A comparative study of the performance of E. coli and K. phaffii for expressing α-cobratoxin.

Three-finger toxins (3FTxs) have traditionally been obtained via venom fractionation of whole venoms from snakes. This method often yields functional toxins, but it can be difficult to obtain pure isoforms, as it is challenging to separate the many different toxins with similar physicochemical properties that generally exist in many venoms. This issue can be circumvented via the use of recombinant expression. However, achieving the correct disulfide bond formation in recombinant toxins is challenging and requires extensive optimization of expression and purification methods to enhance stability and functionality. In this study, we investigated the expression of α-cobratoxin, a well-characterized 3FTx from the monocled cobra (Naja kaouthia), in three different expression systems, namely Escherichia coli BL21 (DE3) cells with the csCyDisCo plasmid, Escherichia coli SHuffle cells, and Komagataella phaffii (formerly known as Pichia pastoris). While none of the tested systems yielded α-cobratoxin identical to the variant isolated from whole venom, the His6-tagged α-cobratoxin expressed in K. phaffii exhibited a comparable secondary structure according to circular dichroism spectra and similar binding properties to the α7 subunit of the nicotinic acetylcholine receptor. The findings presented here illustrate the advantages and limitations of the different expression systems and can help guide researchers who wish to express 3FTxs.

Novel Three-Finger Neurotoxins from Naja melanoleuca Cobra Venom Interact with GABAA and Nicotinic Acetylcholine Receptors.

Cobra venoms contain three-finger toxins (TFT) including α-neurotoxins efficiently binding nicotinic acetylcholine receptors (nAChRs). As shown recently, several TFTs block GABAA receptors (GABAARs) with different efficacy, an important role of the TFTs central loop in binding to these receptors being demonstrated. We supposed that the positive charge (Arg36) in this loop of α-cobratoxin may explain its high affinity to GABAAR and here studied α-neurotoxins from African cobra N. melanoleuca venom for their ability to interact with GABAARs and nAChRs. Three α-neurotoxins, close homologues of the known N. melanoleuca long neurotoxins 1 and 2, were isolated and sequenced. Their analysis on Torpedocalifornica and α7 nAChRs, as well as on acetylcholine binding proteins and on several subtypes of GABAARs, showed that all toxins interacted with the GABAAR much weaker than with the nAChR: one neurotoxin was almost as active as α-cobratoxin, while others manifested lower activity. The earlier hypothesis about the essential role of Arg36 as the determinant of high affinity to GABAAR was not confirmed, but the results obtained suggest that the toxin loop III may contribute to the efficient interaction of some long-chain neurotoxins with GABAAR. One of isolated toxins manifested different affinity to two binding sites on Torpedo nAChR.

Peptide Inhibitors of the α-Cobratoxin-Nicotinic Acetylcholine Receptor Interaction.

Venomous snakebites cause >100 000 deaths every year, in many cases via potent depression of human neuromuscular signaling by snake α-neurotoxins. Emergency therapy still relies on antibody-based antivenom, hampered by poor access, frequent adverse reactions, and cumbersome production/purification. Combining high-throughput discovery and subsequent structure-function characterization, we present simple peptides that bind α-cobratoxin (α-Cbtx) and prevent its inhibition of nicotinic acetylcholine receptors (nAChRs) as a lead for the development of alternative antivenoms. Candidate peptides were identified by phage display and deep sequencing, and hits were characterized by electrophysiological recordings, leading to an 8-mer peptide that prevented α-Cbtx inhibition of nAChRs. We also solved the peptide:α-Cbtx cocrystal structure, revealing that the peptide, although of unique primary sequence, binds to α-Cbtx by mimicking structural features of the nAChR binding pocket. This demonstrates the potential of small peptides to neutralize lethal snake toxins in vitro, establishing a potential route to simple, synthetic, low-cost antivenoms.

Proteomic insights into short neurotoxin-driven, highly neurotoxic venom of Philippine cobra (Naja philippinensis) and toxicity correlation of cobra envenomation in Asia.

