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In this work, the heterodimeric phospholipase A 2 , HDP-2, from viper venom was investigated for its hydrolytic activity in model myelin membranes as well as for its effects on intermembrane exchange of phospholipids (studied by phosphorescence quenching) and on phospholipid polymorphism (studied by 1 H-NMR spectroscopy) to understand the role of sphingomyelin (SM) in the demyelination of nerve fibers. By using well-validated in vitro approaches, we show that the presence of SM in model myelin membranes leads to a significant inhibition of the hydrolytic activity of HDP-2, decreased intermembrane phospholipid exchange, and reduced phospholipid polymorphism. Using AutoDock software, we show that the NH δ+ group of the sphingosine backbone of SM binds to Tyr22(C=O pb δ- ) of HDP-2 via a hydrogen bond which keeps only the polar head of SM inside the HDP-2's active center and positions the sn-2 acyl ester bond away from the active center, thus making it unlikely to hydrolyze the alkyl chains at the sn-2 position. This observation strongly suggests that SM inhibits the catalytic activity of HDP-2 by blocking access to other phospholipids to the active center of the enzyme. Should this observation be verified in further studies, it would offer a tantalizing opportunity for developing effective pharmaceuticals to stop the demyelination of nerve fibers by aberrant PLA 2 s with overt activity - as observed in brain degenerative diseases - by inhibiting SM hydrolysis and/or facilitating SM synthesis in the myelin sheath membrane., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The knowledge of variations in the composition of venoms from different snakes is important from both theoretical and practical points of view, in particular, at developing and selecting an antivenom. Many studies on this topic are conducted with pooled venoms, while the existence and significance of variations in the composition of venoms between individual snakes of the same species are emphasized by many authors. It is important to study both inter- and intra-specific, including intra-population, venom variations, because intra-specific variations in the venom composition may affect the effectiveness of antivenoms as strongly as inter-specific. In this work, based on venom Raman spectroscopy with principal component analysis, we assessed the variations in venoms of individual snakes of the Vipera nikolskii species from two populations and compared these intra-specific variations with inter-specific variations (with regard to the other related species). We demonstrated intra-specific (inter- and intra-population) differences in venom compositions which are smaller than inter-specific variations. We also assessed the compositions of V. nikolskii venoms from two populations to explain inter-population differences. The method used is rapid and requires virtually no preparation of samples, used in extremely small quantities, allowing the venoms of individual snakes to be analyzed. In addition, the method is informative and capable of detecting fairly subtle differences in the composition of venoms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cytotoxins (CTs) are three-finger membrane-active toxins present mainly in cobra venom. Our analysis of the available CT amino acid sequences, literature data on their membrane activity, and conformational equilibria in aqueous solution and detergent micelles allowed us to identify specific amino acid residues which interfere with CT incorporation into membranes. They include Pro9, Ser28, and Asn/Asp45 within the N-terminal, central, and C-terminal loops, respectively. There is a hierarchy in the effect of these residues on membrane activity: Pro9 > Ser28 > Asn/Asp45. Taking into account all the possible combinations of special residues, we propose to divide CTs into eight groups. Group 1 includes toxins containing all of the above residues. Their representatives demonstrated the lowest membrane activity. Group 8 combines CTs that lack these residues. For the toxins from this group, the greatest membrane activity was observed. We predict that when solely membrane activity determines the cytotoxic effects, the activity of CTs from a group with a higher number should exceed that of CTs from a group with a lower number. This classification is supported by the available data on the cytotoxicity and membranotropic properties of CTs. We hypothesize that the special amino acid residues within the loops of the CT molecule may indicate their involvement in the interaction with non-lipid targets.

Evidence to date indicates that activation of nicotinic acetylcholine receptors (nAChRs) can reduce cardiac injury from ischemia and subsequent reperfusion. The use of nAChR agonists in various animal models leads to a reduction in reperfusion injury. Earlier this effect was shown for the agonists of α7 nAChR subtype. In this work, we demonstrated the expression of mRNA encoding α4, α6 and β2 nAChR subunits in the left ventricle of rat heart. In a rat model of myocardial ischemia, we studied the effect of α4β2 nAChR agonists cytisine and varenicline, medicines used for the treatment of nicotine addiction, and found them to significantly reduce myocardium ischemia-reperfusion injury, varenicline manifesting a higher protection. Dihydro-β-erythroidine, antagonist of α4β2 nAChR, as well as methyllycaconitine, antagonist of α7 and α6β2-containing nAChR, prevented protective effect of varenicline. This together with the presence of α4, α6 and β2 subunit mRNA in the left ventricule of rat heart raises the possibility that the varenicline effect is mediated by α4β2 as well as by α7 and/or α6β2-containing receptors. Our results point to a new way for the use of cytisine and varenicline as cardioprotective agents., Competing Interests: Declaration of competing interest The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of the data; in the writing of the manuscript; or in the decision to publish the results., (Copyright © 2023 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Previously, it was shown that the non-conventional toxin WTX from the venom of the cobra Naja kaouthia, when administered intravenously, caused a decrease in blood pressure (BP) and an increase in heart rate (HR) in rats [13]. To identify the site of the toxin molecule responsible for these effects, we studied the influence of synthetic peptide fragments of the WTX on BP and HR in normotensive male Sprague-Dawley rats under general anesthesia induced by Telazol and Xylazine. It was found that peptides corresponding to the WTX central polypeptide loop, stabilized by a disulfide bond, at intravenous injection at concentrations from 0.1 to 1.0 mg/mL caused a dose-dependent decrease in BP, with the HR increasing only in the first 5-10 min after administration. Thus, WTX fragments corresponding to the central polypeptide loop reproduce the decrease in blood pressure caused by the toxin., (© 2023. The Author(s).)

