The effect of membrane composition on the interaction between human CYP51 and its flavonoid inhibitor - luteolin 7,3'-disulfate.

Cytochromes P450 (CYP) are a family of membrane proteins involved in the production of endogenous molecules and the metabolism of xenobiotics. It is well-known that the composition of the membrane can influence the activity and orientation of CYP proteins. However, little is known about how membrane composition affects the ligand binding properties of CYP. In this study, we utilized surface plasmon resonance and fluorescence lifetime analysis to examine the impact of membrane micro-environment composition on the interaction between human microsomal CYP51 (CYP51A1) and its inhibitor, luteolin 7,3'-disulphate (LDS). We observed that membranes containing cholesterol or sphingomyelin exhibited the lowest apparent equilibrium dissociation constant for the CYP51A1-LDS complex. Additionally, the tendency for relation between kinetic parameters of the CYP51A1-LDS complex and membrane viscosity and overall charge was observed. These findings suggest that the specific composition of the membrane, particularly the presence of cholesterol and sphingomyelin, plays a vital role in regulating the interaction between CYP enzymes and their ligands.

Carotenoids from Starfish Patiria pectinifera: Therapeutic Activity in Models of Inflammatory Diseases.

The carotenoids mixture (MC) isolated from the starfish Patiria. pectinifera contains more than 50% astaxanthin, 4-6% each zeaxanthine and lutein, and less pharmacologically active components such as free fatty acids and their glycerides. Astaxanthin, the major component of MC, belongs to the xanthophyll class of carotenoids, and is well known for its antioxidant properties. In this work, in vitro and in vivo studies on the biological activity of MC were carried out. The complex was shown to exhibit anti-inflammatory, anti-allergic and cancer-preventive activity, without any toxicity at a dose of 500 mg/kg. MC effectively improves the clinical picture of the disease progressing, as well as normalizing the cytokine profile and the antioxidant defense system in the in vivo animal models of inflammatory diseases, namely: skin carcinogenesis, allergic contact dermatitis (ACD) and systemic inflammation (SI). In the skin carcinogenesis induced by 7,12-dimethylbenzanthracene, the incidence of papillomas was decreased 1.5 times; 1% MC ointment form in allergic contact dermatitis showed an 80% reduced severity of pathomorphological skin manifestations. Obtained results show that MC from starfish P. pectinifera is an effective remedy for the treatment and prevention of inflammatory processes.

Antitumor Properties of Matrikines of Different Origins: Prospects and Problems of Their Application.

Matrikines (MKs) can be a rich source of functional nutrition components and additional therapy, thereby contributing to human health care and reducing the risk of developing serious diseases, including cancer. Currently, functionally active MKs as products of enzymatic transformation by matrix metalloproteinases (MMPs) are used for various biomedical purposes. Due to the absence of toxic side effects, low species specificity, relatively small size, and presence of various targets at the cell membranes, MKs often exhibit antitumor properties and, therefore, are promising agents for antitumor combination therapy. This review summarizes and analyzes the current data on the antitumor activity of MKs of different origins, discusses the problems and prospects for their therapeutic use, and evaluates the experimental results of studying the antitumor properties of MKs from different echinoderm species generated with the help of a complex of proteolytic enzymes from red king crab Paralithodes camtschatica. Special attention is paid to the analysis of possible mechanisms of the antitumor action of various functionally active MKs, products of the enzymatic activity of various MMPs, and the existing problems for their use in antitumor therapy.

Nicotinic Acetylcholine Receptors Are Novel Targets of APETx-like Toxins from the Sea Anemone Heteractis magnifica.

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.

Comparative Study of the Pharmacological Properties of Luteolin and Its 7,3'-Disulfate.

The global spread of the metabolic syndrome, oncological and viral diseases forces researchers to pay increased attention to the secondary metabolites of marine hydrobionts, which often have a high therapeutic potential in the treatment of these pathologies and are effective components of functional food. The flavone luteolin (LT), as one of the most widely distributed and studied plant metabolites, is distinguished by a diverse spectrum of biological activity and a pleiotropic nature of the mechanism of action at the molecular, cellular and organismal levels. However, there is still practically no information on the spectrum of biological activity of its sulfated derivatives, which are widely represented in seagrasses of the genus Zostera. In the present work, a comparative study of the pharmacological properties of LT and its 7,3'-disulfate was carried out with a brief analysis of the special role of sulfation in the pharmacological activity of flavonoids.

