Yaroslav A. Andreev

Analgesic Compound from Sea Anemone Heteractis crispa Is the First Polypeptide Inhibitor of Vanilloid Receptor 1 (TRPV1)

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)/Kunitz-type 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 (EC50) 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.

Cyanogen bromide cleavage of proteins in salt and buffer solutions.

Protocols for recombinant polypeptide production should provide high yields and be efficient, user friendly, and time saving. To perform cyanogen bromide (CNBr) cleavage of fusion proteins, the majority of researchers first desalted and vacuum-dried samples and then dissolved them in aqueous formic or trifluoroacetic acid. We propose to exclude the desalting step and run CNBr cleavage directly. We show that the commonly used Tris-HCl, sodium phosphate, NaCl, imidazole, and guanidine-HCl do not interfere with the reaction under acidic conditions. Omitting the desalting step does not decrease the final yields of target products, as demonstrated for fusion proteins of different origin and composition.

Bacterial production of latarcin 2a, a potent antimicrobial peptide from spider venom

Natural venoms are promising sources of candidate therapeutics including antibiotics. A recently described potent antimicrobial peptide latarcin 2a (Ltc 2a) from Lachesana tarabaevi spider venom shows a broad-spectrum antibacterial activity. This peptide consists of 26 amino acid residues and therefore its production using chemical synthesis, although trivial, is costly. We describe an easy approach to Ltc 2a production in Escherichia coli using the conventional fusion partner thioredoxin. Latarcin 2a synthetic gene was cloned into the expression vector pET-32b, which was then used to transform E. coli BL21(DE3) strain. His-tagged fusion purification was achieved using metal-chelate affinity chromatography. Since no methionine residues are present in the latarcin 2a sequence, cyanogen bromide could be effectively utilized to separate the target product from the carrier protein. Reverse-phase HPLC was used as the final step of purification; the final yield was approximately 3 mg/L of bacterial culture. To increase the yields, we attempted incorporation of Ltc 2a tandem repeats into the fusion protein; however, production rates greatly decreased due to enhanced fusion toxicity. Moreover, we probed constructs to produce an Ltc 2a dimer and the Ltc 2a propeptide to study their functional properties. Recombinant peptides were produced at appreciable yields and biological tests to determine their activities were performed. Latarcin 2a is the first linear peptide from spider venom and one of the first membrane-active peptides from venomous animals to be biosynthetically produced.

Sea anemone peptide with uncommon β-hairpin structure inhibits acid-sensing ion channel 3 (ASIC3) and reveals analgesic activity

Background: Sea anemone peptides are promising tools for understanding physiological functions of ion channels. Results: A new peptide, Ugr 9-1, was isolated from the sea anemone venom and was shown to inhibit the acid-sensing ion channel 3 (ASIC3) channel. Conclusion: Ugr 9-1 affects the ASIC3 channel, produces analgesic effects, and has a unique spatial structure and mechanism of action. Significance: Ugr 9-1 represents a novel structural fold of natural short peptides modulating neuronal channels. Three novel peptides were isolated from the venom of the sea anemone Urticina grebelnyi. All of them are 29 amino acid peptides cross-linked by two disulfide bridges, with a primary structure similar to other sea anemone peptides belonging to structural group 9a. The structure of the gene encoding the shared precursor protein of the identified peptides was determined. One peptide, π-AnmTX Ugr 9a-1 (short name Ugr 9-1), produced a reversible inhibition effect on both the transient and the sustained current of human ASIC3 channels expressed in Xenopus laevis oocytes. It completely blocked the transient component (IC50 10 ± 0.6 μm) and partially (48 ± 2%) inhibited the amplitude of the sustained component (IC50 1.44 ± 0.19 μm). Using in vivo tests in mice, Ugr 9-1 significantly reversed inflammatory and acid-induced pain. The other two novel peptides, AnmTX Ugr 9a-2 (Ugr 9-2) and AnmTX Ugr 9a-3 (Ugr 9-3), did not inhibit the ASIC3 current. NMR spectroscopy revealed that Ugr 9-1 has an uncommon spatial structure, stabilized by two S-S bridges, with three classical β-turns and twisted β-hairpin without interstrand disulfide bonds. This is a novel peptide spatial structure that we propose to name boundless β-hairpin.

