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Using electrophysiology, the effect of nicotinic acetylcholine receptor (nAChR) ligands on acetylcholine-induced depolarization in the neurons of Helix lucorum snail was studied. It was found that the α-conotoxin PnIA [R9, L10], a selective antagonist of α7 nAChR, and α-cobratoxin (antagonist of α7 and muscle-type nAChR) suppressed neuronal depolarization. Fluorescence microscopy showed staining of the neurons with fluorescently labeled α-bungarotoxin; this staining was reduced by pretreatment with α-cobratoxin. Induced depolarization was also suppressed by α-conotoxin RgIA, a selective inhibitor of α9 nAChR. In contrast to Lymnaea stagnalis nAChR, which are weakly sensitive to neurotoxin II and α-conotoxin GI, antagonists of muscle-type nAChR, H. lucorum receptors were most effectively inhibited by these antagonists. The results obtained, as well as the previously found sensitivity of the receptors studied in this work to muscarinic receptor ligands, indicate an unusual atypical pharmacological profile of H. lucorum nAChR.

Blockade of α6, α3β2, α9α10, and α7 subtypes of nicotinic acetylcholine receptors slows tumor growth in vivo, increases cytotoxic activity of splenocytes from tumor-bearing mice, and, to some extent, reduces the viability of Ehrlich carcinoma cells in vitro. These data indicate that nicotinic acetylcholine receptors are involved in oncogenesis, affecting the survival of tumor cells, inter alia, via modulation of the antitumor immunity.

The data available to date indicate that the activation of nicotinic acetylcholine receptors (nAChR) of α7 type can reduce heart damage resulting from ischemia and subsequent reperfusion. We have studied two new synthetic D-analogs of 6-bromohypaphorine, which are selective agonists of α7 nAChR, in a rat model of myocardial ischemia. Acute myocardial infarction in animals was induced by occlusion of the left coronary artery with its subsequent reperfusion under mechanical lung ventilation. It was found that one of the analogs was more active, and treatment with it at the onset of reperfusion statistically reduced infarct size. This analog also prevented changes in the concentration of potassium and sodium ions in the blood, occurring during occlusion/reperfusion injury. The data obtained indicate that hypaphorine analogs are promising for the development of drugs that reduce the adverse effects of myocardial infarction.

To search for compounds with antiprotozoal activity, effects of snake venoms on the ciliates Tetrahymena pyriformis was studied. T. pyriformis from subkingdom of Protozoa, including the protozoal pathogens, was used as a model organism to select the venoms that are the most active against parasitic protozoans. Various concentrations of venoms were added to the cells, and the cells that survived after 24 h were counted. Among the six snake species from the Viperidae family, the venom of the viper Vipera berus, which completely killed the cells at 49 μg/mL, was the most active. Among four species from the Elapidae family, the previously studied cobra venoms containing cytotoxins with strong antiprotozoal activity as well as the venom of krait Bungarus multicinctus (10 μg/mL) were the most active. The venoms of the pit vipers and Nikolsky's viper did not show any activity at 12.5 mg/mL. Thus, the venoms of V. berus and B. multicinctus are promising for the isolation of new antiprotozoal compounds.

Four dimeric disintegrins were isolated from the venom of the steppe viper V. ursinii using liquid chromatography. Disintegrins prevented adhesion of MCF7 cells to fibronectin, which indicates their interaction with integrin receptors of the V1 type. According to mass spectrometry data, the molar masses of disintegrins are about 14 kDa.

The three-finger snake neurotoxins are selective antagonists of some nicotinic cholinergic receptor subtypes and are used to study these receptors. The peptide neurotoxin azemiopsin, recently isolated from the venom of Azemipos feae, is a selective muscle-type cholinergic receptor blocker. In order to reduce their toxicity and increase resistance under physiological conditions, we have encapsulated these toxins into nanomaterials. The study of nanomaterials after interaction with neurotoxins by the methods of transmission electron microscopy and dynamic light scattering revealed an increase in the size of nanoparticles, which indicates the inclusion of neurotoxins in nanomaterials.

