Pharmacologically Active Peptides of the Sea Anemone Heteractis Crispa and their Biological Templates

It is determined that different venomous terrestrial and marine animals (scorpions, spiders, snakes, centipedes, sea snakes, cone snails, jellyfishes, sea anemones) produce hundreds of biologically active peptides including great number of proteinaceous compounds with large structural and functional diversity [1]. They are used by venomous organisms for protection against their predators and for attacking preys. During the divergent evolution many active peptides belonging to multigene families formed combinatorial libraries representing highly homologous sets of peptide isoforms usually aimed at wide variety of biological targets such as voltagegated sodium and potassium (NaVs and KVs) channels, Transient receptor potential and histamine H1 receptors and many others, all of them are responsible for a physiological and pathophysiological state of an organism, particularly, excitable and non-excitable cells of neuromuscular, neuronal, heart systems etc. [2]. Their dysfunctions and disturbances, as known, result in a number of channelopathies [3]. Many of the venomous peptides being very effective ligands restore the functional activity of different channel subtypes and are currently recognized as pharmacological or therapeutics agents, which are at various stages of preclinical and clinical trials [3].


Introduction
It is determined that different venomous terrestrial and marine animals (scorpions, spiders, snakes, centipedes, sea snakes, cone snails, jellyfishes, sea anemones) produce hundreds of biologically active peptides including great number of proteinaceous compounds with large structural and functional diversity [1]. They are used by venomous organisms for protection against their predators and for attacking preys. During the divergent evolution many active peptides belonging to multigene families formed combinatorial libraries representing highly homologous sets of peptide isoforms usually aimed at wide variety of biological targets such as voltagegated sodium and potassium (NaVs and KVs) channels, Transient receptor potential and histamine H1 receptors and many others, all of them are responsible for a physiological and pathophysiological state of an organism, particularly, excitable and non-excitable cells of neuromuscular, neuronal, heart systems etc. [2]. Their dysfunctions and disturbances, as known, result in a number of channelopathies [3]. Many of the venomous peptides being very effective ligands restore the functional activity of different channel subtypes and are currently recognized as pharmacological or therapeutics agents, which are at various stages of preclinical and clinical trials [3].
One of the most studied marine producers of biologically active compounds with a high pharmacological potential is the sea anemones [4,5], including the specimens of Heteractis genus. Two widespread tropical species, the sea anemone Heteractis crispa (formerly Radianthus macrodactylus) and Heteractis magnifica, are known to produce various biologically active peptides such as three protein types of structurally different toxins: the structural type II neurotoxins modulating some Na V channel subtypes [6], APETx-like peptides blocking proton-activated ASICs channels [7] (both with β-defensin like fold), and α-pore-forming toxins (PFTs, named actinoporins with β-strand+α-helix fold) forming pores in cytoplasmic sphingomyelin-containing membranes [8]. Besides, H. crispa and H. magnifica represent the richest source of serine protease inhibitors (having Kunitz fold) and of α-amylase inhibitors (with β-defensin-like fold) [9][10][11][12][13]. The purpose of this minireview is to demonstrate a scientific and pharmacological potential of a huge number of the produced by H. crispa peptides, which biological targets have been shown by now as a wide range of prey's ion channels, receptors, and serine protease inhibitors.
RTX-I -RTX-V have been shown to be highly toxic for mammalians, their mechanism of an action on different Na V subtypes is currently actively studied in silico and in vitro. We have found that Radianthus neurotoxins modify mammalian Na V 1.1, 1.2, 1.3, 1.6 channel subtypes and insect, BgNa V 1 [19]. These toxins significantly slow down the kinetic of a channel inactivation without changing of their activation and prolong a channel action potential, what leads to a massive release of neurotransmitters from the nerve terminals (the data not published). Due to the positive inotropic effect, the neurotoxins can have a cardio stimulating effect, which can be used in a medical practice in a number of extreme situations. Unfortunately, a significant obstruction for using of these toxins is their high toxicity for mammals. Possibly, obtaining of non-toxic mutant analogs will allow overcoming this obstacle and leading to their subsequent clinical use.

Heteractis APETx2-Like Inhibitors of ASICs Channels
The search and study of ASICs channel ligands represent the great interest for pharmacology and neurophysiology also because these channels are involved in a number of pathological processes including neurological and psychiatric diseases [20]. ASIC-targeting ligands can act on the molecular mechanisms of pain generation.
They specifically affect the channel subtypes functional activity through their significant potentiating or partial inhibiting [21].