The Philippine cobra, Naja philippinensis, is a WHO Category 1 venomous snake of medical importance responsible for fatal envenomation in the northern Philippines. To elucidate the venom proteome and pathophysiology of envenomation, N. philippinensis venom proteins were decomplexed with reverse-phase high-performance liquid chromatography, and protein fractions were subsequently digested with trypsin, followed by nano-liquid chromatography-tandem mass spectrometry analysis and data mining. Three-finger toxins (3FTX, 66.64% of total venom proteins) and phospholipases A2 (PLA2, 22.88%) constitute the main bulk of venom proteome. Other proteins are present at low abundances (<4% each); these include metalloproteinase, serine protease, cobra venom factor, cysteine-rich secretory protein, vespryn, phosphodiesterase, 5' nucleotidase and nerve growth factor. In the three-finger toxin family, the alpha-neurotoxins comprise solely short neurotoxins (SNTX, 44.55%), supporting that SNTX is the principal toxin responsible for neuromuscular paralysis and lethality reported in clinical envenomation. Cytotoxins (CTX) are the second most abundant 3FTX proteins in the venom (21.31%). The presence of CTX correlates with the venom cytotoxic effect, which is more prominent in murine cells than in human cells. From the practical standpoint, SNTX-driven neuromuscular paralysis is significant in N. philippinensis envenomation. Antivenom production and treatment should be tailored accordingly to ensure effective neutralization of SNTX. BIOLOGICAL SIGNIFICANCE: The venom proteome of Naja philippinensis, the Philippine cobra, is unravelled for the first time. Approximately half the protein bulk of the venom is made up of short neurotoxins (44.55% of the total venom proteins). As the only alpha-neurotoxins present in the venom, short neurotoxins are the causative toxins of the post-synaptic blockade and fast-onset neuromuscular paralysis in N. philippinensis envenomation. A substantial amount of cytotoxins (21.31%) was also detected in N. philippinensis venom, supporting that the venom can be cytotoxic although the effect is much weaker in human cells compared to murine cells. The finding is consistent with the low incidence of local tissue necrosis in N. philippinensis envenomation, although this does not negate the need for monitoring and care of bite wound in the patients.

Venomics of Naja sputatrix, the Javan spitting cobra: A short neurotoxin-driven venom needing improved antivenom neutralization.

The venom proteome of Naja sputatrix (Javan spitting cobra) was elucidated through reverse-phase HPLC, nano-ESI-LCMS/MS and data mining. A total of 97 distinct protein forms belonging to 14 families were identified. The most abundant proteins are the three-finger toxins (3FTXs, 64.22%) and phospholipase A2 (PLA2, 31.24%), followed by nerve growth factors (1.82%), snake venom metalloproteinase (1.33%) and several proteins of lower abundance (<1%) including a variety of venom enzymes. At subproteome, the 3FTx is dominated by cytotoxins (48.08%), while short neurotoxins (7.89%) predominate over the long neurotoxins (0.48%) among other neurotoxins of lesser toxicity (muscarinic toxin-like proteins, 5.51% and weak neurotoxins, 2.26%). The major SNTX, CTX and PLA2 toxins were isolated with intravenous median lethal doses determined as 0.13, 1.06 and 0.50µg/g in mice, respectively. SABU, the Indonesia manufactured homologous tri-specific antivenom could neutralize the CTX and PLA2 fraction with moderate potency (potency=0.14-0.16mg toxin per ml antivenom). The SNTX, however, was very poorly neutralized with a potency level of 0.034mg/ml, indicating SNTX as the main limiting factor in antivenom neutralization. The finding helps elucidate the inferior efficacy of SABU reported in neutralizing N. sputatrix venom, and supports the call for antivenom improvement. BIOLOGICAL SIGNIFICANCE: The Javan spitting cobra, Naja sputatrix is by itself a unique species and should not be confused as the equatorial and the Indochinese spitting cobras. The distinction among the spitting cobras was however unclear prior to the revision of cobra systematics in the mid-90's, and results of some earlier studies are now questionable as to which species was implicated back then. The current study successfully profiled the venom proteome of authenticated N. sputatrix, and showed that the venom is made up of approximately 64% three-finger toxins (including neurotoxins and cytotoxins) and 31% phospholipases A2 by total venom proteins. The findings verified that the paralyzing components in the venom i.e. neurotoxins are predominantly the short-chain subtype (SNTX) far exceeding the long-chain subtype (LNTX) which is more abundant in the venoms of monocled cobra and Indian common cobra. The neurotoxicity of N. sputatrix venom is hence almost exclusively SNTX-driven, and effective neutralization of the SNTX is the key to early reversal of paralysis. Unfortunately, as shown through a toxin-specific assay, the immunological neutralization of the SNTX using the Indonesian antivenom (SABU) was extremely weak, implying that SABU has limited therapeutic efficacy in treating N. sputatrix envenomation clinically. From the practical standpoint, actions need to be taken at all levels from laboratory to production and policy making to ensure that the shortcoming is overcome.