Three-finger snake neurotoxins are selective antagonists of some nicotinic acetylcholine receptor subtypes and are widely used to study these receptors. The peptide neurotoxin azemiopsin, recently isolated from the venom of Azemipos feae, is a selective blocker of muscle-type nicotinic acetylcholine receptor. In order to reduce their toxicity and increase resistance under physiological conditions, we have encapsulated these toxins into nanomaterials. The study of nanomaterials after interaction with neurotoxins by the methods of transmission electron microscopy and dynamic light scattering revealed an increase in the size of nanoparticles, which indicates the inclusion of neurotoxins in nanomaterials.

The study of the influence of cobra Naja oxiana cardiotoxins on the contractility of the rat papillary muscles and its rhythm-inotropic characteristics has that the presence of toxins induces a slight contractility decrease in the stimulation frequency range up to 0,1 Hz. In the stimulation frequency range from 0,1 to 0,5 Hz a positive inotropic effect is found. However, the positive inotropic effect is replaced by a negative one with further increase in the frequency up to 3 Hz. In the presence of cardiotoxins, the positive force-frequency relationship in the region of 1-3 Hz, characteristic of healthy rat myocardium, disappears and relationship becomes completely negative. L‑type calcium channel blocker nifedipine does not affect the changes induced by toxins, while a high concentration (10 mM) of calcium prevents the effects of cardiotoxins on the muscle. The results obtained show that the impairment of the force-frequency relationship occurs long before the development of irreversible damage in the myocardium and may be the first sign of the pathological action of cardiotoxins.

A proteomic analysis of the venom of males and females of the Naja kaouthia monocled cobra specimens kept in captivity was carried out. Using the amino acid sequences of proteins of the taxonomic group Serpentes from the UniProt KB database, 875 proteins were identified in the venom samples and the relative content of about 190 of them was determined in each venom sample by the method of label-free quantitative proteomics. The total content of 35 major protein components of the venom of each individual was shown to comprise about 98% of the total amount of venom proteins. Analysis of relative content of the venom proteins in males and females showed a significantly (p < 0.05) higher content of nerve growth factor and natriuretic peptide in the venom of females. Analysis of the profile of posttranslational modifications of N. kaouthia toxins revealed previously unknown sites for phosphorylation, acetylation, and formylation.

The model studies on C6 glioma cells showed that the role of nicotinic acetylcholine receptors (nAChR) in the regulation of the redox balance varies under different cultivation conditions. In a serum-free medium, the inhibitor of nAXP alpha-cobratoxin alleviates the oxidative stress that is manifested in an increase in the activity of superoxide dismutase and catalase, the level of reduced glutathione and in a decrease in the concentration of malonic dialdehyde.

We report that the action of the heterodimeric phospholipases A2 (PLA2s) from Vipera nikolskii, which comprises enzymatically active basic subunit and inactive acidic PLA2 homologue, on the lipid bilayer results in the aggregation and stacking of bilayers. These processes are demonstrated using two independent methods (fluorescence spectroscopy and electron microscopy). Aggregation of bilayers is possible because both subunits of the V. nikolskii heterodimer contain a membrane-binding site (also known as IBS). Thus, when the two IBSs bind to the membrane, the heterodimer acts as a connecting agent. Heterodimers induce aggregation of negatively charged bilayers composed of phosphatidylglycerol and do not induce aggregation of neutral bilayers composed of phosphatidylcholine.

Four dimeric disintegrins were isolated from the venom of the steppe viper V. ursinii using liquid chromatography. Disintegrins prevented adhesion of MCF7 cells to fibronectin, which indicates their interaction with integrin receptors of the αVβ1 type. According to mass spectrometry data, the molar masses of disintegrins are about 14 kDa. The method of peptide mapping established the structure of a new heterodimeric disintegrin weighing 13 995.5 Da and shows that it belongs to the class of RGD/KGD-containing disintegrins.

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.

A new three-finger toxin nakoroxin was isolated from the cobra Naja kaouthia venom, and its complete amino acid sequence was established. Nakoroxin belongs to the group of "orphan" toxins, data on the biological activity of which are practically absent. Nakoroxin shows no cytotoxicity and does not inhibit the binding of α-bungarotoxin to nicotinic acetylcholine receptors of muscle and α7 types. However, it potentiates the binding of α-bungarotoxin to the acetylcholine-binding protein from Lymnaea stagnalis. This is the first toxin with such an unusual property.