Cardiotoxins (CaTx) of the three-finger toxin family are one of the main components of cobra venoms. Depending on the structure of the N-terminal or the central polypeptide loop, they are classified into either group I and II or P- and S-types, respectively, and toxins of different groups or types interact with lipid membranes variably. While their main target in the organism is the cardiovascular system, there is no data on the effects of CaTxs from different groups or types on cardiomyocytes. To evaluate these effects, a fluorescence measurement of intracellular Ca 2+ concentration and an assessment of the rat cardiomyocytes' shape were used. The obtained results showed that CaTxs of group I containing two adjacent proline residues in the N-terminal loop were less toxic to cardiomyocytes than group II toxins and that CaTxs of S-type were less active than P-type ones. The highest activity was observed for Naja oxiana cobra cardiotoxin 2, which is of P-type and belongs to group II. For the first time, the effects of CaTxs of different groups and types on the cardiomyocytes were studied, and the data obtained showed that the CaTx toxicity to cardiomyocytes depends on the structures both of the N-terminal and central polypeptide loops.

Protozoal infections are a world-wide problem. The toxicity and somewhat low effectiveness of the existing drugs require the search for new ways of protozoa suppression. Snake venom contains structurally diverse components manifesting antiprotozoal activity; for example, those in cobra venom are cytotoxins. In this work, we aimed to characterize a novel antiprotozoal component(s) in the Bungarus multicinctus krait venom using the ciliate Tetrahymena pyriformis as a model organism. To determine the toxicity of the substances under study, surviving ciliates were registered automatically by an original BioLaT-3.2 instrument. The krait venom was separated by three-step liquid chromatography and the toxicity of the obtained fractions against T. pyriformis was analyzed. As a result, 21 kDa protein toxic to Tetrahymena was isolated and its amino acid sequence was determined by MALDI TOF MS and high-resolution mass spectrometry. It was found that antiprotozoal activity was manifested by β-bungarotoxin (β-Bgt) differing from the known toxins by two amino acid residues. Inactivation of β-Bgt phospholipolytic activity with p -bromophenacyl bromide did not change its antiprotozoal activity. Thus, this is the first demonstration of the antiprotozoal activity of β-Bgt, which is shown to be independent of its phospholipolytic activity.

In aqueous solutions, cobra cytotoxins (CTX), three-finger folded proteins, exhibit conformational equilibrium between conformers with either cis or trans peptide bonds in the N-terminal loop (loop-I). The equilibrium is shifted to the cis form in toxins with a pair of adjacent Pro residues in this loop. It is known that CTX with a single Pro residue in loop-I and a cis peptide bond do not interact with lipid membranes. Thus, if a cis peptide bond is present in loop-I, as in a Pro-Pro containing CTX, this should weaken its lipid interactions and likely cytotoxic activities. To test this, we have isolated seven CTX from Naja naja and N. haje cobra venoms. Antibacterial and cytotoxic activities of these CTX, as well as their capability to induce calcein leakage from phospholipid liposomes, were evaluated. We have found that CTX with a Pro-Pro peptide bond indeed exhibit attenuated membrane-perturbing activity in model membranes and lower cytotoxic/antibacterial activity compared to their counterparts with a single Pro residue in loop-I., Competing Interests: The authors declare no conflicts of interest.

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., (© 2022. Pleiades Publishing, Ltd.)