Interaction of three-finger proteins from snake venoms and from mammalian brain with the cys-loop receptors and their models.

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

Geographical venom variations of the Southeast Asian monocled cobra (Naja kaouthia): venom-induced neuromuscular depression and antivenom neutralization.

The Southeast Asian monocled cobras (Naja kaouthia) exhibit geographical variations in their venom proteomes, especially on the composition of neurotoxins. This study compared the neuromuscular depressant activity of the venoms of N. kaouthia from Malaysia (NK-M), Thailand (NK-T) and Vietnam (NK-V), and the neutralization of neurotoxicity by a monospecific antivenom. On chick biventer cervicis nerve-muscle preparation, all venoms abolished the indirect twitches, with NK-T venom being the most potent (shortest t90, time to 90% twitch inhibition), followed by NK-V and NK-M. Acetylcholine and carbachol failed to reverse the blockade, indicating irreversible/pseudo-irreversible post-synaptic neuromuscular blockade. KCl restored the twitches variably (NK-M preparation being the least responsive), consistent with different degree of muscle damage. The findings support that NK-T venom has the most abundant curarimimetic alpha-neurotoxins, while NK-M venom contains more tissue-damaging cytotoxins. Pre-incubation of tissue with N. kaouthia monovalent antivenom (NKMAV) prevented venom-induced twitch depression, with the NK-T preparation needing the largest antivenom dose. NKMAV added after the onset of neuromuscular depression could only halt the inhibitory progression but failed to restore full contraction. The findings highlight the urgency of early antivenom administration to sequester as much circulating neurotoxins as possible, thereby hastening toxin elimination from the circulation. In envenomed mice, NKMAV administered upon the first neurological sign neutralized the neurotoxic effect, with the slowest full recovery noticed in the NK-T group. This is consistent with the high abundance of neurotoxins in the NK-T venom, implying that a larger amount or repeated dosing of NKMAV may be required in NK-T envenomation.

Nicotinic acetylcholine receptor α7 subunit is involved in the cobratoxin-induced antinociception in an animal model of neuropathic pain.

In this study we report that cobratoxin (CbTX), a long-chain postsynaptic α-neurotoxin isolated from the Thailand cobra, Naja naja kaouthia, has antinociceptive effect in rats with neuropathic pain. The neuropathic pain model was established in rats with partial sciatic nerve ligature (PSNL) method. The pain response was examined behaviorally with mechanical paw withdrawal and thermal paw withdrawal method. Different doses (0.56, 1.12 and 4.50 µg/kg) of CbTX were injected intrathecally. Injection of CbTX resulted in a significant dose-dependent antinociception as evidenced by increased mechanical withdrawal threshold and thermal withdrawal latency. CbTX also induces a significant dose-dependent inhibition of pain-evoked unit discharges of thalamic parafascicular neurons. Both the behavioral mechanical and thermal antinociception and the inhibition of pain-evoked discharges of neurons in thalamic parafascicular nucleus in PSNL model could be mimicked by PUN282987, selective α7 nicotinic AChR (α7 nAChR) agonist and reversed by methyllycaconitine (MLA) selective α7 nAChR antagonist. In summary, these results suggested that AChR α7 subunit was involved in the antinociceptive action of CbTX for neuropathic pain and might be the candidate target for analgesic drug design.

Complex between α-bungarotoxin and an α7 nicotinic receptor ligand-binding domain chimaera.

