Previously, we found that the venom of the pit viper Gloydius saxatilis inhibited the muscle-type nicotinic acetylcholine receptor (nAChR). Using liquid chromatography, a protein glosaxin was isolated from the venom that inhibited the binding of the α-bungarotoxin to the nAChR of muscle type from Torpedo californica. The amino acid sequence of the isolated protein was analyzed by high resolution mass spectrometry. Subsequent bioinformatic analysis showed that it is homologous to the amino acid sequences of disintegrin-like proteins, consisting of non-catalytic domains of type PIII metalloproteinases from the venom of pit vipers of genus Gloydius. A study of the biological activity of the isolated protein showed that it inhibits the binding of α-bungarotoxin to Torpedo californica nAChR with IC50 = 51 μM. The protein also inhibited acetylcholine-induced functional responses of the human neuronal nAChR of α3β2 subtype. This is the first evidence of the ability of proteins consisting of non-catalytic domains of snake venom type PIII metalloproteinase to inhibit the nAChR.

An Erratum to this paper has been published: https://doi.org/10.1134/S1607672923050095

Objective: Although main components of the venoms from Viperidae snakes are hemotoxins, several studies indicate the presence of neurotoxins in these venoms. We previously found that the venom of pit viper Gloydius saxatilis inhibited the muscle-type nicotinic acetylcholine receptor (nAChR). The objective of present work is to isolate and characterize a neurotoxic protein from this venom. Methods: The protein was isolated by liquid chromatography and characterized using high resolution mass-spectrometry. Results and Discussion: The isolated protein called glosaxin inhibited the binding of the α-bungarotoxin to the nAChR of muscle type from Torpedo californica. Investigation of the amino acid sequence of the isolated protein by high resolution mass spectrometry and the subsequent bioinformatic analysis showed that it is homologous to the amino acid sequences of disintegrin-like proteins, consisting of non-catalytic domains of class PIII metalloproteinases from the venom of pit vipers of genus Gloydius. Glosaxin was shown to inhibit the binding of α-bungarotoxin to T. californica nAChR with IC50 = 51 μM. It also inhibited ACh-induced functional responses of the human neuronal nAChR of α3β2 subtype. Conclusions: This is the first evidence for the ability of proteins consisting of non-catalytic domains of snake venom class PIII metalloproteinase to inhibit the nAChR.

The effects of extracts of ten plant species from Russia and five species from Vietnam on the growth and survival of ciliates Tetrahymena pyriformis were studied. T. pyriformis belongs to the subkingdom Protozoa, which also includes pathogens of protozoan infections. Extraction of dried plants was carried out with acidic and alkaline aqueous solutions, as well as with an aqueous ethanol. Various amounts of extracts were added to the ciliate cells, and the number of cells survived after incubation for 1 and 24 h was recorded. We found that our samples of several plants, including wormwood, harmala, and licorice, similarly to those studied earlier, exhibit antiprotozoal activity, which may indicate that the secondary metabolites are the same in plants from different regions. Using the ciliate T. pyriformis as a model organism, the presence of antiprotozoal activity in extracts of lilac, chondrilla, cinquefoil, hop, and elm was shown for the first time.

— In the 1960s and 1970s, the Institute of Chemistry of Natural Compounds developed a topochemical approach for designing new biologically active peptide compounds, the applicability of which to the creation of inhibitors and effective substrates of proteolytic enzymes was shown by the author of this review under the direct supervision of V.T. Ivanov. The next task was to establish the conformation of protein neurotoxins from snake venoms and to study the topography of their binding to the target, the nicotinic acetylcholine receptor (nAChR) from the electric organ of the Torpedo marmorata ray. With selectively labeled derivatives containing one fluorescent or spin label on established amino acid residues, neurotoxin residues in contact with nAChR were identified for the first time. Later, in collaboration with the laboratory of V.T. Ivanov, new analogs of α-conotoxins (peptide neurotoxins from venomous Conus mollusks), were synthesized including their photoactivated derivatives, which showed the participation of all Torpedo nAChR subunits in the binding of α-conotoxins. In the final part, the review briefly presents the recent achievements of the Department of Molecular Neuroimmune Signaling (headed by V.I. Tsetlin) concerning the isolation and synthesis of new peptide and protein neurotoxins and the study of how they work.