Multigene PFTs of H. crispa and their Biological Template
Native pore-forming toxins (α-PFT) or actinoporins (20kDa) previously obtained from the whole body of H. crispa and Oulactis orientalis (Figure 3a) [8] along with the peptide toxins described above have an important scientific potential because they have the unusual spatial structure [26] (Figure 3b), which stipulates the formation of functionally active protein-lipid pores due to an evolutionary tuned mechanism of interaction with biological targets, cytoplasmic membranes (Figure 3c) [27]. The duality of the nature of actinoporins interaction with targets allows researchers to use them as molecular tools for studying of the cytoplasmic membranes topology and mechanisms of functioning [28]. Recent years successful attempts have been made to create therapeutic agents with anti-tumor, antimicrobial, anti-parasitic activity on the basis of actinoporin structures [29][30][31]. We have established the presence of H. crispa actinoporin multigene family consisting of 47 representatives [32]. It was shown that the cytotoxic (hemolytic) activity of several native and recombinant members derived from the DNA sequences correlates with the hydrophobicity of their amphiphilic N-termini (28 aa) and the direction of their dipole moments [32]. (c) The model of FraC pore-formation: an actinoporin monomer binds a membrane and promotes protein-protein interaction between two ones producing the formation of a dimer; then, as a result of dimer interaction with monomers or dimer(s) and due to the insertion of extended actinoporin N-ends in the lipid bilayer, an prepare is first formed, and then a functional pore is created [28].
At the same time, actinoporin binding affinity to biological targets, cytoplasmic membranes, is assigned not only by the interaction of actinoporin POC-binding site with membrane sphingomyelin and phosphatidylcholine, but also by alternative ones of the actinoporin RGD-motive with membrane integrins [33].
Earlier we showed that the cytotoxic effect (IC 50

Sea Anemone Kunitz-Type Peptides as Blockers of TRPV1
Today vanilloid receptor TRPV1 is known as a key player of many important physiological processes occurring in almost all organs and tissues. Being the sensors of temperature, mechanical and chemical stimuli (including noxious ones), this receptor is involved in the development of different pain states, which, as rule, accompanied by inflammatory pain associated with diseases of cardiovascular, gastrointestinal, urinary, respiratory, central nervous systems [35]. In this connection, the search and study of action mechanisms of the TRPV1 modulators (both agonists and antagonists) and their alternative role as anti-inflammatory agents, in addition to analgesic one, represent one of the most important tasks of life sciences [36]. To date, most known TRPV1-targeting agonists and antagonists are the low-molecular compounds, many of which undergo, despite the hyperthermia effect, preclinical and clinical trials [37].
Recently, we have shown that rHCRG21 is the most effective peptide antagonist of TRPV1 [41]. "So, APHC1 inhibited ~32% of capsaicininduced currents at more than 200 nM [38], while APHC3 had a lower inhibitory effect (25%) even at higher concentrations more than 300 nM [45]. Unlike them, rHCRG21 has been shown to be a full blocker of TRPV1 (IC 50 6,9 μM, maximal inhibition 95%) [41]. APHC1-APHC3, so called "analgesic peptides", show in vivo an analgesic activity at doses of 0.01-0.1 mg/kg [38,39,45] due to their inhibition of TRPV1. With that, these TRPV1 blockers demonstrate a significant analgesic effect without hyperthermia [39,45]." Some other representatives of the combinatorial library, HCGS1.10, HCGS 1.19, HCGS 1.20, HCGS1.36 [10], have been shown to possess in vivo a pronounced analgesic effect in the model of thermal pain stimulation ("tail withdrawal") [47]. In addition, when studying an anti-inflammatory activity on RAW 264.7 macrophages in the presence of lipopolysaccharide (LPS), rHCGS1.20 peptide shows an anti-inflammatory activity at the concentration of 10μM, it significantly reduces the level of NO by 25% [50]. The peptides HCRG1 and HCRG2 reduce the expression of inflammatory mediators, interleukin-1β (proIL-1β) in LPS-activated J774A.1 macrophage, as well as the secretion of interleukin-6 (IL-6) and the tumor necrosis factor TNF-α. However, these peptides do not reduce LPS-induced formation of NO [12]. Thus, the Kunitz-type peptides of H. crispa are promising analgesics and anti-inflammatory agents for scientific researches and pharmacology.

Conclusion
The submitted minireview and the data presented in it do not contradict the long-established view that peptides from the sea anemone venom are highly selective, potent, and relatively safe compounds as potential therapeutic agents. This work was partially supported by research projects RFBR projects No 18-04-00631, No 18-38-00387, and "ERA"_RUS_ST2017-228.