Cytotoxins (CTXs), an essential class of the non-enzymatic three-finger toxin family, are ubiquitously present in cobra venoms. These low-molecular-mass toxins, contributing to about 40 to 60% of the cobra venom proteome, play a significant role in cobra venom-induced toxicity, more prominently in dermonecrosis. Structurally, CTXs contain the conserved three-finger hydrophobic loops; however, they also exhibit a certain degree of structural diversity that dictates their biological activities. In their mechanism, CTXs mediate toxicity by affecting cell membrane structures and membrane-bound proteins and activating apoptotic and necrotic cell death pathways. Notably, some CTXs are also responsible for depolarizing neurons and heart muscle membranes, thereby contributing to the cardiac failure frequently observed in cobra-envenomed victims. Consequently, they are also known as cardiotoxins (CdTx). Studies have shown that cobra venom CTXs form cognate complexes with other components that potentiate the toxic effects of the venom's individual component. This review focuses on the pharmacological mechanism of cobra venom CTXs and their complexes, highlighting their significance in cobra venom-induced pathophysiology and toxicity. Furthermore, the potency of commercial antivenoms in reversing the adverse effects of cobra venom CTXs and their complexes in envenomed victims has also been discussed.

Background: The cardiovascular system is one of the first systems to be affected by snake toxins; but not many toxins exert a direct effect on the heart. Cobra venom cardiotoxins are among those few toxins that attack the heart. Although the two cardiotoxin types (S and P) differ in their central-loop structure, it is not known whether they differ in their effect on the mammalian heart. We compared the effects of S- and P-type cardiotoxins, CTХ-1 and CTХ-2, respectively, from the cobra Naja oxiana , on the isolated rat heart ., Methods: An isolated rat heart perfused according to the Langendorff technique was used in this study to investigate the activity of cardiotoxins CTX-1 and CTX-2. The following parameters were registered: the left ventricular developed pressure, calculated as the difference between systolic and diastolic pressure in the left ventricle, the end-diastolic pressure, the heart rate, time to maximal end-diastolic pressure (heart contracture), and time to depression of the heart contraction., Results: Both cardiotoxins at the concentration of 5 μg/mL initially produce a slight increase in systolic intraventricular pressure, followed by its rapid decrease with a simultaneous increase in diastolic intraventricular pressure until reaching contracture. CTX-2 blocks cardiac contractions faster than CTX-1; in its presence the maximum diastolic pressure is reached faster and the magnitude of the developed contracture is higher., Conclusion: The P-type cardiotoxin CTX-2 more strongly impairs rat heart functional activity than the S-type cardiotoxin CTX-1, as expressed in its faster blockage of cardiac contractions as well as in more rapid development and greater magnitude of contracture in its presence., Competing Interests: Competing interests : The authors declare that they have no competing interests.

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., (© 2022. Pleiades Publishing, Ltd.)

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., (© 2022. Pleiades Publishing, Ltd.)

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 IC 50 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., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2022 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Cobra cytotoxins (CTs) belong to the three-fingered protein family and possess membrane activity. Here, we studied cytotoxin 13 from Naja naja cobra venom (CT13Nn). For the first time, a spatial model of CT13Nn with both "water" and "membrane" conformations of the central loop (loop-2) were determined by X-ray crystallography. The "water" conformation of the loop was frequently observed. It was similar to the structure of loop-2 of numerous CTs, determined by either NMR spectroscopy in aqueous solution, or the X-ray method. The "membrane" conformation is rare one and, to date has only been observed by NMR for a single cytotoxin 1 from N. oxiana (CT1No) in detergent micelle. Both CT13Nn and CT1No are S-type CTs. Membrane-binding of these CTs probably involves an additional step-the conformational transformation of the loop-2. To confirm this suggestion, we conducted molecular dynamics simulations of both CT1No and CT13Nn in the Highly Mimetic Membrane Model of palmitoiloleoylphosphatidylglycerol, starting with their "water" NMR models. We found that the both toxins transform their "water" conformation of loop-2 into the "membrane" one during the insertion process. This supports the hypothesis that the S-type CTs, unlike their P-type counterparts, require conformational adaptation of loop-2 during interaction with lipid membranes.

Cardiotoxins (CaTxs) are a group of snake toxins that affect the cardiovascular system (CVS). Two types (S and P) of CaTxs are known, but the exact differences in the effects of these types on CVS have not been thoroughly studied. We investigated cellular mechanisms of action on CVS for Naja oxiana cobra CaTxs CTX-1 (S-type) and CTX-2 (P-type) focusing on the papillary muscle (PM) contractility and contraction of aortic rings (AR) supplemented by pharmacological analysis. It was found that CTX-1 and CTX-2 exerted dose-dependent effects manifested in PM contracture and AR contraction. CTX-2 impaired functions of PM and AR more strongly than CTX-1. Effects of CaTxs on PM were significantly reduced by nifedipine, an L-type Ca 2+ channel blocker, and by KB-R7943, an inhibitor of reverse-mode Na + /Ca 2+ exchange. Furthermore, 2-aminoethoxydiphenyl borate, an inhibitor of store-operated calcium entry, partially restored PM contractility damaged by CaTxs. The CaTx influence on AR contracture was significantly reduced by nifedipine and KB-R7943. The involvement of reverse-mode Na + /Ca 2+ exchange in the effect of CaTxs on the rat aorta was shown for the first time. The results obtained indicate that CaTx effects on CVS are mainly associated with disturbance of transporting systems responsible for the Ca 2+ influx.

