Specific Amino Acid Residues in the Three Loops of Snake Cytotoxins Determine Their Membrane Activity and Provide a Rationale for a New Classification of These Toxins.

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.

Analysis of intra-specific variations in the venom of individual snakes based on Raman spectroscopy.

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.

Anti-smoking drugs cytisine and varenicline reduce cardiac reperfusion injury in rat model of myocardial ischemia.

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 ß2 nAChR subunits in the left ventricle of rat heart. In a rat model of myocardial ischemia, we studied the effect of α4ß2 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-ß-erythroidine, antagonist of α4ß2 nAChR, as well as methyllycaconitine, antagonist of α7 and α6ß2-containing nAChR, prevented protective effect of varenicline. This together with the presence of α4, α6 and ß2 subunit mRNA in the left ventricule of rat heart raises the possibility that the varenicline effect is mediated by α4ß2 as well as by α7 and/or α6ß2-containing receptors. Our results point to a new way for the use of cytisine and varenicline as cardioprotective agents.

Molecular Mechanisms of Animal Toxins, Venoms and Antivenoms.

In many animals belonging to different taxa, venoms evolved as a means of defense and/or a means of attack/hunting [...].

Effects of Cobra Cardiotoxins on Intracellular Calcium and the Contracture of Rat Cardiomyocytes Depend on Their Structural Types.

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 Ca2+ 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.

The Potassium Channel Blocker ß-Bungarotoxin from the Krait Bungarus multicinctus Venom Manifests Antiprotozoal Activity.

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 ß-bungarotoxin (ß-Bgt) differing from the known toxins by two amino acid residues. Inactivation of ß-Bgt phospholipolytic activity with p-bromophenacyl bromide did not change its antiprotozoal activity. Thus, this is the first demonstration of the antiprotozoal activity of ß-Bgt, which is shown to be independent of its phospholipolytic activity.

Current Insights in the Mechanisms of Cobra Venom Cytotoxins and Their Complexes in Inducing Toxicity: Implications in Antivenom Therapy.

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.

S- and P-type cobra venom cardiotoxins differ in their action on isolated rat heart.

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, CTÐ¥-1 and CTÐ¥-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.

Effects of Cardiotoxins from Naja oxiana Cobra Venom on Rat Heart Muscle and Aorta: A Comparative Study of Toxin-Induced Contraction Mechanisms.

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 Ca2+ channel blocker, and by KB-R7943, an inhibitor of reverse-mode Na+/Ca2+ 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+/Ca2+ 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 Ca2+ influx.

Variability in the Spatial Structure of the Central Loop in Cobra Cytotoxins Revealed by X-ray Analysis and Molecular Modeling.

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.

Membrane-Disrupting Activity of Cobra Cytotoxins Is Determined by Configuration of the N-Terminal Loop.

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.

Snake Toxins Labeled by Green Fluorescent Protein or Its Synthetic Chromophore are New Probes for Nicotinic acetylcholine Receptors.

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-BF2) 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-BF2 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.

Snake venom phospholipase A2s exhibit strong virucidal activity against SARS-CoV-2 and inhibit the viral spike glycoprotein interaction with ACE2.

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 A2s (PLA2s) from snake venoms against viruses from different families but there was no data for coronaviruses. Here we show that PLA2s from snake venoms protect Vero E6 cells against SARS-CoV-2 cytopathic effects. PLA2s showed low cytotoxicity to Vero E6 cells with some activity at micromolar concentrations, but strong antiviral activity at nanomolar concentrations. Dimeric PLA2 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, PLA2s 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 PLA2s on ß-coronaviruses. Thus, snake PLA2s 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.

The omega-loop of cobra cytotoxins tolerates multiple amino acid substitutions.

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 13C, 15N 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.

α-Conotoxins and α-Cobratoxin Promote, while Lipoxygenase and Cyclooxygenase Inhibitors Suppress the Proliferation of Glioma C6 Cells.

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, ß2 and ß4 subunits of nAChR were expressed in C6 glioma cells. This is the first indication for involvement of nAChRs in mechanisms of glioma cell proliferation.

Three-finger proteins from snakes and humans acting on nicotinic receptors: Old and new.

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.

Spatial Structure and Activity of Synthetic Fragments of Lynx1 and of Nicotinic Receptor Loop C Models.

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

Snake C-type lectin-like proteins inhibit nicotinic acetylcholine receptors.

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.