Polymorphonuclear neutmphilic granulocytes (PMNs) are extremely important in defense of the organism against infections and in inflammatory processes including neuroinflammation and pain sensation. Different subtypes of nicotinic acetylcholine receptors (nAChRs) are involved in modulation of PMN activities. Earlier we determined expression of alpha 2-7, alpha 9, beta 3, beta 4 subunits and regulatory role of alpha 7 and alpha 3 beta 2 nAChR subtypes in functions of inflammatory PMNs. Other authors detected mRNA of alpha 9 subunit in bone marrow neutrophils (BM-PMNs). Murine BM-PMNs coming out from the bone marrow, where they develop, to blood were characterized as mature. There was no data for alpha 10 and for the presence of functionally active alpha 9 alpha 10 nAChRs in BM-PMNs. Here we detected for the first time mRNA expression of the alpha 10 nAChR subunit in BM-PMNs and confirmed the expression of mRNA for alpha 9 nAChR. With the help of alpha-conotoxins RgIA and Vc1.1, highly selective antagonists of alpha 9 alpha 10 nAChRs, we have revealed participation of alpha 9 and/or alpha 9 alpha 10 nAChRs in regulation of cytosolic Ca2+ concentration, cell adhesion, and in generation of reactive oxygen species (ROS). Nicotine, choline, RgIA, and Vc1.1 induced Ca2+ transients in BM-PMNs, enhanced cell adhesiveness and decreased production of ROS indicating involvement of alpha 9, possibly co-assembled with alpha 10, nAChRs in the BM-PMN activity for recruitment and cytotoxicity.

Among the brain tumors, glioma is the most common. In general, different biochemical mechanisms, involving nicotinic acetylcholine receptors (nAChRs) and the arachidonic acid cascade are involved in oncogenesis. Although the engagement of the latter in survival and proliferation of rat C6 glioma has been shown, there are practically no data about the presence and the role of nAChRs in C6 cells. In this work we studied the effects of nAChR antagonists, marine snail α-conotoxins and snake α-cobratoxin, on the survival and proliferation of C6 glioma cells. The effects of the lipoxygenase and cyclooxygenase inhibitors either alone or together with α-conotoxins and α-cobratoxin were studied in parallel. It was found that α-conotoxins and α-cobratoxin promoted the proliferation of C6 glioma cells, while nicotine had practically no effect at concentrations below 1 µL/mL. Nordihydroguaiaretic acid, a nonspecific lipoxygenase inhibitor, and baicalein, a 12-lipoxygenase inhibitor, exerted antiproliferative and cytotoxic effects on C6 cells. nAChR inhibitors weaken this effect after 24 h cultivation but produced no effects at longer times. Quantitative real-time polymerase chain reaction showed that mRNA for α4, α7, β2 and β4 subunits of nAChR were expressed in C6 glioma cells. This is the first indication for involvement of nAChRs in mechanisms of glioma cell proliferation.

Lynx1, membrane-bound protein co-localized with the nicotinic acetylcholine receptors (nAChRs) and regulates their function, is a three-finger protein (TFP) made of three β-structural loops, similarly to snake venom α-neurotoxin TFPs. Since the central loop II of α-neurotoxins is involved in binding to nAChRs, we have recently synthesized the fragments of Lynx1 central loop, including those with the disulfide between Cys residues introduced at N- and C-termini, some of them inhibiting muscle-type nAChR similarly to the whole-size water-soluble Lynx1 (ws-Lynx1). Literature shows that the main fragment interacting with TFPs is the C-loop of both nAChRs and acetylcholine binding proteins (AChBPs) while some ligand-binding capacity is preserved by analogs of this loop, for example, by high-affinity peptide HAP. Here we analyzed the structural organization of these peptide models of ligands and receptors and its role in binding. Thus, fragments of Lynx1 loop II, loop C from the Lymnaea stagnalis AChBP and HAP were synthesized in linear and Cys-cyclized forms and structurally (CD and NMR) and functionally (radioligand assay on Torpedo nAChR) characterized. Connecting the C- and N-termini by disulfide in the ws-Lynx1 fragment stabilized its conformation which became similar to the loop II within the 1H-NMR structure of ws-Lynx1, the activity being higher than for starting linear fragment but lower than for peptide with free cysteines. Introduced disulfides did not considerably change the structure of HAP and of loop C fragments, the former preserving high affinity for α-bungarotoxin, while, surprisingly, no binding was detected with loop C and its analogs.