The Sea Anemone Neurotoxins Modulating Sodium Channels: An Insight at Structure and Functional Activity after Four Decades of Investigation.

Many human cardiovascular and neurological disorders (such as ischemia, epileptic seizures, traumatic brain injury, neuropathic pain, etc.) are associated with the abnormal functional activity of voltage-gated sodium channels (VGSCs/NaVs). Many natural toxins, including the sea anemone toxins (called neurotoxins), are an indispensable and promising tool in pharmacological researches. They have widely been carried out over the past three decades, in particular, in establishing different NaV subtypes functional properties and a specific role in various pathologies. Therefore, a large number of publications are currently dedicated to the search and study of the structure-functional relationships of new sea anemone natural neurotoxins-potential pharmacologically active compounds that specifically interact with various subtypes of voltage gated sodium channels as drug discovery targets. This review presents and summarizes some updated data on the structure-functional relationships of known sea anemone neurotoxins belonging to four structural types. The review also emphasizes the study of type 2 neurotoxins, produced by the tropical sea anemone Heteractis crispa, five structurally homologous and one unique double-stranded peptide that, due to the absence of a functionally significant Arg14 residue, loses toxicity but retains the ability to modulate several VGSCs subtypes.

Deep-Sea Anemones Are Prospective Source of New Antimicrobial and Cytotoxic Compounds.

The peculiarities of the survival and adaptation of deep-sea organisms raise interest in the study of their metabolites as promising drugs. In this work, the hemolytic, cytotoxic, antimicrobial, and enzyme-inhibitory activities of tentacle extracts from five species of sea anemones (Cnidaria, orders Actiniaria and Corallimorpharia) collected near the Kuril and Commander Islands of the Far East of Russia were evaluated for the first time. The extracts of Liponema brevicorne and Actinostola callosa demonstrated maximal hemolytic activity, while high cytotoxic activity against murine splenocytes and Ehrlich carcinoma cells was found in the extract of Actinostola faeculenta. The extracts of Corallimorphus cf. pilatus demonstrated the greatest activity against Ehrlich carcinoma cells but were not toxic to mouse spleen cells. Sea anemones C. cf. pilatus and Stomphia coccinea are promising sources of antimicrobial and antifungal compounds, being active against Gram-positive bacteria Bacillus subtilis, Staphylococcus aureus, and yeast Candida albicans. Moreover, all sea anemones contain α-galactosidase inhibitors. Peptide mass fingerprinting of L. brevicorne and C. cf. pilatus extracts provided a wide range of peptides, predominantly with molecular masses of 4000-5900 Da, which may belong to a known or new structural class of toxins. The obtained data allow concluding that deep-sea anemones are a promising source of compounds for drug discovery.

Sea Anemone Kunitz-Type Peptides Demonstrate Neuroprotective Activity in the 6-Hydroxydopamine Induced Neurotoxicity Model.

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.

Kunitz-Type Peptides from the Sea Anemone Heteractis crispa Demonstrate Potassium Channel Blocking and Anti-Inflammatory Activities.