Fluorescent derivatives are widely used to study the structure and functions of proteins. Quantum dots (QDs), fluorescent semiconductor nanocrystals, have a high quantum yield and are much more resistant to bleaching compared to organic dyes. Conjugates of α-neurotoxins with QDs were used for visualization of human α7 acetylcholine receptors heterologously expressed in GH4C1 pituitary adenoma cells. Specific staining of cells by the conjugated toxins was observed.

Nonconventional three-finger toxin BMLCL was isolated from B. multicinctus venom, and its interaction with different subtypes of nicotinic acetylcholine receptor (nAChR) was studied. It was found that BMLCL is able to interact with high efficiency with both α7 and muscle type nAChRs.

Genes encoding two three-finger toxins TFT-AF and TFT-VN, nucleotide sequences of which were earlier determined by cloning cDNA from venom glands of vipers Azemiops feae and Vipera nikolskii, respectively, were expressed for the first time in E. coli cells. The biological activity of these toxins was studied by electrophysiological techniques, calcium imaging, and radioligand analysis. It was shown for the first time that viper three-finger toxins are antagonists of nicotinic acetylcholine receptors of neuronal and muscle type.

A role of nicotinic acetylcholine receptors (nAChR) in the development of Parkinson's disease (PD) has been investigated using two mouse models corresponding to the presymptomatic stage and the early symptomatic stage of PD. Quantitative determination of nAChR in the striatum and substantia nigra (SN) was performed using the radioactive derivatives of epibatidine, -conotoxin MII, and -bungarotoxin as ligands. The number of ligand-binding sites changed differently depending on their location in the brain, the stage of the disease and the receptor subtype. Epibatidine binding decreased in the striatum to 66% and 70% at the presymptomatic and early symptomatic stages, respectively, whereas in SN a 160% increase was registered at the presymptomatic stage. The -conotoxin MII binding on striatal dopaminergic axonal terminals at the presymptomatic stage decreased by 20% and at the symptomatic stage it demonstrated a further decrease. The increase in -bungarotoxin binding at the presymptomatic stage and a decrease at the early symptomatic stage was observed in the striatum. In SN, the level of -bungarotoxin binding decreased at the presymptomatic stage and kept constant at the symptomatic stage. The significant decrease in the expression of Chrna4 and Chrna6 genes encoding 4 and 6 nAChR subunits was observed in SN at the early symptomatic stage, while a 13-fold increase in expression of the Chrna7 gene encoding the 7 nAChR subunit was detected at the presymptomatic stage. The data obtained suggest possible involvement of nAChR in compensatory mechanisms at early PD stages.Issledovana rol' nikotinovykh atsetilkholinovykh retseptorov (nAKhR) na rannikh stadiiakh bolezni Parkinsona (BP) na éksperimental'nykh modeliakh u mysheĭ, sootvetstvuiushchikh dosimptomnoĭ i ranneĭ simptomnoĭ stadiiam BP. Kolichestvennoe opredelenie nAKhR v preparatakh striatuma i chernoĭ substantsii (ChS) provodili s pomoshch'iu ligandov, selektivnykh dlia razlichnykh podtipov nAKhR: radioaktivnykh proizvodnykh épibatidina, -konotoksina MII i -bungarotoksina. Kolichestvo vyiavlennykh ligand-sviazyvaiushchikh saĭtov zaviselo ot ikh tipa, lokalizatsii v golovnom mozge i stadii zabolevaniia. V striatume sviazyvanie épibatidina na dosimptomnoĭ i ranneĭ simptomnoĭ stadiiakh snizheno na 66% i 70% sootvetstvenno, a v ChS – povysheno do 160% na dosimptomnoĭ stadii po sravneniiu s kontrolem. Sviazyvanie -konotoksina MII na terminaliakh aksonov dofaminergicheskikh neĭronov na dosimptomnoĭ stadii bylo snizheno na 20%, i snizhenie ikh kolichestva prodolzhilos' na ranneĭ simptomnoĭ stadii. Sviazyvanie -bungarotoksina v striatume povysheno na dosimptomnoĭ i snizheno na ranneĭ simptomnoĭ stadiiakh, a v ChS umen'shalos' uzhe na dosimptomnoĭ stadii i ostavalos' postoiannym. Znachitel'noe snizhenie ékspressii genov neĭronal'nykh nAKhR Chrna4 i Chrna6, kodiruiushchikh 4 i 6 sub"edinitsy nAKhR, otmecheno v ChS na ranneĭ simptomnoĭ stadii, a dlia gena Chrna7, kodiruiushchego 7 sub"edinitsu, obnaruzheno 13-kratnoe uvelichenie na dosimptomnoĭ stadii. Poluchennye dannye ob izmeneniiakh urovnia mRNK ili funktsional'nykh kholinoretseptorov svidetel'stvuiut o vozmozhnom uchastii nAKhR v kompensatornykh mekhanizmakh na rannikh stadiiakh BP.