Polymorphonuclear neutrophilic granulocytes (PMNs) are extremely important in defense of the organism against infections and in inflammatory processes including neuroinflammation and pain sensation. Different subtypes of nicotinic acetylcholine receptors (nAChRs) are involved in modulation of PMN activities. Earlier we determined expression of α2-7, α9, β3, β4 subunits and regulatory role of α7 and α3β2 nAChR subtypes in functions of inflammatory PMNs. Other authors detected mRNA of α9 subunit in bone marrow neutrophils (BM-PMNs). Murine BM-PMNs coming out from the bone marrow, where they develop, to blood were characterized as mature. There was no data for α10 and for the presence of functionally active α9α10 nAChRs in BM-PMNs. Here we detected for the first time mRNA expression of the α10 nAChR subunit in BM-PMNs and confirmed the expression of mRNA for α9 nAChR. With the help of α-conotoxins RgIA and Vc1.1, highly selective antagonists of α9α10 nAChRs, we have revealed participation of α9 and/or α9α10 nAChRs in regulation of cytosolic Ca2+ concentration, cell adhesion, and in generation of reactive oxygen species (ROS). Nicotine, choline, RgIA, and Vc1.1 induced Ca2+ transients in BM-PMNs, enhanced cell adhesiveness and decreased production of ROS indicating involvement of α9, possibly co-assembled with α10, nAChRs in the BM-PMN activity for recruitment and cytotoxicity.

Several biochemical mechanisms, including the arachidonic acid cascade and activation of nicotinic acetylcholine receptors (nAChRs), are involved in increased tumor survival. Combined application of inhibitors acting on these two pathways may result in a more pronounced antitumor effect. Here, we show that baicalein (selective 12-lipoxygenase inhibitor), nordihydroguaiaretic acid (non-selective lipoxygenase inhibitor), and indomethacin (non-selective cyclooxygenase inhibitor) are cytotoxic to Ehrlich carcinoma cells in vitro. Marine snail α-conotoxins PnIA, RgIA and ArIB11L16D, blockers of α3β2/α6β2, α9α10 and α7 nAChR subtypes, respectively, as well as α-cobratoxin, a blocker of α7 and muscle subtype nAChRs, exhibit low cytotoxicity, but enhance the antitumor effect of baicalein 1.4-fold after 24 h and that of nordihydroguaiaretic acid 1.8–3.9-fold after 48 h of cell cultivation. α-Conotoxin MII, a blocker of α6-containing and α3β2 nAChR subtypes, increases the cytotoxic effect of indomethacin 1.9-fold after 48 h of cultivation. In vivo, baicalein, α-conotoxins MII and PnIA inhibit Ehrlich carcinoma growth and increase mouse survival; these effects are greatly enhanced by the combined application of α-conotoxin MII with indomethacin or conotoxin PnIA with baicalein. Thus, we show, for the first time, antitumor synergism of α-conotoxins and arachidonic acid cascade inhibitors.

Snake venom α-neurotoxins, invaluable pharmacological tools, bind with high affinity to distinct subtypes of nicotinic acetylcholine receptor. The combinatorial high-affinity peptide (HAP), homologous to the C-loop of α1 and α7 nAChR subunits, binds biotinylated α-bungarotoxin (αBgt) with nanomolar affinity and might be a protection against snake-bites. Since there are no data on HAP interaction with other toxins, we checked its binding of α-cobratoxin (αCtx), similar to αBgt in action on nAChRs. Using radioiodinated αBgt, we confirmed a high affinity of HAP for αBgt, the complex formation is supported by mass spectrometry and gel chromatography, but only weak binding was registered with αCtx. A combination of protein intrinsic fluorescence measurements with the principal component analysis of the spectra allowed us to measure the HAP-αBgt binding constant directly (29 nM). These methods also confirmed weak HAP interaction with αCtx (>10000 nM). We attempted to enhance it by modification of HAP structure relying on the known structures of α-neurotoxins with various targets and applying molecular dynamics. A series of HAP analogues have been synthesized, HAP[L9E] analogue being considerably more potent than HAP in αCtx binding (7000 nM). The proposed combination of experimental and computational approaches appears promising for analysis of various peptide-protein interactions.

