Venomous animals from distinct phyla such as spiders, scorpions, snakes, cone snails, or sea anemones produce small toxic proteins interacting with a variety of cell targets. Their bites often cause pain. One of the ways of pain generation is the activation of TRPV1 channels. Screening of 30 different venoms from spiders and sea anemones for modulation of TRPV1 activity revealed inhibitors in tropical sea anemone Heteractis crispa venom. Several separation steps resulted in isolation of an inhibiting compound. This is a 56-residue-long polypeptide named APHC1 that has a Bos taurus trypsin inhibitor (BPTI)/Kunitztype fold, mostly represented by serine protease inhibitors and ion channel blockers. APHC1 acted as a partial antagonist of capsaicin-induced currents (32 ؎ 9% inhibition) with half-maximal effective concentration (EC 50 ) 54 ؎ 4 nM. In vivo, a 0.1 mg/kg dose of APHC1 significantly prolonged tail-flick latency and reduced capsaicin-induced acute pain. Therefore, our results can make an important contribution to the research into molecular mechanisms of TRPV1 modulation and help to solve the problem of overactivity of this receptor during a number of pathological processes in the organism.During the evolutionary process, different poisonous animals combined a set of bioactive compounds in their venoms used mainly to paralyze prey and/or as a defense against predators (1, 2). Bites of these creatures may induce inflammation, pain, tissue necrosis, allergic reactions, and neurotoxic effects such as convulsions, paralysis, respiratory failure, and cardiovascular stroke (3). Numerous toxic peptides are found within these venoms, and some of them can discriminate between closely related cellular targets that make them attractive for drug development and scientific use (4). Molecules accounting for lethal and inflammation effects of venoms have been extensively characterized, but less is known about the properties of other compounds. We concentrated on searching the compounds able to reduce TRPV1 2 conductivity. These receptors are expressed in mammalians in small and medium size dorsal root ganglion neurons and are localized in peripheral and central neuronal system (5-7). At present, it is accepted that TRPV1 receptors are molecular integrators of pain stimulus and initiate neuronal response during inflammation. Experiments with knock-out mice lacking the gene of vanilloid receptor clearly demonstrate its role in pain perception (8, 9). Since vanilloid receptor had been disclosed and cloned in 1997, it became an object of numerous investigations as a potential target for novel drugs against pain of different origin (10). As recently reported, vanillotoxins from a tarantula Psalmopoeus cambridgei directly activate TRPV1 in micromolar concentrations, causing pain effect in the same way as capsaicin does (11). Venoms of several jellyfish also seem to interact with TRPV1, knocking down its desensitization (12). A number of small molecules were synthesized that selectively inhibit TRPV1 in nanomolar concentration ...

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.

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