Polypeptide Modulators of TRPV1 Produce Analgesia without Hyperthermia

Transient receptor potential vanilloid 1 receptors (TRPV1) play a significant physiological role. The study of novel TRPV1 agonists and antagonists is essential. Here, we report on the characterization of polypeptide antagonists of TRPV1 based on in vitro and in vivo experiments. We evaluated the ability of APHC1 and APHC3 to inhibit TRPV1 using the whole-cell patch clamp approach and single cell Ca2+ imaging. In vivo tests were performed to assess the biological effects of APHC1 and APHC3 on temperature sensation, inflammation and core body temperature. In the electrophysiological study, both polypeptides partially blocked the capsaicin-induced response of TRPV1, but only APHC3 inhibited acid-induced (pH 5.5) activation of the receptor. APHC1 and APHC3 showed significant antinociceptive and analgesic activity in vivo at reasonable doses (0.01–0.1 mg/kg) and did not cause hyperthermia. Intravenous administration of these polypeptides prolonged hot-plate latency, blocked capsaicin- and formalin-induced behavior, reversed CFA-induced hyperalgesia and produced hypothermia. Notably, APHC3’s ability to inhibit the low pH-induced activation of TRPV1 resulted in a reduced behavioural response in the acetic acid-induced writhing test, whereas APHC1 was much less effective. The polypeptides APHC1 and APHC3 could be referred to as a new class of TRPV1 modulators that produce a significant analgesic effect without hyperthermia.

Novel mode of action of plant defense peptides - hevein-like antimicrobial peptides from wheat inhibit fungal metalloproteases.

The multilayered plant immune system relies on rapid recognition of pathogen‐associated molecular patterns followed by activation of defense‐related genes, resulting in the reinforcement of plant cell walls and the production of antimicrobial compounds. To suppress plant defense, fungi secrete effectors, including a recently discovered Zn‐metalloproteinase from Fusarium verticillioides, named fungalysin Fv‐cmp. This proteinase cleaves class IV chitinases, which are plant defense proteins that bind and degrade chitin of fungal cell walls. In this study, we investigated plant responses to such pathogen invasion, and discovered novel inhibitors of fungalysin. We produced several recombinant hevein‐like antimicrobial peptides named wheat antimicrobial peptides (WAMPs) containing different amino acids (Ala, Lys, Glu, and Asn) at the nonconserved position 34. An additional Ser at the site of fungalysin proteolysis makes the peptides resistant to the protease. Moreover, an equal molar concentration of WAMP‐1b or WAMP‐2 to chitinase was sufficient to block the fungalysin activity, keeping the chitinase intact. Thus, WAMPs represent novel protease inhibitors that are active against fungal metalloproteases. According to in vitro antifungal assays WAMPs directly inhibited hyphal elongation, suggesting that fungalysin plays an important role in fungal development. A novel molecular mechanism of dynamic interplay between host defense molecules and fungal virulence factors is suggested.

Molecules to selectively target receptors for treatment of pain and neurogenic inflammation

Receptors that are involved in generation and transduction of pain signals attract much interest from the scientific and corporate communities. Good commercial prospects for successful development of effective analgesic drugs stimulate significantly the research. This article provides a brief overview of the key molecular targets, i.e. cell receptors, inhibition of which can lead to analgesia. Today transient receptor potential (TRP), purinergic (P2X) receptors and acidsensing ion channels (ASIC) are considered to be the most important proteins for perception of pain stimuli. These ionotropic receptors also participate in the development of inflammation; their hyperactivity leads to many pathological conditions and is closely associated with acute and inflammatory pain. Development of molecules capable to selectively modulate these receptors, their in vitro and in vivo effects, as well as perspectives for practical application described in patents and research articles are reviewed in this paper.

Genes encoding 4-Cys antimicrobial peptides in wheat Triticum kiharae Dorof. et Migush.: multimodular structural organization, instraspecific variability, distribution and role in defence