Feae's viper Azemipos feae belongs to the Azemiopinae subfamily of the Viperidae family. The effects of Viperidae venoms are mostly coagulopathic with limited neurotoxicity manifested by phospholipases A2. From A. feae venom, we have earlier isolated azemiopsin, a novel neurotoxin inhibiting the nicotinic acetylcholine receptor. To characterize other A. feae toxins, we applied label-free quantitative proteomics, which revealed 120 unique proteins, the most abundant being serine proteinases and phospholipases A2. In total, toxins representing 14 families were identified, among which bradykinin-potentiating peptides with unique amino acid sequences possessed biological activity in vivo. The proteomic analysis revealed also basal (commonly known as non-conventional) three-finger toxins belonging to the group of those possessing neurotoxic activity. This is the first indication of the presence of three-finger neurotoxins in viper venom. In parallel, the transcriptomic analysis of venom gland performed by Illumina next-generation sequencing further revealed 206 putative venom transcripts. Together, the study unveiled the venom proteome and venom gland transciptome of A. feae, which in general resemble those of other snakes from the Viperidae family. However, new toxins not found earlier in viper venom and including three-finger toxins and unusual bradykinin-potentiating peptides were discovered.

Neurotoxins are among the main components of scorpion and snake venoms. Scorpion neurotoxins affect voltage‐gated ion channels, while most snake neurotoxins target ligand‐gated ion channels, mainly nicotinic acetylcholine receptors (nAChRs). We report that scorpion venoms inhibit α‐bungarotoxin binding to both muscle‐type nAChR from Torpedo californica and neuronal human α7 nAChR. Toxins inhibiting nAChRs were identified as OSK‐1 (α‐KTx family) from Orthochirus scrobiculosus and HelaTx1 (κ‐KTx family) from Heterometrus laoticus, both being blockers of voltage‐gated potassium channels. With an IC50 of 1.6 μm, OSK1 inhibits acetylcholine‐induced current through mouse muscle‐type nAChR heterologously expressed in Xenopus oocytes. Other well‐characterized scorpion toxins from these families also bind to Torpedo nAChR with micromolar affinities. Our results indicate that scorpion neurotoxins present target promiscuity.

The proteins of the Ly6 family have a three-finger folding as snake venom α-neurotoxins, targeting nicotinic acetylcholine receptors (nAChRs), and some of them, like mammalian secreted Ly6/uPAR protein (SLURP1) and membrane-attached Ly-6/neurotoxin (Lynx1), also interact with distinct nAChR subtypes. We believed that synthetic fragments of these endogenous proteins might open new ways for drug design because nAChRs are well-known targets for developing analgesics and drugs against neurodegenerative diseases. Since interaction with nAChRs was earlier shown for synthetic fragments of the α-neurotoxin central loop II, we synthesized a 15-membered fragment of human Lynx1, its form with two Cys residues added at the N- and C-termini and forming a disulfide, as well as similar forms of human SLURP1, SLURP2, and of Drosophila sleepless protein (SSS). The IC50 values measured in competition with radioiodinated α-bungarotoxin for binding to the membrane-bound Torpedo californica nAChR were 4.9 and 7.4 µM for Lynx1 and SSS fragments, but over 300 µM for SLURP1 or SLURP2 fragments. The affinity of these compounds for the α7 nAChR in the rat pituitary tumor-derived cell line GH4C1 was different: 13.1 and 147 µM for SSS and Lynx1 fragments, respectively. In competition for the ligand-binding domain of the α9 nAChR subunit, SSS and Lynx1 fragments had IC50 values of about 40 µM, which correlates with the value found for the latter with the rat α9α10 nAChR expressed in the Xenopus oocytes. Thus, the activity of these synthetic peptides against muscle-type and α9α10 nAChRs indicates that they may be useful in design of novel myorelaxants and analgesics.