The Kunitz/BPTI peptide family includes unique representatives demonstrating various biological activities. Electrophysiological screening of peptides HCRG1 and HCRG2 from the sea anemone Heteractis crispa on six Kv1.x channel isoforms and insect Shaker IR channel expressed in Xenopus laevis oocytes revealed their potassium channels blocking activity. HCRG1 and HCRG2 appear to be the first Kunitz-type peptides from sea anemones blocking Kv1.3 with IC50 of 40.7 and 29.7 nM, respectively. In addition, peptides mainly vary in binding affinity to the Kv1.2 channels. It was established that the single substitution, Ser5Leu, in the TRPV1 channel antagonist, HCRG21, induces weak blocking activity of Kv1.1, Kv1.2, and Kv1.3. Apparently, for the affinity and selectivity of Kunitz-fold toxins to Kv1.x isoforms, the number and distribution along their molecules of charged, hydrophobic, and polar uncharged residues, as well as the nature of the channel residue at position 379 (Tyr, Val or His) are important. Testing the compounds in a model of acute local inflammation induced by the introduction of carrageenan administration into mice paws revealed that HCRG1 at doses of 0.1-1 mg/kg reduced the volume of developing edema during 24 h, similar to the effect of the nonsteroidal anti-inflammatory drug, indomethacin, at a dose of 5 mg/kg. ELISA analysis of the animals blood showed that the peptide reduced the synthesis of TNF-α, a pro-inflammatory mediator playing a leading role in the development of edema in this model.

Marine Polyhydroxynaphthoquinone, Echinochrome A: Prevention of Atherosclerotic Inflammation and Probable Molecular Targets.

The effect of low doses of echinochrome A (EchA), a natural polyhydroxy-1,4-naphthoquinone pigment from the sea urchin Scaphechinus mirabilis, has been studied in clinical trials, when it was used as an active substance of the drug Histochrome® and biologically active supplement Thymarin. Several parameters of lipid metabolism, antioxidant status, and the state of the immune system were analyzed in patients with cardiovascular diseases (CVD), including contaminating atherosclerosis. It has been shown that EchA effectively normalizes lipid metabolism, recovers antioxidant status and reduces atherosclerotic inflammation, regardless of the method of these preparations' administrations. Treatment of EchA has led to the stabilization of patients, improved function of the intracellular matrix and decreased epithelial dysfunction. The increased expression of surface human leukocyte antigen DR isotype (HLA-DR) receptors reflects the intensification of intercellular cooperation of immune cells, as well as an increase in the efficiency of processing and presentation of antigens, while the regulation of CD95 + expression levels suggests the stimulation of cell renewal processes. The immune system goes to a different level of functioning. Computer simulations suggest that EchA, with its aromatic structure of the naphthoquinone nucleus, may be a suitable ligand of the cytosolic aryl cell receptor, which affects the response of the immune system and causes the rapid expression of detoxification enzymes such as CYP and DT diaphorase, which play a protective role with CVD. Therefore, EchA possesses not only an antiradical effect and antioxidant activity, but is also a SOD3 mimetic, producing hydrogen peroxide and controlling the expression of cell enzymes through hypoxia-inducible factors (HIF), peroxisome proliferator-activated receptors (PPARs) and aryl hydrocarbon receptor (AhR).

APETx-Like Peptides from the Sea Anemone Heteractis crispa, Diverse in Their Effect on ASIC1a and ASIC3 Ion Channels.

Currently, five peptide modulators of acid-sensing ion channels (ASICs) attributed to structural class 1b of sea anemone toxins have been described. The APETx2 toxin is the first and most potent ASIC3 inhibitor, so its homologs from sea anemones are known as the APETx-like peptides. We have discovered that two APETx-like peptides from the sea anemone Heteractis crispa, Hcr 1b-3 and Hcr 1b-4, demonstrate different effects on rASIC1a and rASIC3 currents. While Hcr 1b-3 inhibits both investigated ASIC subtypes with IC50 4.95 ± 0.19 µM for rASIC1a and 17 ± 5.8 µM for rASIC3, Hcr 1b-4 has been found to be the first potentiator of ASIC3, simultaneously inhibiting rASIC1a at similar concentrations: EC50 1.53 ± 0.07 µM and IC50 1.25 ± 0.04 µM. The closest homologs, APETx2, Hcr 1b-1, and Hcr 1b-2, previously demonstrated the ability to inhibit hASIC3 with IC50 63 nM, 5.5, and 15.9 µM, respectively, while Hcr 1b-2 also inhibited rASIC1a with IC50 4.8 ± 0.3 µM. Computer modeling allowed us to describe the peculiarities of Hcr 1b-2 and Hcr 1b-4 interfaces with the rASIC1a channel and the stabilization of the expanded acidic pocket resulting from peptides binding which traps the rASIC1a channel in the closed state.