The COVID-19 pandemic caused by SARS-CoV-2 requires new treatments both to alleviate the symptoms and to prevent the spread of this disease. Previous studies demonstrated good antiviral and virucidal activity of phospholipase A 2 s (PLA 2 s) from snake venoms against viruses from different families but there was no data for coronaviruses. Here we show that PLA 2 s from snake venoms protect Vero E6 cells against SARS-CoV-2 cytopathic effects. PLA 2 s showed low cytotoxicity to Vero E6 cells with some activity at micromolar concentrations, but strong antiviral activity at nanomolar concentrations. Dimeric PLA 2 from the viper Vipera nikolskii and its subunits manifested especially potent virucidal effects, which were related to their phospholipolytic activity, and inhibited cell-cell fusion mediated by the SARS-CoV-2 spike glycoprotein. Moreover, PLA 2 s interfered with binding both of an antibody against ACE2 and of the receptor-binding domain of the glycoprotein S to 293T/ACE2 cells. This is the first demonstration of a detrimental effect of PLA 2 s on β-coronaviruses. Thus, snake PLA 2 s are promising for the development of antiviral drugs that target the viral envelope, and could also prove to be useful tools to study the interaction of viruses with host cells., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Fluorescence can be exploited to monitor intermolecular interactions in real time and at a resolution up to a single molecule. It is a method of choice to study ligand-receptor interactions. However, at least one of the interacting molecules should possess good fluorescence characteristics, which can be achieved by the introduction of a fluorescent label. Gene constructs with green fluorescent protein (GFP) are widely used to follow the expression of the respective fusion proteins and monitor their function. Recently, a small synthetic analogue of GFP chromophore ( p -HOBDI-BF 2 ) was successfully used for tagging DNA molecules, so we decided to test its applicability as a potential fluorescent label for proteins and peptides. This was done on α-cobratoxin (α-CbTx), a three-finger protein used as a molecular marker of muscle-type, neuronal α7 and α9/α10 nicotinic acetylcholine receptors (nAChRs), as well as on azemiopsin, a linear peptide neurotoxin selectively inhibiting muscle-type nAChRs. An activated N-hydroxysuccinimide ester of p -HOBDI-BF 2 was prepared and utilized for toxin labeling. For comparison we used a recombinant α-CbTx fused with a full-length GFP prepared by expression of a chimeric gene. The structure of modified toxins was confirmed by mass spectrometry and their activity was characterized by competition with iodinated α-bungarotoxin in radioligand assay with respective receptor preparations, as well as by thermophoresis. With the tested protein and peptide neurotoxins, introduction of the synthetic GFP chromophore induced considerably lower decrease in their affinity for the receptors as compared with full-length GFP attachment. The obtained fluorescent derivatives were used for nAChR visualization in tissue slices and cell cultures., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kasheverov, Kuzmenkov, Kudryavtsev, Chudetskiy, Shelukhina, Barykin, Ivanov, Siniavin, Ziganshin, Baranov, Tsetlin, Vassilevski and Utkin.)

The first toxin to give rise to the three-finger protein (TFP) family was α-bungarotoxin (α-Bgt) from Bungarus multicinctus krait venom. α-Bgt was crucial for research on nicotinic acetylcholine receptors (nAChRs), and in this Review article we focus on present data for snake venom TFPs and those of the Ly6/uPAR family from mammalians (membrane-bound Lynx1 and secreted SLURP-1) interacting with nAChRs. Recently isolated from Bungarus candidus venom, αδ-bungarotoxins differ from α-Bgt: they bind more reversibly and distinguish two binding sites in Torpedo californica nAChR. Naja kaouthia α-cobratoxin, classical blocker of nAChRs, was shown to inhibit certain GABA-A receptor subtypes, whereas α-cobratoxin dimer with 2 intermolecular disulfides has a novel type of 3D structure. Non-conventional toxin WTX has additional 5th disulfide not in the central loop, as α-Bgt, but in the N-terminal loop, like all Ly6/uPAR proteins, and inhibits α7 and Torpedo nAChRs. A water-soluble form of Lynx1, ws-Lynx1, was expressed in E. coli, its 1 H-NMR structure and binding to several nAChRs determined. For SLURP-1, similar information was obtained with its recombinant analogue rSLURP-1. A common feature of ws-Lynx1, rSLURP-1, and WTX is their activity against nAChRs and muscarinic acetylcholine receptors. Synthetic SLURP-1, identical to the natural protein, demonstrated some differences from rSLURP-1 in distinguishing nAChR subtypes. The loop II fragment of the Lynx1 was synthesized having the same µM affinity for the Torpedo nAChR as ws-Lynx1. This review illustrates the productivity of parallel research of nAChR interactions with the two TFP groups., (© 2020 International Society for Neurochemistry.)

