reduce the toxic effects of ATP. The influx of calcium, as well as the fluorescent dye YO-PRO-1, was significantly suppressed by the studied peptides. Immunofluorescence experiments confirmed that the peptides reduce the P2X7 expression level in neuronal Neuro-2a cells. Two selected active peptides, HCRG1 and HCGS1.10, were found to specifically interact with the extracellular domain of P2X7 and formed stable complexes with the receptor in surface plasmon resonance experiments. The molecular docking approach allowed us to establish the putative binding sites of the most active HCRG1 peptide on the extracellular domain of the P2X7 homotrimer and propose a mechanism for regulating its function. Thus, our work demonstrates the ability of the Kunitz-type peptides to prevent neuronal death by affecting signaling through the P2X7 receptor.

α-Amylase is a generally acknowledged molecular target of a distinct class of antidiabetic drugs named α-glucosidase inhibitors. This class of medications is scarce and rather underutilized, and treatment with current commercial drugs is accompanied by unpleasant adverse effects. However, mammalian α-amylase inhibitors are abundant in nature and form an extensive pool of high-affinity ligands that are available for drug discovery. Individual compounds and natural extracts and preparations are promising therapeutic agents for conditions associated with impaired starch metabolism, e.g., diabetes mellitus, obesity, and other metabolic disorders. This review focuses on the structural diversity and action mechanisms of active natural products with inhibitory activity toward mammalian α-amylases, and emphasizes proteinaceous inhibitors as more effective compounds with significant potential for clinical use.

Diabetes mellitus is a serious threat to human health in both developed and developing countries. Optimal disease control requires the use of a diet and a combination of several medications, including oral hypoglycemic agents such as alpha-glucosidase inhibitors. Currently, the arsenal of available drugs is insufficient, which determines the relevance of studying new potent alpha-amylase inhibitors. We implemented the recombinant production of sea anemone derived alpha-amylase inhibitor magnificamide in Escherichia coli. Peptide was isolated by a combination of liquid chromatography techniques. Its folding and molecular weight was proved by H-1 NMR and mass spectrometry. The K-i value of magnificamide against human pancreatic alpha-amylase is 3.1 nM according to Morrison equation for tight binding inhibitors. Our study of the thermodynamic characteristics of binding of magnificamide to human salivary and pancreatic alpha-amylases by isothermal titration calorimetry showed the presence of different binding mechanisms with Kd equal to 0.11 mu M and 0.1 nM, respectively. Experiments in mice with streptozotocin-induced diabetes mimicking diabetes mellitus type 1 were used to study the efficiency of magnificamide against postprandial hyperglycemia. It was found that at a dose of 0.005 mg kg(-1), magnificamide effectively blocks starch breakdown and prevents the development of postprandial hyperglycemia in T1D mice. Our results demonstrated the therapeutic potential of magnificamide for the control of postprandial hyperglycemia.

The nicotinic acetylcholine receptors (nAChRs) are prototypical ligand-gated ion channels, provide cholinergic signaling, and are modulated by various venom toxins and drugs in addition to neurotransmitters. Here, four APETx-like toxins, including two new toxins, named Hmg 1b-2 Metox and Hmg 1b-5, were isolated from the sea anemone Heteractis magnifica and characterized as novel nAChR ligands and acid-sensing ion channel (ASIC) modulators. All peptides competed with radiolabeled α-bungarotoxin for binding to Torpedo californica muscle-type and human α7 nAChRs. Hmg 1b-2 potentiated acetylcholine-elicited current in human α7 receptors expressed in Xenopus laevis oocytes. Moreover, the multigene family coding APETx-like peptides library from H. magnifica was described and in silico surface electrostatic potentials of novel peptides were analyzed. To explain the 100% identity of some peptide isoforms between H. magnifica and H. crispa, 18S rRNA, COI, and ITS analysis were performed. It has been shown that the sea anemones previously identified by morphology as H. crispa belong to the species H. magnifica.

A number of studies confirmed the involvement of transient receptor potential vanilloid (TRPV) and acid-sensing (ASIC) ion channels in the physiological processes associated with the development of anxiety disorders. This makes their ligands new potential anxiolytic agents. We examined the efficacy of two peptides from the sea anemone Heteractis crispa, Hcr 1b-2 and HCRG21, affecting ASIC1a and TRPV1 channels, respectively, in the open field and elevated plus maze tests. According to the obtained data, HCRG21 significantly decreases both the level of anxiety and stimulates the activity of animals at doses of 0.01–1 mg/kg, whereas Hcr 1b-2 has a weak anxiolytic effect only at a dose of 0.1 mg/kg. The pharmacodynamic study showed that the HCRG21 has an anxiolytic effect for 2 h, and its effectiveness is higher than that of the reference drug.