A new multigene HCIQ subfamily from the sea anemone Heteractis crispa encodes Kunitz-peptides exhibiting neuroprotective activity against 6-hydroxydopamine.

The Kunitz/BPTI-type peptides are ubiquitous in numerous organisms including marine venomous animals. The peptides demonstrate various biological activities and therefore they are the subject of a number of investigations. We have discovered a new HCIQ subfamily belonging to recently described multigene HCGS family of Heteractis crispa Kunitz-peptides. The uniqueness of this subfamily is that the HCIQ precursors contain a propeptide terminating in Lys-Arg (endopeptidase cleavage site) the same as in the neuro- and cytotoxin ones. Moreover, the HCIQ genes contain two introns in contrast to HCGS genes with one intron. As a result of Sanger and amplicon deep sequencings, 24 HCIQ isoforms were revealed. The recombinant peptides for the most prevalent isoform (HCIQ2c1) and for the isoform with the rare substitution Gly17Glu (HCIQ4c7) were obtained. They can inhibit trypsin with Ki 5.2 × 10-8 M and Ki 1.9 × 10-7 M, respectively, and interact with some serine proteinases including inflammatory ones according to the SPR method. For the first time, Kunitz-peptides have shown to significantly increase neuroblastoma cell viability in an in vitro 6-OHDA-induced neurotoxicity model being a consequence of an effective decrease of ROS level in the cells.

New Insights into the Type II Toxins from the Sea Anemone Heteractis crispa.

Toxins modulating NaV channels are the most abundant and studied peptide components of sea anemone venom. Three type-II toxins, δ-SHTX-Hcr1f (= RpII), RTX-III, and RTX-VI, were isolated from the sea anemone Heteractis crispa. RTX-VI has been found to be an unusual analog of RTX-III. The electrophysiological effects of Heteractis toxins on nine NaV subtypes were investigated for the first time. Heteractis toxins mainly affect the inactivation of the mammalian NaV channels expressed in the central nervous system (NaV1.1-NaV1.3, NaV1.6) as well as insect and arachnid channels (BgNaV1, VdNaV1). The absence of Arg13 in the RTX-VI structure does not prevent toxin binding with the channel but it has changed its pharmacological profile and potency. According to computer modeling data, the δ-SHTX-Hcr1f binds within the extracellular region of the rNaV1.2 voltage-sensing domain IV and pore-forming domain I through a network of strong interactions, and an additional fixation of the toxin at the channel binding site is carried out through the phospholipid environment. Our data suggest that Heteractis toxins could be used as molecular tools for NaV channel studies or insecticides rather than as pharmacological agents.

Magnificamide, a ß-Defensin-Like Peptide from the Mucus of the Sea Anemone Heteractis magnifica, Is a Strong Inhibitor of Mammalian α-Amylases.

Sea anemones' venom is rich in peptides acting on different biological targets, mainly on cytoplasmic membranes and ion channels. These animals are also a source of pancreatic α-amylase inhibitors, which have the ability to control the glucose level in the blood and can be used for the treatment of prediabetes and type 2 diabetes mellitus. Recently we have isolated and characterized magnificamide (44 aa, 4770 Da), the major α-amylase inhibitor of the sea anemone Heteractis magnifica mucus, which shares 84% sequence identity with helianthamide from Stichodactyla helianthus. Herein, we report some features in the action of a recombinant analog of magnificamide. The recombinant peptide inhibits porcine pancreatic and human saliva α-amylases with Ki's equal to 0.17 ± 0.06 nM and 7.7 ± 1.5 nM, respectively, and does not show antimicrobial or channel modulating activities. We have concluded that the main function of magnificamide is the inhibition of α-amylases; therefore, its functionally active recombinant analog is a promising agent for further studies as a potential drug candidate for the treatment of the type 2 diabetes mellitus.

Multigene Family of Pore-Forming Toxins from Sea Anemone Heteractis crispa.