— Polypeptide SE-33 (SETRPVLNRLFDKIRQVIRKFEKGIKEKSKRFF), which is a retro analog of natural antimicrobial protein cathelicidin LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES), was synthesized by the method of solid-phase peptide synthesis. Similar to the natural peptide, polypeptide SE-33 forms an amphipathic alpha helix but has an inverted amino acid sequence compared to cathelicidin. It has been shown the physicochemical properties of polypeptide SE-33 are similar to those of the natural compound. In vitro experiments have shown that polypeptide SE-33 exerts a bactericidal effect on the cells of Staphylococcus aureus Wood 46, which is comparable with the effect of cathelicidin LL-37, as well as pronounced antifungal activity against the clinical isolates of Candida albicans , Cryptococcus neoformans , Rhodotorula mucilaginosa , Trichosporon cutaneum , Geotrichum sp. The MICs of polypeptide SE-33 for different fungal strains were in the range of 31.2 to 1024 μg/mL. Polypeptide SE-33 demonstrated high activity in vivo in the model of vulvovaginal candidiasis (VVC) in mice comparable with that of pimafucin. In the absence of side effects and signs of pathology, polypeptide SE-33 in all doses tested (1, 10 and 50 mg/mL) statistically reduced the vaginal load of the mice compared to the placebo group. The pronounced antibacterial and antifungal activity of polypeptide SE-33, as well as the absence of a toxic effect in the VVC model in mice, suggest polypeptide SE-33 as a promising antimicrobial agent.

AIM:Spider venom is a rich source of antibacterial peptides, whose hemolytic activity is often excessive.METHODOLOGY:How to get rid of it? Using latarcins from Lachesana tarabaevi and oxyopinin Oxt 4a from Oxyopes takobius spider venoms we performed coarse-grained molecular dynamics simulations of these peptides in the presence of lipid bilayers, mimicking erythrocyte membranes. This identified hemolytically active fragments within Oxt 4a and latarcins. Then, we synthesized five 20-residue peptides, containing different parts of the Oxt 4a and latarcin-1 sequence, carrying mutations within the identified regions.CONCLUSION:The antibacterial and hemolytic tests suggested that the three of the synthesized peptides demonstrated substantial decrease in hemolytic activity, retaining, or even exceeding antibacterial potential of the parent peptides.

Azemiopsin (Az), a linear peptide from the Azemiops feae viper venom, contains no disulfide bonds, is a high-affinity and selective inhibitor of nicotinic acetylcholine receptor (nAChR) of muscle type and may be considered as potentially applicable nondepolarizing muscle relaxant. In this study, we investigated its preclinical profile in regard to in vitro and in vivo efficacy, acute and chronic toxicity, pharmacokinetics, allergenic capacity, immunotoxicity and mutagenic potency. The peptide effectively inhibited (IC50 ~ 19 nM) calcium response of muscle nAChR evoked by 30 μM (EC100) acetylcholine but was less potent (IC50 ~ 3 μM) at α7 nAChR activated by 10 μM (EC50) acetylcholine and had a low affinity to α4β2 and α3-containing nAChR, as well as to GABAA or 5HT3 receptors. Its muscle relaxant effect was demonstrated at intramuscular injection to mice at doses of 30–300 µg/kg, 30 µg/kg being the initial effective dose and 90 µg/kg—the average effective dose. The maximal muscle relaxant effect of Az was achieved in 10 min after the administration and elimination half-life of Az in mice was calculated as 20–40 min. The longest period of Az action observed at a dose of 300 µg/kg was 55 min. The highest acute toxicity (LD50 510 μg/kg) was observed at intravenous injection of Az, at intramuscular or intraperitoneal administration it was less toxic. The peptide showed practically no immunotoxic, allergenic or mutagenic capacity. Overall, the results demonstrate that Az has good drug-like properties for the application as local muscle relaxant and in its parameters, is not inferior to the relaxants currently used. However, some Az modification might be effective to extend its narrow therapeutic window, a typical characteristic and a weak point of all nondepolarizing myorelaxants.