Snake venoms, as complex mixtures of peptides and proteins, affect various vital systems of the organism. One of the main targets of the toxic components from snake venoms is the cardiovascular system. Venom proteins and peptides can act in different ways, exhibiting either cardiotoxic or cardioprotective effects. The principal classes of these compounds are cobra cardiotoxins, phospholipases A2, and natriuretic, as well as bradykinin-potentiating peptides. There is another group of proteins capable of enhancing angiogenesis, which include, e.g., vascular endothelial growth factors possessing hypotensive and cardioprotective activities. Venom proteins and peptides exhibiting cardiotropic and vasoactive effects are promising candidates for the design of new drugs capable of preventing or constricting the development of pathological processes in cardiovascular diseases, which are currently the leading cause of death worldwide. For example, a bradykinin-potentiating peptide from Bothrops jararaca snake venom was the first snake venom compound used to create the widely used antihypertensive drugs captopril and enalapril. In this paper, we review the current state of research on snake venom components affecting the cardiovascular system and analyse the mechanisms of physiological action of these toxins and the prospects for their medical application., (Copyright ® 2021 National Research University Higher School of Economics.)

Cobra cytotoxins (CTs), the three-fingered proteins, feature high amino acid sequence homology in the beta-strands and variations in the loop regions. We selected a pair of cytotoxins from Naja kaouthia crude venom to clarify the sequence-structure relationships. Using chromatography and mass spectroscopy, we separated and identified the mixture of cytotoxins 2 and 3, differentiated by the only Val 41/Ala 41 substitution. Here, using natural abundance 13 C, 15 N NMR-spectroscopy we performed chemical shift assignments of the signals of the both toxins in aqueous solution in the major and minor forms. Combining NOE and chemical shift data, the toxins' spatial structure was determined. Finally, we proved that the tip of the "finger"-2, or the loop-2 of cytotoxins adopts the shape of an omega-loop with a tightly-bound water molecule in its cavity. Comparison with other NMR and X-ray structures of cytotoxins possessing different amino acid sequences reveals spatial similarity in this family of proteins, including the loop-2 region, previously considered to be flexible., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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 1 H-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.

Excitatory α7 neuronal nicotinic receptors (nAChR) are widely expressed in the central and peripheral nervous and immune systems and are important for learning, memory, and immune response regulation. Specific α7 nAChR ligands, including positive allosteric modulators are promising to treat cognitive disorders, inflammatory processes, and pain. One of them, PNU-120596, highly increased the neuron response to α7 agonists and retarded desensitization, showing selectivity for α7 as compared to heteromeric nAChRs, but was not examined at the inhibitory ligand-gated channels. We studied PNU-120596 action on anion-conducting channels using voltage-clamp techniques: it slightly potentiated the response of human glycine receptors expressed in PC12 cells, of rat GABA A receptors in cerebellar Purkinje cells and mouse GABA A Rs heterologously expressed in Xenopus oocytes. On the contrary, PNU-120596 exerted an inhibitory effect on the receptors mediating anion currents in Lymnaea stagnalis neurons: two nAChR subtypes, GABA and glutamate receptors. Acceleration of the current decay, contrary to slowing down desensitization in mammalian α7 nAChR, was observed in L. stagnalis neurons predominantly expressing one of the two nAChR subtypes. Thus, PNU-120596 effect on these anion-selective nAChRs was just opposite to the action on the mammalian cation-selective α7 nAChRs. A comparison of PNU-120596 molecule docked to the models of transmembrane domains of the human α7 AChR and two subunits of L. stagnalis nAChR demonstrated some differences in contacts with the amino acid residues important for PNU-120596 action on the α7 nAChR. Thus, our results show that PNU-120596 action depends on a particular subtype of these Cys-loop receptors., (© 2020 International Society for Neurochemistry.)

Venoms of viperid snakes affect mostly hemostasis, while C-type lectin-like proteins (CTLPs), one of the main components of viperid venoms, act as anticoagulants, procoagulants, or agonists/antagonists of platelet activation. However, we have shown earlier that CTLPs from the saw-scaled viper Echis multisquamatus , called emunarecins EM1 and EM2, were able to inhibit nicotinic acetylcholine receptors (nAChRs) in neurons of a pond snail ( Lymnaea stagnalis ). Here we analysed the structure of the emunarecins by mass spectrometry and report that EM1 and EM2 inhibit fluorescent α-bungarotoxin binding to both muscle-type nAChRs from Torpedo californica and human neuronal α7 nAChRs. EM1 at 23µM and EM2 at 9µM almost completely prevented fluorecsent α-bungarotoxin binding to muscle-type nAChRs. Interaction with human neuronal α7 nAChR was weaker; EM1 at the concentration of 23µM blocked the α-bungarotoxin binding only by about 40% and EM2 at 9µM by about 20%. The efficiency of the EM2 interaction with nAChRs was comparable to that of a non-conventional toxin, WTX, from Naja kaouthia cobra venom. Together with the data obtained earlier, these results show that CTLPs may represent new nAChR ligands., (© Copyright The Author(s).)