Sea anemones produce pore-forming toxins, actinoporins, which are interesting as tools for cytoplasmic membranes study, as well as being potential therapeutic agents for cancer therapy. This investigation is devoted to structural and functional study of the Heteractis crispa actinoporins diversity. Here, we described a multigene family consisting of 47 representatives expressed in the sea anemone tentacles as prepropeptide-coding transcripts. The phylogenetic analysis revealed that actinoporin clustering is consistent with the division of sea anemones into superfamilies and families. The transcriptomes of both H. crispa and Heteractis magnifica appear to contain a large repertoire of similar genes representing a rapid expansion of the actinoporin family due to gene duplication and sequence divergence. The presence of the most abundant specific group of actinoporins in H. crispa is the major difference between these species. The functional analysis of six recombinant actinoporins revealed that H. crispa actinoporin grouping was consistent with the different hemolytic activity of their representatives. According to molecular modeling data, we assume that the direction of the N-terminal dipole moment tightly reflects the actinoporins' ability to possess hemolytic activity.

New APETx-like peptides from sea anemone Heteractis crispa modulate ASIC1a channels.

Sea anemones are an abundant source of various biologically active peptides. The hydrophobic 20% ethanol fraction of tropical sea anemone Heteractis crispa was shown to contain at least 159 peptide compounds including neurotoxins, proteinase and α-amylase inhibitors, as well as modulators of acid-sensing ion channels (ASICs). The three new peptides, π-AnmTX Hcr 1b-2, -3, and -4 (41 aa) (short names Hcr 1b-2, -3, -4), identified by a combination of reversed-phase liquid chromatography and mass spectrometry were found to belong to the class 1b sea anemone neurotoxins. The amino acid sequences of these peptides were determined by Edman degradation and tandem mass spectrometry. The percent of identity of Hcr 1b-2, -3, and -4 with well-known ASIC3 inhibitors Hcr 1b-1 from H. crispa and APETx2 from Anthopleura elegantissima is 95-78% and 46-49%, respectively. Electrophysiological experiments on homomeric ASIC channels expressed in Xenopus laevis oocytes establish that these peptides are the first inhibitors of ASIC1a derived from sea anemone venom. The major peptide, Hcr 1b-2, inhibited both rASIC1a (IC50 4.8 ±â€¯0.3 µM; nH 0.92 ±â€¯0.05) and rASIC3 (IC50 15.9 ±â€¯1.1 µM; nH 1.0 ±â€¯0.05). The maximum inhibition at saturating peptide concentrations reached 64% and 81%, respectively. In the model of acid-induced muscle pain Hcr 1b-2 was also shown to exhibit an antihyperalgesic effect, significantly reducing of the pain threshold of experimental animals.

Peptide fingerprinting of the sea anemone Heteractis magnifica mucus revealed neurotoxins, Kunitz-type proteinase inhibitors and a new ß-defensin α-amylase inhibitor.

Sea anemone mucus, due to its multiple and vital functions, is a valuable substance for investigation of new biologically active peptides. In this work, compounds of Heteractis magnifica mucus were separated by multistage liquid chromatography and resulting fractions were analyzed by MALDI-TOF MS. Peptide maps constructed according to the molecular masses and hydrophobicity showed presence of 326 both new and known peptides. Several major peptides from mucus were identified, including the sodium channel toxin RpII isolated earlier from H. magnifica, and four Kunitz-type proteinase inhibitors identical to H. crispa ones. Kunitz-type transcript diversity was studied and sequences of mature peptides were deduced. New ß-defensin α-amylase inhibitor, a homolog of helianthamide from Stichodactyla helianthus, was isolated and structurally characterized. Overall, H. magnifica is a source of biologically active peptides with great pharmacological potential. BIOLOGICAL SIGNIFICANCE: Proteinase and α-amylase inhibitors along with toxins are major components of H. magnifica mucus which play an important role in the successful existence of sea anemones. Obtained peptide maps create a basis for more accurate identification of peptides during future transcriptomic/genomic studies of sea anemone H. magnifica.

Kunitz-Type Peptide HCRG21 from the Sea Anemone Heteractis crispa Is a Full Antagonist of the TRPV1 Receptor.