Cardiotoxins (cytotoxins, CT) are β-structured proteins isolated from the venom of cobra. They consist of 59-61 amino acid residues, whose antiparallel chains form three 'fingers'. In contrast to neurotoxins with an overall similar fold, CTs are amphiphilic. The amphiphilicity is caused by positively charged lysine and arginine residues flanking the tips of the loops that consist primarily of hydrophobic amino acids. A similar distribution of amino acid residues is typical for linear (without disulfide bonds) cationic cytolytic peptides from the venoms of other snakes and insects. Many of them are now considered to be lead compounds in combatting bacterial infec- tions and cancer. In the present review, we summarize the data on the antibacterial activity of CTs and compare it to the activity of linear peptides. K ey W ord S antibacterial activity, lipopolysaccharide, peptidoglycan, plasma membrane, three-finger cardiotox- ins (cytotoxins), cytolytic cationic peptides. abbre V iation S AMP - antimicrobial peptide; GAG - glucosaminoglycan; CL - cardiolipin; LPS - lipopolysac- charide, LTA - lipoteichoic acid; XRA - X-ray analysis; PG - phosphatidylglycerol; PE - phosphatidylethanola- mine; CT - cytotoxin (cardiotoxin) from snake venom, NMR - nuclear magnetic resonance.

Phospholipases A2 (PLA2s) are the most abundant family of snake venom proteins and play a significant role in prey envenomation. Their content in venoms is rather high. PLA2s not only have enzyme activity but exhibit other types of biological activities including neurotoxicity. We have earlier shown that a protein bitanarin from the venom of the puff adder Bitis arietans is capable to block the responses of Lymnaea stagnalis neurons to acetylcholine and represents an active PLA2 at the same time. Further investigation of PLA2s isolated from the venoms of snakes of two families revealed their capability to interact with nicotinic acetylcholine receptors (nAChRs): PLA2 from Vipera ursinii (Viperidae family), Naja kaouthia, and Bungarus fasciatus (Elapidae family) suppressed acetylcholine-induced current in identified neurons of L. staganlis. The effect was evident at PLA2 concentration in the range of tens micromoles. The data obtained suggest the presence in a PLA2 molecule of a site interacting with nAChR and a possible involvement of nAChR block in toxic action of PLA2s.

Compounds activating γ-aminobutyric acid type A receptor were isolated from the toad Bufo bufo venom as a result of chromatographic separation. Analysis of the structure of these compounds by mass spectrometry and nuclear magnetic resonance showed that they are arginine derivatives of dicarboxylic acids and represent suberylarginine, pimeloylarginine, and adipoylarginine.

Cytotoxins or cardiotoxins is a group of polycationic toxins from cobra venom belonging to the ‘three-finger’ protein superfamily (Ly6/uPAR family) which includes small β-structural proteins (60–90 residues) with high disulfide bond content (4–5 disulfides). Due to a high cytotoxic activity for cancer cells, cytotoxins are considered as potential anticancer agents. Development of the high-throughput production methods is required for the prospective applications of cytotoxins. Here, efficient approach for bacterial production of recombinant analogue of cytotoxin I from N. oxiana containing additional N-terminal Met-residue (rCTX1) was developed. rCTX1 was produced in the form of E. coli inclusion bodies. Refolding in optimized conditions provided ∼6 mg of correctly folded protein from 1 L of bacterial culture. Cytotoxicity of rCTX1 for C6 rat glioma cells was found to be similar to the activity of wild type CTX1. The milligram quantities of 13C,15N-labeled rCTX1 were obtained. NMR study confirmed the similarity of the spatial structures of recombinant and wild-type toxins. Additional Met residue does not perturb the overall structure of the three-finger core. The analysis of available data for different Ly6/uPAR proteins of snake and human origin revealed that efficiency of their folding in vitro is correlated with the number of proline residues in the third loop and the surface area of hydrophobic residues buried within the protein interior. The obtained data indicate that hydrophobic core is important for the folding of proteins with high disulfide bond content. Developed expression method opens new possibilities for structure-function studies of CTX1 and other related three-finger proteins.