— Many different receptors and ion channels that regulate ion currents are involved in the control of the cardiovascular system (CVS). The functioning of the CVS occurs with the involvement of the processes of nervous and humoral regulation; in both cases, acetylcholine receptors of different families and subtypes as well as of different localization take part in these processes. It was demonstrated that acetylcholine receptors are directly located on the cell membranes of the heart and blood vessels; and this review considers the regulation of CVS functions with the involvement of only those acetylcholine receptors that are located in the tissue of the heart and blood vessels. In general, both muscarinic and nicotinic acetylcholine receptors are widely present in the tissues of the CVS. At the same time, muscarinic acetylcholine receptors are generally involved in the regulation of vascular tone and contractility of the heart, while nicotinic acetylcholine receptors are mainly involved in the regulation of a number of important pathophysiological processes directly affecting the functioning of the CVS. The regulation of the functioning of acetylcholine receptors can be considered as an addition to the existing methods of treating CVS diseases, including such diseases as atherosclerosis and heart failure. The use of blockers and activators of acetylcholine receptors for the study and/or treatment of pathological conditions of CVS is discussed.

— In screening the venoms of various snake species, we found that the venom of the Madagascar cat-eyed snake Madagascarophis colubrinus competes with α-bungarotoxin for binding to the nicotinic acetylcholine receptor from Torpedo californica . Using liquid chromatography, a peptide that inhibits the binding of α‑bungartoxin to this receptor was isolated from the venom and named macoluxin. The amino acid sequence of this 23-amino acid peptide was determined by automatic Edman degradation. Comparison with amino acid sequences of known proteins showed that the macoluxin sequence is homologous to the α-helical region of the sequence of snake venom metalloproteinases. The peptide was synthesized by solid-phase peptide synthesis, and the study of its biological activity showed that it inhibits the binding of α-bungarotoxin to the Torpedo receptor with an IC 50 of 47 μM. Macoluxin also reversibly inhibited acetylcholine-induced currents in the muscle-type nicotinic acetylcholine receptor. This is the first data on the presence of a peptide that can inhibit the nicotinic acetylcholine receptor in the venom of rear-fanged snakes.

Оligoarginines were recently discovered (Lebedev et al., 2019 Nov) as a novel class of nicotinic acetylcholine receptors (nAChRs) inhibitors, octaoligoarginine R8 showing a relatively high affinity (44 nM) for the α9/α10 nAChR. Since the inhibition of α9/α10 nAChR by α-conotoxin RgIA and its analogs is a possible way to drugs against neuropathic pain, here in a mice model we compared R8 with α-conotoxin RgIA in the effects on the chemotherapy-induced peripheral neuropathy (CIPN), namely on the long-term oxaliplatin induced neuropathy. Tests of cold allodynia, hot plate, Von Frey and grip strength analysis revealed for R8 and α-conotoxin RgIA similar positive effects, expressed most prominently after two weeks of administration. Histological analysis of the dorsal root ganglia sections showed for R8 and RgIA a similar partial correction of changes in the nuclear morphology of neurons. Since α9/α10 nAChR might be not the only drug target for R8, we analyzed the R8 action on rat TRPV1 and TRPA1, well-known nociceptive receptors. Against rTRPV1 at 25 μM there was no inhibition, while for rTRPA1 IC50 was about 20 μM. Thus, involvement of rTRPA1 cannot be excluded, but in view of the R8 much higher affinity for α9/α10 nAChR the latter seems to be the main target and the easily synthesized R8 can be considered as a potential candidate for a drug design.

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.

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.

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 antioxidant activity and protective effect in the toxicity model of H2O2 were studied for arachidonic (AA-CHOL), docosahexaenoic (DHA-CHOL), linoleic (Ln-CHOL), and oleic (Ol-CHOL) fatty acids, as well as arachidonoyl dicholine (AA-diCHOL) and O-arachidonoyl bistetramethylaminoisopropanol (ABTAP). AA-CHOL, DHA-CHOL and Ln-CHOL provided a 20% increase in cell survival. AA-CHOL, AA-diCHOL, Ol-CHOL, and ABTAP had a radical-scavenging effect in the ABTS test, approximately equal to the activity of a standard radical scavenger Trolox.

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.

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.

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.