To identify high-affinity interactions between long-chain α-neurotoxins and nicotinic receptors, we determined the crystal structure of the complex between α-btx (α-bungarotoxin) and a pentameric ligand-binding domain constructed from the human α7 AChR (acetylcholine receptor) and AChBP (acetylcholine-binding protein). The complex buries ~2000 Ų (1 Å=0.1 nm) of surface area, within which Arg³6 and Phe³² from finger II of α-btx form a π-cation stack that aligns edge-to-face with the conserved Tyr¹84 from loop-C of α7, while Asp³° of α-btx forms a hydrogen bond with the hydroxy group of Tyr¹84. These inter-residue interactions diverge from those in a 4.2 Å structure of α-ctx (α-cobratoxin) bound to AChBP, but are similar to those in a 1.94 Å structure of α-btx bound to the monomeric α1 extracellular domain, although compared with the monomer-bound complex, the α-btx backbone exhibits a large shift relative to the protein surface. Mutational analyses show that replacing Tyr¹84 with a threonine residue abolishes high-affinity α-btx binding, whereas replacing with a phenylalanine residue maintains high affinity. Comparison of the α-btx complex with that coupled to the agonist epibatidine reveals structural rearrangements within the binding pocket and throughout each subunit. The overall findings highlight structural principles by which α-neurotoxins interact with nicotinic receptors.

Identification of α-cobratoxin in equine plasma by LC-MS/MS for doping control.

Cobra venom (Naja kaouthia) contains a toxin called α-cobratoxin (α-Cbtx). This toxin is a natural protein containing 71 amino acids (MW 7821 Da) with a reported analgesic potency greater than morphine. In 2007, in USA, this substance was found in the barns of a thoroughbred trainer and since then till date, the lack of a detection of this molecule has remained a recurring problem for the horseracing industry worldwide. To solve this problem, the first method for the detection of α-cobratoxin in equine plasma has now been developed. Plasma sample (3 mL) was treated with ammonium sulfate at the isoelectric point of α-Cbtx, and the pellet was dissolved in a phosphate buffer and mixed with methanol for precipitation. The supernatant was then concentrated prior to its extraction on WCX SPE cartridges. The eluate was concentrated with two consecutive filtration steps before the trypsin digestion. The samples were analyzed using a LC-MS/MS Q Exactive instrument at 70,000 resolution on the product ions of the doubly charged precursor of the target peptide ((24)TWCDAFCSIR(33)). The method was validated (n = 18) at 5 µg/L (640 pmol/L) according to the Association of Official Racing Chemists (AORC) requirements. The lower limit of detection was 1 µg/L (130 pmol/L). The present method has made it possible for us to confirm the presence of α-Cbtx in a horse plasma sample 24 h post the administration of α-Cbtx. Thus, the present method provides the first sensitive, specific, and reliable analytical method to confirm the presence of α-Cbtx in equine plasma.

Dimeric α-cobratoxin X-ray structure: localization of intermolecular disulfides and possible mode of binding to nicotinic acetylcholine receptors.

In Naja kaouthia cobra venom, we have earlier discovered a covalent dimeric form of α-cobratoxin (αCT-αCT) with two intermolecular disulfides, but we could not determine their positions. Here, we report the αCT-αCT crystal structure at 1.94 Å where intermolecular disulfides are identified between Cys(3) in one protomer and Cys(20) of the second, and vice versa. All remaining intramolecular disulfides, including the additional bridge between Cys(26) and Cys(30) in the central loops II, have the same positions as in monomeric α-cobratoxin. The three-finger fold is essentially preserved in each protomer, but the arrangement of the αCT-αCT dimer differs from those of noncovalent crystallographic dimers of three-finger toxins (TFT) or from the κ-bungarotoxin solution structure. Selective reduction of Cys(26)-Cys(30) in one protomer does not affect the activity against the α7 nicotinic acetylcholine receptor (nAChR), whereas its reduction in both protomers almost prevents α7 nAChR recognition. On the contrary, reduction of one or both Cys(26)-Cys(30) disulfides in αCT-αCT considerably potentiates inhibition of the α3ß2 nAChR by the toxin. The heteromeric dimer of α-cobratoxin and cytotoxin has an activity similar to that of αCT-αCT against the α7 nAChR and is more active against α3ß2 nAChRs. Our results demonstrate that at least one Cys(26)-Cys(30) disulfide in covalent TFT dimers, similar to the monomeric TFTs, is essential for their recognition by α7 nAChR, although it is less important for interaction of covalent TFT dimers with the α3ß2 nAChR.