Two low-molecular-weight compounds were isolated from the parotid gland secret of the toad Bufo bufo, which by absorption spectra and HPLC-MS/MS chromatography data correspond to di- and trimethyl derivatives of serotonin (5-hydorxytryptamine): bufotenine (confirmed by counter synthesis) and bufotenidine (5-HTQ). In experiments on competitive radioligand binding, these compounds showed a higher affinity and selectivity for neuronal α7 nicotinic acetylcholine receptors compared with the muscular cholinergic receptors. The most efficient compound in terms of binding value was bufotenine, the efficiency of 5-HTQ was an order of magnitude lower, and the minimal activity was exhibited by serotonin.

International audience; With the use of surface plasmon resonance (SPR) it was shown that ws-Lynx1, a water-soluble analog of the three-finger membrane-bound protein Lynx1, that modulates the activity of brain nicotinic acetylcholine receptors (nAChRs), interacts with the acetylcholine-binding protein (AChBP) with high affinity, K D = 62 nM. This result agrees with the earlier demonstrated competition of ws-Lynx1 with radioiodinated α-bungarotoxin for binding to AChBP. For the first time it was shown that ws-Lynx1 binds to GLIC, prokaryotic Cys-loop receptor (K D = 1.3 μM). On the contrary, SPR revealed that α-cobratoxin, a three-finger protein from cobra venom, does not bind to GLIC. Obtained results indicate that SPR is a promising method for analysis of topography of ws-Lynx1 binding sites using its mutants and those of AChBP and GLIC.

Cobra venom factor (CVF) depletes the complement system of the blood by forming stable convertase C3/C5 of the alternative pathway. We found that CVF from the Thailand cobra venom slows down the growth of subcutaneous Ehrlich carcinoma (EC) in mice at a dose of 1.7 nmol/g. Previously, we described a similar effect for the nerve growth factor (NGF) from the venom of this cobra. However, these factors did not exhibit either synergy or additive effect. On the contrary, they neutralized the antitumor effect of each other when they were administered simultaneously. Therefore, on the one hand, the NGF antitumor effect against EC manifests itself under the conditions of inflammation, and normal functioning of the complement system is necessary for this effect to occur. On the other hand, suppression of the humoral immune system leads to a slowdown of the EC growth, but administration of NGF prevents this.

Serine proteinases and Kunitz-type inhibitors are widely represented in the venoms of snakes belonging to different genera. During the studies of the venoms of snakes inhabiting Russia, we have cloned cDNAs coding for novel proteins of these families. A novel serine proteinase that we named nikobin was identified in the venom gland of the Nikolsky viper. The amino acid sequence of nikobin deduced from the cDNA sequence slightly differs from those of the serine proteinases found in other snakes, displaying 15 unique amino acid substitutions. This is the first serine proteinase from a viper of the Vipera genus for which the complete amino acid sequence has been determined. A cDNA coding for a Kunitz-type inhibitor has also been cloned. The deduced amino acid sequence of the inhibitor displays overall homology to the already known sequences of analogous proteins from vipers of the Vipera genus. However, several unusual amino acid substitutions that can cause a change of the inhibitor activity have been detected.