Snake venoms possess lethal activities against different organisms, ranging from bacteria to higher vertebrates. Several venoms were shown to be active against protozoa, however, data about the anti-protozoan activity of cobra and viper venoms are very scarce. We tested the effects of venoms from several snake species on the ciliate Tetrahymena pyriformis . The venoms tested induced T. pyriformis immobilization, followed by death, the most pronounced effect being observed for cobra Naja sumatrana venom. The active polypeptides were isolated from this venom by a combination of gel-filtration, ion exchange and reversed-phase HPLC and analyzed by mass spectrometry. It was found that these were cytotoxins of the three-finger toxin family. The cytotoxins from several cobra species were tested and manifested toxicity for infusorians. Light microscopy revealed that, because of the cytotoxin action, the infusorians' morphology was changed greatly, from teardrop-like to an almost spherical shape, this alteration being accompanied by a leakage of cell contents. Fluorescence microscopy showed that the fluorescently labelled cytotoxin 2 from cobra N. oxiana was localized mainly at the membrane of killed infusorians, indicating that cytotoxins may kill T. pyriformis by causing membrane rupture. This work is the first evidence of the antiprotozoal activity of cobra venom cytotoxins, as demonstrated by the example of the ciliate T. pyriformis .

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.

Immune response during sepsis is characterized by hyper-inflammation followed by immunosuppression. The crucial role of macrophages is well-known for both septic stages, since they are involved in immune homeostasis and inflammation, their dysfunction being implicated in immunosuppression. The cholinergic anti-inflammatory pathway mediated by macrophage α7 nicotinic acetylcholine receptor (nAChR) represents possible drug target. Although α7 nAChR activation on macrophages reduces the production of proinflammatory cytokines, the role of these receptors in immunological changes at the cellular level is not fully understood. Using α7 nAChR selective agonist PNU 282,987, we investigated the influence of α7 nAChR activation on the expression of cytokines and, for the first time, of the macrophage membrane markers: cluster of differentiation 14 (CD14), human leukocyte antigen-DR (HLA-DR), CD11b, and CD54. Application of PNU 282,987 to THP-1Mϕ (THP-1 derived macrophages) cells led to inward ion currents and Ca 2+ increase in cytoplasm showing the presence of functionally active α7 nAChR. Production of cytokines tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-10 was estimated in classically activated macrophages (M1) and treatment with PNU 282,987 diminished IL-10 expression. α7 nAChR activation on THP-1Mϕ, THP-1M1, and monocyte-derived macrophages (MDMs) increased the expression of HLA-DR, CD54, and CD11b molecules, but decreased CD14 receptor expression, these effects being blocked by alpha (α)-bungarotoxin. Thus, PNU 282,987 enhances the macrophage-mediated immunity via α7 nAChR by regulating expression of their membrane receptors and of cytokines, both playing an important role in preventing immunosuppressive states.

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.

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.

Antibacterial activity of the three-finger toxins from cobra venom, including cytotoxin 3 from N. kaouthia, cardiotoxin-like basic polypeptide A5 from N. naja (CLBP), and alpha-neurotoxin from N. oxiana venom, was investigated. All toxins failed to influence Gram-negative bacteria. The most pronounced activity against Bacillus subtilis was demonstrated by CLBP. The latter is ascribed to the presence of additional Lys-residues within the membrane-binding motif of this toxin., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Snake venoms are the complex mixtures of different compounds manifesting a wide array of biological activities. The venoms of kraits (genus Bungarus, family Elapidae) induce mainly neurological symptoms; however, these venoms show a cytotoxicity against cancer cells as well. This study was conducted to identify in Bungarus fasciatus venom an active compound(s) exerting cytotoxic effects toward MCF7 human breast cancer cells and A549 human lung cancer cells., Methods: The crude venom of B. fasciatus was separated by gel-filtration on Superdex HR 75 column and reversed phase HPLC on C18 column. The fractions obtained were screened for cytotoxic effect against MCF7, A549, and HK2 cell lines using colorimetric assay with the tetrazolium dye MTT- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. The primary structure of active protein was established by ultra high resolution LC-MS/MS. The molecular mechanism of the isolated protein action on MCF7 cells was elucidated by flow cytometry., Results: MTT cell viability assays of cancer cells incubated with fractions isolated from B. fasciatus venom revealed a protein with molecular mass of about 13 kDa possessing significant cytotoxicity. This protein manifested the dose and time dependent cytotoxicity for MCF7 and A549 cell lines while showed no toxic effect on human normal kidney HK2 cells. In MCF7, flow cytometry analysis revealed a decrease in the proportion of Ki-67 positive cells. As Ki-67 protein is a cellular marker for proliferation, its decline indicates the reduction in the proliferation of MCF7 cells treated with the protein. Flow cytometry analysis of MCF7 cells stained with propidium iodide and Annexin V conjugated with allophycocyanin showed that a probable mechanism of cell death is apoptosis. Mass spectrometric studies showed that the cytotoxic protein was phospholipase A 2 . The amino acid sequence of this enzyme earlier was deduced from cloned cDNA, and in this work it was isolated from the venom as a protein for the first time. It is also the first krait phospholipase A 2 manifesting the cytotoxicity for cancer cells., Competing Interests: The authors declare that they have no competing interests., (© 2019 Tran et al.)