Neurotoxin II bound to acetylcholine receptors in native membranes studied by dynamic nuclear polarization NMR.

Methods enabling structural studies of membrane-integrated receptor systems without the necessity of purification provide an attractive perspective in membrane protein structural and molecular biology. This has become feasible in principle since the advent of dynamic nuclear polarization (DNP) magic-angle-spinning NMR spectroscopy, which delivers the required sensitivity. In this pilot study, we observed well-resolved solid-state NMR spectra of extensively (13)C-labeled neurotoxin II bound to the nicotinic acetylcholine receptor (nAChR) in native membranes. We show that TOTAPOL, a biradical required for DNP, is localized at membrane and protein surfaces. The concentration of active, membrane-attached biradical decreases with time, probably because of reactive components of the membrane preparation. An optimal distribution of active biradical has strong effects on the NMR data. The presence of inactive TOTAPOL in membrane-proximal situations but active biradical in the surrounding water/glycerol "glass" leads to well-resolved spectra, yet a considerable enhancement (ε = 12) is observed. The resulting spectra of a protein ligand bound to its receptor are paving the way for further DNP investigations of proteins embedded in native membrane patches.

Virtual screening against alpha-cobratoxin.

alpha-Cobratoxin (Cbtx), the neurotoxin isolated from the venom of the Thai cobra Naja kaouthia , causes paralysis by preventing acetylcholine (ACh) binding to nicotinic acetylcholine receptors (nAChRs). In the current study, the region of the Cbtx molecule that is directly involved in binding to nAChRs is used as the target for anticobratoxin drug design. The crystal structure (1YI5) of Cbtx in complex with the acetylcholine binding protein (AChBP), a soluble homolog of the extracellular binding domain of nAChRs, was selected to prepare an alpha-cobratoxin active binding site for docking. The amino acid residues (Ser182-Tyr192) of the AChBP structure, the binding site of Cbtx, were used as the positive control to validate the prepared Cbtx active binding site (root mean square deviation < 1.2 A). Virtual screening of the National Cancer Institute diversity set, a library of 1990 compounds with nonredundant pharmacophore profiles, using AutoDock against the Cbtx active site, revealed 39 potential inhibitor candidates. The adapted in vitro radioligand competition assays using [(3)H]epibatidine and [(125)I]bungarotoxin against the AChBPs from the marine species, Aplysia californica (Ac), and from the freshwater snails, Lymnaea stagnalis (Ls) and Bolinus truncates (Bt), revealed 4 compounds from the list of inhibitor candidates that had micromolar to nanomolar interferences for the toxin binding to AChBPs. Three hits (NSC42258, NSC121865, and NSC134754) can prolong the survival time of the mice if administered 30 min before injection with Cbtx, but only NSC121865 and NSC134754 can prolong the survival time if injected immediately after injection with Cbtx. These inhibitors serve as novel templates/scaffolds for the development of more potent and specific anticobratoxin.

Rediocides A and G as potential antitoxins against cobra venom.

Rediocides A and G, the principle components of Trigonostemon reidioides (Kurz) Craib, which is known as Lotthanong in Thai, were investigated for a detoxification mechanism against Naja kaouthia venom by in silico, in vitro, and in vivo methods. Molecular dockings of alpha-cobratoxin with rediocides A and G were performed, and the binding energies were found to be -14.17 and -14.14 kcal/mol, respectively. Rediocides bind to alpha-cobratoxin at the same location as alpha-cobratoxin binds to the nicotinic acetylcholine receptor (nAChR), i.e., at the Asp27, Phe29, Arg33, Gly34, Lys35, and Val37 residues. alpha-Cobratoxin cannot bind to nAChR, because some of its binding sites are occupied with rediocides. From in vitro SDS-PAGE, it was found that rediocides can diminish the bands of alpha-cobratoxin. In the presence of acetylcholine-binding protein (AChBP), it was apparent that rediocides can bind both alpha-cobratoxin and AChBP. From an in vivo test, it was found that injection of rediocides at 0.5 mg/kg immediately after an alpha-cobratoxin dose of three times LD(50) cannot prolong the survival time of mice. However, rediocide can prolong the survival time, if it is injected 30 min before the injection of alpha-cobratoxin. The in vitro SDS-PAGE and the in vivo results support the in silico detoxification mechanism of rediocides against cobra venom at a molecular level.