The cytotoxic properties of cytotoxins (CTs) from snake venom are mediated by their interaction with the cell membrane. The hydrophobic pattern containing the tips of loops I-III and flanked by polar residues is known to be a membrane-binding motif of CTs. However, this is not enough to explain the difference in activity among various CTs which are similar in sequence and in 3D structure. The mechanism of further CT-membrane interaction leading to pore formation and cell death still remains unknown. Published experimental data on the specific interaction between CT and low molecular weight anionic components (sulphatide) of the bilayer point to the existence of corresponding ligand binding sites on the surface of toxin molecules. In this work we study the membrane-lytic properties of CT I, CT II (Naja oxiana), and CT 4 (Naja kaouthia), which belong to different structural and functional types (P- and S-type) of CTs, by measuring the intensity of a fluorescent dye, calcein released from liposomes containing a phosphatidylserine (PS) lipid as an anionic component. Using molecular docking simulations, we find and characterize three sites in CT molecules that can potentially bind the PS polar head. Based on the data obtained, we suggest a hypothesis that CTs can specifically interact with one or more of the anionic lipids (in particular, with PS) contained in the membrane, thus facilitating the interaction between CTs and the lipid bilayer of a cell membrane.

The gene for the "weak" toxin of Naja kaouthia venom was expressed in Escherichia coli. "Weak" toxin is a specific inhibitor of nicotine acetylcholine receptor, but mechanisms of interaction of similar neurotoxins with receptors are still unknown. Systems previously elaborated for neurotoxin II from venom of the cobra Naja oxiana were tested for bacterial production of "weak" toxin from N. kaouthia venom. Constructs were designed for cytoplasmic production of N. kaouthia "weak" toxin in the form of a fused polypeptide chain with thioredoxin and for secretion with the leader peptide STII. However, it became possible to obtain "weak" toxin in milligram amounts only within cytoplasmic inclusion bodies. Different approaches for refolding of the toxin were tested, and conditions for optimization of the yield of the target protein during refolding were investigated. The resulting protein was characterized by mass spectrometry and CD and NMR spectroscopy. Experiments on competitive inhibition of (125)I-labeled alpha-bungarotoxin binding to the Torpedo californica electric organ membranes containing the muscle-type nicotine acetylcholine receptor (alpha1(2)beta1gammadelta) showed the presence of biological activity of the recombinant "weak" toxin close to the activity of the natural toxin (IC(50) = 4.3 +/- 0.3 and 3.0 +/- 0.5 microM, respectively). The interaction of the recombinant toxin with alpha7 type human neuronal acetylcholine receptor transfected in the GH(4)C(1) cell line also showed the presence of activity close to that of the natural toxin (IC(50) 31 +/- 5.0 and 14.8 +/- 1.3 microM, respectively). The developed bacterial system for production of N. kaouthia venom "weak" toxin was used to obtain (15)N-labeled analog of the neurotoxin.

Cobra cytotoxins, small proteins of three-fingered toxin family, unspecifically damage membranes in different cells and artificial vesicles. However, the molecular mechanism of this damage is not yet completely understood. We used steered molecular dynamics simulations to study the interaction of cardiotoxin A3 from Naja atra cobra venom with hydrated 1-palmitoyl-2-oleoyl-1-sn-3-phosphatidylcholine (POPC) bilayer. The studied system included one cytotoxin molecule, 64 lipid molecules (32 molecules in each monolayer) and 2500 water molecules. It was found that the toxin interacted with zwitterionic bilayer formed by POPC. During first nanosecond of simulation the toxin molecule was oriented toward membrane surface by loops' basement including cytotoxin regions Cys14-Asn19 and Cys38-Ser46. This orientation was stable enough and was not changed during next 6 ns of simulation. The obtained data suggest that cytotoxin molecule cannot penetrate into membrane composed of zwitterionic lipids without some auxiliary interaction.