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 IC 50 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., (© 2019 Federation of European Biochemical Societies.)

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.

It is well-known that drugs administered into an organism intravenously or through the gastrointestinal tract are degraded by enzymes of the body, reducing their therapeutic effect. One of the ways to decrease this undesirable process is through the inclusion of drugs in nanomaterials. Earlier strong anticoagulant activity was demonstrated for dipeptide IleTrp (IW) and adenosine (Ado). In this work, the effect of inclusion in nanomaterials on the biological activity of IW and Ado was studied. For this purpose, Ado and IW were incorporated into thermosensitive nanogel composed of pluronic P123-grafted heparin. The prepared nanocarrier was characterized by transmission electron microscopy, dynamic light scattering, and ζ-potential. Biological activity was determined by measuring the bleeding time from mouse tail in vivo and the time of clot formation in vitro. It was found that encapsulation of Ado and IW into nanomaterial significantly increased their effects, resulting in an increase in the bleeding time from mouse tail and clot formation time. Thus, inclusion of low molecular weight anticoagulants Ado and IW into nanomaterials may be considered a way to increase their biological activity.

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 IC 50 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 IC 50 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.

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 rhythmoinotropic characteristics has shown 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 the 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.

αδ-Bungarotoxins, a novel group of long-chain α-neurotoxins, manifest different affinity to two agonist/competitive antagonist binding sites of muscle-type nicotinic acetylcholine receptors (nAChRs), being more active at the interface of α-δ subunits. Three isoforms (αδ-BgTx-1-3) were identified in Malayan Krait ( Bungarus candidus ) from Thailand by genomic DNA analysis; two of them (αδ-BgTx-1 and 2) were isolated from its venom. The toxins comprise 73 amino acid residues and 5 disulfide bridges, being homologous to α-bungarotoxin (α-BgTx), a classical blocker of muscle-type and neuronal α7, α8, and α9α10 nAChRs. The toxicity of αδ-BgTx-1 (LD 50  = 0.17-0.28 µg/g mouse, i.p. injection) is essentially as high as that of α-BgTx. In the chick biventer cervicis nerve-muscle preparation, αδ-BgTx-1 completely abolished acetylcholine response, but in contrast with the block by α-BgTx, acetylcholine response was fully reversible by washing. αδ-BgTxs, similar to α-BgTx, bind with high affinity to α7 and muscle-type nAChRs. However, the major difference of αδ-BgTxs from α-BgTx and other naturally occurring α-neurotoxins is that αδ-BgTxs discriminate the two binding sites in the Torpedo californica and mouse muscle nAChRs showing up to two orders of magnitude higher affinity for the α-δ site as compared with α-ε or α-γ binding site interfaces. Molecular modeling and analysis of the literature provided possible explanations for these differences in binding mode; one of the probable reasons being the lower content of positively charged residues in αδ-BgTxs. Thus, αδ-BgTxs are new tools for studies on nAChRs., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Enzyme-responsive liposomes release their cargo in response to pathologically increased levels of enzymes at the target site. We report herein an assembly of phospholipase A2-responsive liposomes based on colchicinoid lipid prodrugs incorporated into lipid bilayer of the nanosized vesicles. The liposomes were constructed to addresses two important issues: (i) the lipid prodrugs were designed to fit the structure of the enzyme binding site; and (ii) the concept of lateral pressure profile was used to design lipid prodrugs that introduce almost no distortions into the lipid bilayer packing, thus ensuring that corresponding liposomes are stable. The colchicinoid agents exhibit antiproliferative activity in subnanomolar range of concentrations.