Sea anemone venoms comprise multifarious peptides modulating biological targets such as ion channels or receptors. The sequence of a new Kunitz-type peptide, HCRG21, belonging to the Heteractis crispa RG (HCRG) peptide subfamily was deduced on the basis of the gene sequence obtained from the Heteractis crispa cDNA. HCRG21 shares high structural homology with Kunitz-type peptides APHC1-APHC3 from H. crispa, and clusters with the peptides from so named "analgesic cluster" of the HCGS peptide subfamily but forms a separate branch on the NJ-phylogenetic tree. Three unique point substitutions at the N-terminus of the molecule, Arg1, Gly2, and Ser5, distinguish HCRG21 from other peptides of this cluster. The trypsin inhibitory activity of recombinant HCRG21 (rHCRG21) was comparable with the activity of peptides from the same cluster. Inhibition constants for trypsin and α-chymotrypsin were 1.0 × 10-7 and 7.0 × 10-7 M, respectively. Electrophysiological experiments revealed that rHCRG21 inhibits 95% of the capsaicin-induced current through transient receptor potential family member vanilloid 1 (TRPV1) and has a half-maximal inhibitory concentration of 6.9 ± 0.4 µM. Moreover, rHCRG21 is the first full peptide TRPV1 inhibitor, although displaying lower affinity for its receptor in comparison with other known ligands. Macromolecular docking and full atom Molecular Dynamics (MD) simulations of the rHCRG21-TRPV1 complex allow hypothesizing the existence of two feasible, intra- and extracellular, molecular mechanisms of blocking. These data provide valuable insights in the structural and functional relationships and pharmacological potential of bifunctional Kunitz-type peptides.

Glycosides from edible sea cucumbers stimulate macrophages via purinergic receptors.

Since ancient times, edible sea cucumbers have been considered a jewel of the seabed and used in Asian folk medicine for stimulation of resistance against different diseases. However, the power of this sea food has not been established on a molecular level. A particular group of triterpene glycosides was found to be characteristic metabolites of the animals, responsible for this biological action. Using one of them, cucumarioside A2-2 (CA2-2) from the edible Cucumaria japonica species as an example as well as inhibitory analysis, patch-clamp on single macrophages, small interfering RNA technique, immunoblotting, SPR analysis, computer modeling and other methods, we demonstrate low doses of CA2-2 specifically to interact with P2X receptors (predominantly P2X4) on membranes of mature macrophages, enhancing the reversible ATP-dependent Ca2+ intake and recovering Ca2+ transport at inactivation of these receptors. As result, interaction of glycosides of this type with P2X receptors leads to activation of cellular immunity.

New Kunitz-Type HCRG Polypeptides from the Sea Anemone Heteractis crispa.

Sea anemones are a rich source of Kunitz-type polypeptides that possess not only protease inhibitor activity, but also Kv channels toxicity, analgesic, antihistamine, and anti-inflammatory activities. Two Kunitz-type inhibitors belonging to a new Heteractis crispa RG (HCRG) polypeptide subfamily have been isolated from the sea anemone Heteractis crispa. The amino acid sequences of HCRG1 and HCRG2 identified using the Edman degradation method share up to 95% of their identity with the representatives of the HCGS polypeptide multigene subfamily derived from H. crispa cDNA. Polypeptides are characterized by positively charged Arg at the N-terminus as well as P1 Lys residue at their canonical binding loop, identical to those of bovine pancreatic trypsin inhibitor (BPTI). These polypeptides are shown by our current evidence to be more potent inhibitors of trypsin than the known representatives of the HCGS subfamily with P1Thr. The kinetic and thermodynamic characteristics of the intermolecular interactions between inhibitors and serine proteases were determined by the surface plasmon resonance (SPR) method. Residues functionally important for polypeptide binding to trypsin were revealed using molecular modeling methods. Furthermore, HCRG1 and HCRG2 possess anti-inflammatory activity, reducing tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) secretions, as well as proIL-1ß expression in lipopolysaccharide (LPS)-activated macrophages. However, there was no effect on nitric oxide (NO) generation.