A novel family of antifungal peptides was discovered in the wheat Triticum kiharae Dorof. et Migusch. Two members of the family, designated Tk‐AMP‐X1 and Tk‐AMP‐X2, were completely sequenced and shown to belong to the α‐hairpinin structural family of plant peptides with a characteristic C1XXXC2‐X(n)‐C3XXXC4 motif. The peptides inhibit the spore germination of several fungal pathogens in vitro. cDNA and gene cloning disclosed unique structure of genes encoding Tk‐AMP‐X peptides. They code for precursor proteins of unusual multimodular structure, consisting of a signal peptide, several α‐hairpinin (4‐Cys) peptide domains with a characteristic cysteine pattern separated by linkers and a C‐terminal prodomain. Three types of precursor proteins, with five, six or seven 4‐Cys peptide modules, were found in wheat. Among the predicted family members, several peptides previously isolated from T. kiharae seeds were identified. Genes encoding Tk‐AMP‐X precursors have no introns in the protein‐coding regions and are upregulated by fungal pathogens and abiotic stress, providing conclusive evidence for their role in stress response. A combined PCR‐based and bioinformatics approach was used to search for related genes in the plant kingdom. Homologous genes differing in the number of peptide modules were discovered in phylogenetically‐related Triticum and Aegilops species, including polyploid wheat genome donors. Association of the Tk‐AMP‐X genes with A, B/G or D genomes of hexaploid wheat was demonstrated. Furthermore, Tk‐AMP‐X‐related sequences were shown to be widespread in the Poaceae family among economically important crops, such as barley, rice and maize.

Novel antifungal α-hairpinin peptide from Stellaria media seeds: structure, biosynthesis, gene structure and evolution

Plant defense against disease is a complex multistage system involving initial recognition of the invading pathogen, signal transduction and activation of specialized genes. An important role in pathogen deterrence belongs to so-called plant defense peptides, small polypeptide molecules that present antimicrobial properties. Using multidimensional liquid chromatography, we isolated a novel antifungal peptide named Sm-AMP-X (33 residues) from the common chickweed (Stellaria media) seeds. The peptide sequence shows no homology to any previously described proteins. The peculiar cysteine arrangement (C1X3C2XnC3X3C4), however, allocates Sm-AMP-X to the recently acknowledged α-hairpinin family of plant defense peptides that share the helix-loop-helix fold stabilized by two disulfide bridges C1–C4 and C2–C3. Sm-AMP-X exhibits high broad-spectrum activity against fungal phytopathogens. We further showed that the N- and C-terminal “tail” regions of the peptide are important for both its structure and activity. The truncated variants Sm-AMP-X1 with both disulfide bonds preserved and Sm-AMP-X2 with only the internal S–S-bond left were progressively less active against fungi and presented largely disordered structure as opposed to the predominantly helical conformation of the full-length antifungal peptide. cDNA and gene cloning revealed that Sm-AMP-X is processed from a unique multimodular precursor protein that contains as many as 12 tandem repeats of α-hairpinin-like peptides. Structure of the sm-amp-x gene and two related pseudogenes sm-amp-x-ψ1 and sm-amp-x-ψ2 allows tracing the evolutionary scenario that led to generation of such a sophisticated precursor protein. Sm-AMP-X is a new promising candidate for engineering disease resistance in plants.

Lignan from Thyme Possesses Inhibitory Effect on ASIC3 Channel Current

Background: ASIC3 channels contribute to pain stimuli perception. Results: A new compound (sevanol) was isolated from thyme extract and was shown to inhibit ASIC3 current components. Conclusion: Sevanol could play a considerable role in thyme analgesic properties. Significance: The data on the structure and potency of sevanol can make a contribution to understanding the function of ASIC3 and can be helpful for developing other pharmacological substances targeting ASIC3. A novel compound was identified in the acidic extract of Thymus armeniacus collected in the Lake Sevan region of Armenia. This compound, named “sevanol,” to our knowledge is the first low molecular weight natural molecule that has a reversible inhibition effect on both the transient and the sustained current of human ASIC3 channels expressed in Xenopus laevis oocytes. Sevanol completely blocked the transient component (IC50 353 ± 23 μm) and partially (∼45%) inhibited the amplitude of the sustained component (IC50 of 234 ± 53 μm). Other types of acid-sensing ion channel (ASIC) channels were intact to sevanol application, except ASIC1a, which showed more than six times less affinity to it as compared with the inhibitory action on the ASIC3 channel. To elucidate the structure of sevanol, the set of NMR spectra in two solvents (d6-DMSO and D2O) was collected, and the complete chemical structure was confirmed by liquid chromatography-mass spectrometry with electrospray ionization (LC-ESI+-MS) fragmentation. This compound is a new lignan built up of epiphyllic acid and two isocitryl esters in positions 9 and 10. In vivo administration of sevanol (1–10 mg/kg) significantly reversed thermal hyperalgesia induced by complete Freunds adjuvant injection and reduced response to acid in a writhing test. Thus, we assume the probable considerable role of sevanol in known analgesic and anti-inflammatory properties of thyme.