Three-finger toxins (TFTs) comprise one of largest families of snake venom toxins. While they are principal to and the most toxic components of the venoms of the Elapidae snake family, their presence has also been detected in the venoms of snakes from other families. The first TFT, α-bungarotoxin, was discovered almost 50 years ago and has since been used widely as a specific marker of the α7 and muscle-type nicotinic acetylcholine receptors. To date, the number of TFT amino acid sequences deposited in the UniProt Knowledgebase free-access database is more than 700, and new members are being added constantly. Although structural variations among the TFTs are not numerous, several new structures have been discovered recently; these include the disulfide-bound dimers of TFTs and toxins with nonstandard pairing of disulfide bonds. New types of biological activities have also been demonstrated for the well-known TFTs, and research on this topic has become a hot topic of TFT studies. The classic TFTs α-bungarotoxin and α-cobratoxin, for example, have now been shown to inhibit ionotropic receptors of γ-aminobutyric acid, and some muscarinic toxins have been shown to interact with adrenoceptors. New, unexpected activities have been demonstrated for some TFTs as well, such as toxin interaction with interleukin or insulin receptors and even TFT-activated motility of sperm. This minireview provides a summarization of the data that has emerged in the last decade on the TFTs and their activities., Competing Interests: Conflict-of-interest statement: Utkin YN declares no conflict of interest related to this publication.

Background: Aging is a common and inevitable stage in the life cycle of higher organisms. Different organs, including the central nervous system, are affected by aging in different ways. Many processes are involved in aging, and neurodegeneration is one of the aging processes in which the central nervous system is engaged. Brain degeneration during normal aging underlies cognitive disorders experienced by older people. Not all molecular mechanisms associated with age-related neurodegeneration are fully understood; however, there is a whole range of data on the participation of nicotinic acetylcholine receptors in the processes of aging and neurodegeneration. Two main subtypes of nicotinic acetylcholine receptor α7 and α4β2 present in the central nervous system are affected by these processes. The loss of these receptor subtypes during normal aging is one of the reasons for the cognitive impairments. The decrease in nicotinic acetylcholine receptors is also very important for the pathogenesis of age-related neurodegenerative diseases. Thus, the drugs enhancing receptor functions may be considered promising for the treatment of cognitive dysfunction in the aged people., Conclusion: To achieve healthy longevity, the molecular processes that occur during aging should be established. In this regard, the participation and role of nicotinic acetylcholine receptors in the brain aging and degeneration are considered in this review., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Cobra cytotoxins (CTs) belong to the three-fingered protein family. They are classified into S- and P-types, the latter exhibiting higher membrane-perturbing capacity. In this work, we investigated the interaction of CTs with phospholipid bilayers, using coarse-grained (CG) and full-atom (FA) molecular dynamics (MD). The object of this work is a CT of an S-type, cytotoxin I (CT1) from N.oxiana venom. Its spatial structure in aqueous solution and in the micelles of dodecylphosphocholine (DPC) were determined by 1 H-NMR spectroscopy. Then, via CG- and FA MD-computations, we evaluated partitioning of CT1 molecule into palmitoyloleoylphosphatidylcholine (POPC) membrane, using the toxin spatial models, obtained either in aqueous solution, or detergent micelle. The latter model exhibits minimal structural changes upon partitioning into the membrane, while the former deviates from the starting conformation, loosing the tightly bound water molecule in the loop-2. These data show that the structural changes elicited by CT1 molecule upon incorporation into DPC micelle take place likely in the lipid membrane, although the mode of the interaction of this toxin with DPC micelle (with the tips of the all three loops) is different from its mode in POPC membrane (primarily with the tip of the loop-1 and both the tips of the loop-1 and loop-2).

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.

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 (IC 50 ~ 19 nM) calcium response of muscle nAChR evoked by 30 μM (EC 100 ) acetylcholine but was less potent (IC 50 ~ 3 μM) at α7 nAChR activated by 10 μM (EC 50 ) acetylcholine and had a low affinity to α4β2 and α3-containing nAChR, as well as to GABA A 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 (LD 50 510 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., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript and in the decision to publish the results.

Introduction: The brain is the most complicated organ in a vertebrate's organism. In a human, it contains about two hundred billions of neurons and non-neuronal cells. To understand the mechanisms of the brain functions is the great challenge for the researchers. Much is already done on this way; however, it remains a lot to do still, and to get deeper knowledge, new approaches should be developed. One of this is to use benefits that nanotechnology brings in this area. Nanotechnology opens up unique opportunities, not only for material science research, but also for biology, medicine, and many other disciplines. There are several kinds of nanoparticles that can be applied in brain studies, Quantum Dots (QD) being so far most often used. QD are semiconductor light emitting nanocrystals with nanometer-sized structures of unique optical properties. They have bright fluorescence, are resistant to bleaching and able of emitting fluorescent light of different wavelengths. These properties make QD perfect tools for visualization of brain structures and mechanisms underlying its functions. Due to unique QD properties, even single molecules under study can be observed. Moreover QDs can be used for brain-targeted drug delivery., Conclusion: In this review, the application of quantum dots for the brain research is considered and benefits that it can bring are discussed., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)