Weak toxins are the "three-fingered" snake venoms toxins grouped together by having an additional disulfide in the N-terminal loop I. In general, weak toxins have low toxicity, and biological targets have been identified for some of them only, recently by detecting the effects on the nicotinic acetylcholine receptors (nAChR). Here the methods of docking and molecular dynamics simulations are used for comparative modeling of the complexes between four weak toxins of known spatial structure (WTX, candoxin, bucandin, gamma-bungarotoxin) and nAChRs. WTX and candoxin are those toxins whose blocking of the neuronal alpha7- and muscle-type nAChR has been earlier shown in binding assays and electrophysiological experiments, while for the other two toxins no such activity has been reported. Only candoxin and WTX are found here to give stable solutions for the toxin-nAChR complexes. These toxins appear to approach the binding site similarly to short alpha-neurotoxins, but their final position resembles that of alpha-cobratoxin, a long alpha-neurotoxin, in the complex with the acetylcholine-binding protein. The final spatial structures of candoxin and WTX complexes with the alpha7 neuronal or muscle-type nAChR are very similar and do not provide immediate answer why candoxin has a much higher affinity than WTX, but both of them share a virtually irreversible mode of binding to one or both these nAChR subtypes. Possible explanation comes from docking and MD simulations which predict fast kinetics of candoxin association with nAChR, no gross changes in the toxin conformation (with smaller toxin flexibility on alpha7 nAChR), while slow WTX binding to nAChR is associated with slow irreversible rearrangement both of the tip of the toxin loop II and of the binding pocket residues locking finally the toxin molecule. Computer modeling showed that the additional disulfide in the loop I is not directly involved in receptor binding of WTX and candoxin, but it stabilizes the structure of loop I which plays an important role in toxin delivery to the binding site. In summary, computer modeling visualized possible modes of binding for those weak toxins which interact with the nAChR, provided no solutions for those weak toxins whose targets are not the nAChRs, and demonstrated that the additional disulfide in loop I cannot be a sound criteria for joining all weak toxins into one group; the conclusion about the diversity of weak toxins made from computer modeling is in accord with the earlier phylogenetic analysis.

Multiple injections of conotoxin MII, a blocker of alfa3-s2 and alfa6-containing subtypes of nicotinic acetylcholine receptors (n-AChRs), as well as conotoxin ArIB11L16D, a blocker of alfa7 subtype n-AChR, at a dose of 1 nmol/kg reduce both the lactate dehydrogenase level in tumor cells and the inflammatory leukocyte infiltration in tumor tissue in mice bearing Ehrlich carcinoma. The first stage of pathomorphism was detected in the tumor tissue after the treatment with the ArIB11L16D conotoxin, whereas the second stage was observed after the treatment with conotoxins RgIA and MII. Only MII injections led to a significant reduction in tumor growth. Our results show the involvement of n-AChRs in the regulation of metabolic processes and cell-cell interactions related to carcinogenesis and tumor-associated inflammation.

Cytotoxins are positively charged polypeptides that constitute about 60% of all proteins in cobra venom; they have a wide spectrum of biological activities. By CD spectroscopy, cytotoxins CT1 and CT2 Naja oxiana, CT3 Naja kaouthia, and CT1 and CT2 Naja haje were shown to have similar secondary structure in an aqueous environment, with dominating beta-sheet structure, and to vary in the twisting angle of the beta-sheet and the conformation of disulfide groups. Using dodecylphosphocholine micelles and liposomes, CT1 and CT2 Naja oxiana were shown to incorporate into lipid structures without changes in the secondary structure of the peptides. The binding of CT1 and CT2 Naja oxiana with liposomes was associated with an increase in the beta-sheet twisting and a sign change of the dihedral angle of one disulfide group. The cytotoxins were considerably different in cytotoxicity and cooperativity of the effect on human promyelocytic leukemia cells HL60, mouse myelomonocytic cells WEHI-3, and human erythroleukemic cells K562. The most toxic CT2 Naja oxiana and CT3 Naja kaouthia possessed low cooperativity of interaction (Hill coefficient h = 0.6-0.8), unlike 10-20-fold less toxic CT1 and CT2 Naja haje (h = 1.2-1.7). CT1 Naja oxiana has an intermediate position on the cytotoxicity scale and is characterized by h = 0.5-0.8. The cytotoxins under study induced necrosis of HL60 cells and failed to activate apoptosis. The differences in cytotoxicity are supposed to be related not with features of the secondary structure of the peptides, but with interactions of side chains of variable amino acid residues with lipids and/or membrane proteins.

A sensitive nonradioactive method for detection of substances interacting with the neuronal α7-type nicotinic acetylcholine receptor (AChR) was proposed. The method uses biotinylated α-cobratoxin (Bt-CTX) and is based on the ability of the N-terminal ligand-binding extracellular domain (LBED) of AChR to interact with α-cobratoxin (CTX) as does the whole receptor. LBED produced by heterologous expression of a gene fragment of the α7 subunit of AChR from the rat brain in Escherichia coli cells was sorbed in wells of a 96-well plate and incubated with Bt-CTX. The specifically bound Bt-CTX was determined by staining with streptavidin–peroxidase complex. The ability of other compounds to interact with α7-AChR was checked according to the degree with which they inhibit the Bt-CTX binding to LBED. Nicotine, carbamylcholine, d-tubocurarin, anabaseine, conotoxin ImI, and neurotoxin II were used as model compounds. The sensitivity of this method was comparable with that of the radioligand method (up to 10 pmol).