The paper presents the results of analysis of the effect of a high-affinity peptide (HAP) homologous to a fragment of the nicotinic acetylcholine receptor (nAChR) on the absorption and fluorescence spectra of thiazole orange and thioflavin T complexes with cucurbit[8]uril in aqueous solution. In the presence of HAP, a change in the absorption spectra of the dye complexes and a drop in the fluorescence intensity occur; for thiazole orange, the fluorescence intensity is restored to the initial level in the presence of α-bungarotoxin capable of high-affinity binding to nAChR. The proposed method make it possible to detect the presence of α-bungarotoxin.

Despite some success for small molecules, elucidating structure–function relationships for biologically active peptides — the ligands for various targets in the organism — remains a great challenge and calls for the development of novel approaches. Some of us recently proposed the Protein Surface Topography (PST) approach, which benefits from a simplified representation of biomolecules’ surface as projection maps, which enables the exposure of the structure–function dependencies. Here, we use PST to uncover the “activity pattern” in α-conotoxins — neuroactive peptides that effectively target nicotinic acetylcholine receptors (nAChRs). PST was applied in order to design several variants of the α-conotoxin PnIA, which were synthesized and thoroughly studied. Among the best was PnIA[R9, L10], which exhibits nanomolar affinity for the α7 nAChR, selectivity and a slow wash-out from this target. Importantly, these mutations could hardly be delineated by “standard” structure-based drug design. The proposed combination of PST with a set of experiments proved very efficient for the rational construction of new bioactive molecules.

Phospholipase A(2) (named bitanarin) possessing capability to block nicotinic acetylcholine receptors (nAChRs) was isolated earlier (Vulfius et al., 2011) from puff adder Bids arietans venom. Further studies indicated that low molecular weight fractions of puff adder venom inhibit nAChRs as well. In this paper, we report on isolation from this venom and characterization of three novel peptides called baptides 1, 2 and 3 that reversibly block nAChRs. To isolate the peptides, the venom of B. arietans was fractionated by gel-filtration and reversed phase chromatography. The amino acid sequences of peptides were established by de novo sequencing using MALDI mass spectrometry. Baptide 1 comprised 7, baptides 2 and 3 10 amino acid residues, the latter being acetylated at the N-terminus. This is the first indication for the presence of such post-translational modification in snake venom proteins. None of the peptides contain cysteine residues. For biological activity studies the peptides were prepared by solid phase peptide synthesis. Baptide 3 and 2 blocked acetylcholine-elicited currents in isolated Lymnaea stagnalis neurons with IC50 of about 50 mu M and 250 mu M, respectively. In addition baptide 2 blocked acetylcholine-induced currents in muscle nAChR heterologously expressed in Xenopus oocytes with IC50 of about 3 mu M. The peptides did not compete with radioactive alpha-bungarotoxin for binding to Torpedo and alpha 7 nAChRs at concentration up to 200 mu M that suggests non-competitive mode of inhibition. Calcium imaging studies on alpha 7 and muscle nAChRs heterologously expressed in mouse neuroblastoma Neuro2a cells showed that on alpha 7 receptor baptide 2 inhibited acetylcholine-induced increasing intracellular calcium concentration with IC50 of 20.6 +/- 3.93 mu M. On both alpha 7 and muscle nAChRs the suppression of maximal response to acetylcholine by about 50% was observed at baptide 2 concentration of 25 mu M, the value being close to IC50 on alpha 7 nAChR. These data are in accord with non-competitive inhibition as follows from a-bungarotoxin binding experiments. The described peptides are the shortest peptides without disulfide bridges isolated from animal venom and capable to inhibit nAChR by non-competitive way. (C) 2016 Published by Elsevier Ltd.