The use of a high-fat diet, along with streptozotocin administration, can provide more profound insight into the mechanism of development of complications in diabetes, as well as their treatment. High-fat diet given over 3 weeks before intraperitoneal injection of streptozotocin in a dose of 40 mg/kg promoted the appearance of hyperglycemia in Wistar rats. The biochemical analysis of blood serum revealed increased levels of urea, triglycerides, cholesterol, AST, ALT, and concentration of inorganic phosphates and K + ions in the high-fat diet group in comparison with the control. Both the biochemical analysis of the blood and histological analysis showed more pronounced abnormalities in rats receiving high-fat diet in comparison with animals receiving standard ration. These changes are the early markers for the development of nephropathy, impaired liver function, and microvascular disorders typical of patients with diabetes mellitus.

Currently the TRPV1 (transient receptor potential vanilloid type 1) channel is considered to be one of the main targets for pro-inflammatory mediators including TNF-α. Similarly, the inhibition of TRPV1 activity in the peripheral nervous system affects pro-inflammatory mediator production and enhances analgesia in total. In this study, the analgesic and anti-inflammatory effects of HCRG21, the first peptide blocker of TRPV1, were demonstrated in a mice model of carrageenan-induced paw edema. HCRG21 in doses of 0.1 and 1 mg/kg inhibited edema formation compared to the control, demonstrated complete edema disappearance in 24 h in a dose of 1 mg/kg, and effectively reduced the productionof TNF-α in both doses examined. ELISA analysis of blood taken 24 h after carrageenan administration showed a dramatic cytokine value decrease to 25 pg/mL by HCRG21 versus 100 pg/mL in the negative control group, which was less than the TNF-α level in the intact group (40 pg/mL). The HCRG21 demonstrated potent analgesic effects on the models of mechanical and thermal hyperalgesia in carrageenan-induced paw edema. The HCRG21 relief effect was comparable to that of indomethacin taken orally in a dose of 5 mg/kg, but was superior to this nonsteroidal anti-inflammatory drug (NSAID) in duration (which lasted 24 h) in the mechanical sensitivity experiment. The results confirm the existence of a close relationship between TRPV1 activity and TNF-α production once again, and prove the superior pharmacological potential of TRPV1 blockers and the HCRG21 peptide in particular.

Although TRPV1 ion channel has been attracting researchers’ attention for many years, its functions in animal organisms, the principles of regulation, and the involvement in pathological processes have not yet been fully clarified. Mutagenesis experiments and structural studies have identified the structural features of the channel and binding sites for its numerous ligands; however, these studies are far from conclusion. This review summarizes recent achievements in the TRPV1 research with special focus on structural and functional studies of the channel and on its ligands, which are extremely diverse in their nature and interaction specificity to TRPV1. Particular attention was given to the effects of numerous endogenous agonists and antagonists that can fine-tune the channel sensitivity to its usual activators, such as capsaicin, heat, acids, or their combination. In addition to the pain sensing not covered in this review, the TRPV1 channel was found to be involved in the regulation of many important physiological and pathological processes and, therefore, can be considered as a promising therapeutic target in the treatment of various diseases, such as pneumonia, ischemia, diabetes, epilepsy, schizophrenia, psoriasis, etc.

Kunitz-type peptides from venomous animals have been known to inhibit different proteinases and also to modulate ion channels and receptors, demonstrating analgesic, anti-inflammatory, anti-histamine and many other biological activities. At present, there is evidence of their neuroprotective effects. We have studied eight Kunitz-type peptides of the sea anemone Heteractis crispa to find molecules with cytoprotective activity in the 6-OHDA-induced neurotoxicity model on neuroblastoma Neuro-2a cells. It has been shown that only five peptides significantly increase the viability of neuronal cells treated with 6-OHDA. The TRPV1 channel blocker, HCRG21, has revealed the neuroprotective effect that could be indirect evidence of TRPV1 involvement in the disorders associated with neurodegeneration. The pre-incubation of Neuro-2a cells with HCRG21 followed by 6-OHDA treatment has resulted in a prominent reduction in ROS production compared the untreated cells. It is possible that the observed effect is due to the ability of the peptide act as an efficient free-radical scavenger. One more leader peptide, InhVJ, has shown a neuroprotective activity and has been studied at concentrations of 0.01–10.0 µM. The target of InhVJ is still unknown, but it was the best of all eight homologous peptides in an absolute cell viability increment on 38% of the control in the 6-OHDA-induced neurotoxicity model. The targets of the other three active peptides remain unknown.