Selective cloning of the cDNA coding for a weak neurotoxin (WTX) from cobra N. kaouthia including the 5'- and 3'-non-translated regions (NTR) is described. The known amino acid sequence of WTX was used together with the nucleotide sequence of a weak neurotoxin NNAM2 from cobra Naja atra, to design WTX-specific primers for direct amplification of an internal WTX cDNA fragment by RT- PCR. The sequence of the complete WTX cDNA was determined in sequencing runs on internal PCR products, cloned 3'- and 5'-RACE-fragments and several full-length cDNA clones. The cDNA coding sequence is in excellent agreement with the previously determined WTX amino acid sequence, has a high homology with other known weak toxin cDNAs, whereas even higher homology (up to 96%) with several classes of 3-finger toxins was detected in the 59 bp 3'-NTR consensus sequence. A possible function of the highly conserved nucleotide sequence elements is discussed.

Two photoactivatable analogues of α-conotoxin GI with the benzoylphenylalanine residue (Bpa) substituted for His10 or Tyr11 were synthesized using the method of solid-phase peptide synthesis. In addition, α-conotoxin MI was chemically modified by placing an azidobenzoyl or a benzoylbenzoyl photo label at Nα of Gly1 or Ne of Lys10. All the photoactivatable analogues were purified by HPLC, their structures were confirmed by MALDI MS, and the label positions in their molecules were localized by MS of their trypsinolysis fragments. All the analogues interacted with the nicotinic acetylcholine receptor (AChR) from Torpedo californica as efficiently as the native α-conotoxins, with the differences in the inhibition constants being within one order of magnitude under the same conditions. [125I] Derivatives prepared from all the analogues retained the ability to be bound by AChR and were used in the photoinduced AChR crosslinking. All the AChR subunits were found to be crosslinked to the photoactivatable analogues, with the linking depending on both the chemical nature of label and its position in the α-conotoxin molecule.

The influence of cobra neurotoxins on the Cl-dependent responses to acetylcholine (ACh) of Lymnaea neurons was studied by the voltage-clamp technique. It was found that a short chain neurotoxin II (NT II), a long chain cobratoxin (CTX) and weak neurotoxin (WTX) diminished the ACh-induced currents, the block being concentration-dependent and competitive. The IC 50 values of 130 nM for CTX, 11 μM for NT II, and 67 μM for WTX were determined. The block induced by NT II was quickly reversible upon toxin washout, whereas the action of CTX and WTX was only partially reversible even after an hour of intensive washing. The data obtained suggest that acetylcholine receptors (AChRs) in Lymnaea neurons have common features with cation-selective α7 AChRs of vertebrates and one type of Aplysia Cl-conducting AChRs.

With the purpose of studying structure-function relationships among weak neurotoxins (called so because of their low toxicity), we have isolated a toxin (WTX) from the venom of cobra Naja kaouthia using a combination of gel-filtration and ion-exchange chromatography. The amino acid sequence of the isolated toxin was determined by means of Edman degradation and MALDI mass spectrometry, the primary structure obtained being confirmed by 1H-NMR in the course of spatial structure analysis. The WTX sequence differs slightly from that of the toxin CM-9a isolated earlier from the same venom (Joubert and Taljaard, Hoppe-Seyler's Z. Physiol. Chem., 361 (1980) 425). The differences include an extra residue (Trp36) between Ser35 and Arg37 as well as interchanging of two residues (Tyr52 and Lys50) in the C-terminal part of the toxin molecule. These changes improve the alignment that can be made with other weak neurotoxin sequences. An extended sequence comparison reveals that WTX is the first case of a tryptophan-containing weak neurotoxin isolated from cobra venom. WTX was found to compete with radioiodinated alpha-bungarotoxin for binding to the membrane-bound nicotinic acetylcholine receptor from Torpedo californica.