Loop 3 of short neurotoxin II is an additional interaction site with membrane-bound nicotinic acetylcholine receptor as detected by solid-state NMR spectroscopy.

The contact area of neurotoxin II from Naja naja oxiana when interacting with the membrane-bound nicotinic acetylcholine receptor from Torpedo californica was determined by solid-state, magic-angle spinning NMR spectroscopy. For this purpose, the carbon signals for more than 90% of the residues of the bound neurotoxin were assigned. Differences between the solution and solid-state chemical shifts of the free and bound form of the toxin are confined to distinct surface regions. Loop II of the short toxin was identified as the main interaction site. In addition, loop III of neurotoxin II shows several strong responses defining an additional interaction site. A comparison with the structures of alpha-cobratoxin bound to the acetylcholine-binding protein from snail species Lymnaea stagnalis and Aplysia californica, and of alpha-bungarotoxin bound to an extracellular domain of an alpha-subunit of the receptor reveals different contact areas for long and short alpha-neurotoxins.

Naturally occurring disulfide-bound dimers of three-fingered toxins: a paradigm for biological activity diversification.

Disulfide-bound dimers of three-fingered toxins have been discovered in the Naja kaouthia cobra venom; that is, the homodimer of alpha-cobratoxin (a long-chain alpha-neurotoxin) and heterodimers formed by alpha-cobratoxin with different cytotoxins. According to circular dichroism measurements, toxins in dimers retain in general their three-fingered folding. The functionally important disulfide 26-30 in polypeptide loop II of alpha-cobratoxin moiety remains intact in both types of dimers. Biological activity studies showed that cytotoxins within dimers completely lose their cytotoxicity. However, the dimers retain most of the alpha-cobratoxin capacity to compete with alpha-bungarotoxin for binding to Torpedo and alpha7 nicotinic acetylcholine receptors (nAChRs) as well as to Lymnea stagnalis acetylcholine-binding protein. Electrophysiological experiments on neuronal nAChRs expressed in Xenopus oocytes have shown that alpha-cobratoxin dimer not only interacts with alpha7 nAChR but, in contrast to alpha-cobratoxin monomer, also blocks alpha3beta2 nAChR. In the latter activity it resembles kappa-bungarotoxin, a dimer with no disulfides between monomers. These results demonstrate that dimerization is essential for the interaction of three-fingered neurotoxins with heteromeric alpha3beta2 nAChRs.

Effect of cobratoxin binding on the normal mode vibration within acetylcholine binding protein.

Recent crystal structures of the acetylcholine binding protein (AChBP) have revealed surprisingly small structural alterations upon ligand binding. Here we investigate the extent to which ligand binding may affect receptor dynamics. AChBP is a homologue of the extracellular component of ligand-gated ion channels (LGICs). We have previously used an elastic network normal-mode analysis to propose a gating mechanism for the LGICs and to suggest the effects of various ligands on such motions. However, the difficulties with elastic network methods lie in their inability to account for the modest effects of a small ligand or mutation on ion channel motion. Here, we report the successful application of an elastic network normal mode technique to measure the effects of large ligand binding on receptor dynamics. The present calculations demonstrate a clear alteration in the native symmetric motions of a protein due to the presence of large protein cobratoxin ligands. In particular, normal-mode analysis revealed that cobratoxin binding to this protein significantly dampened the axially symmetric motion of the AChBP that may be associated with channel gating in the full nAChR. The results suggest that alterations in receptor dynamics could be a general feature of ligand binding.

Docking of alpha-cobratoxin suggests a basal conformation of the nicotinic receptor.

We investigate the interactions between the long chain alpha-cobratoxin (Cbtx) and the nicotinic acetylcholine receptor using a rigid body docking procedure. The method, (i) reproduces the binding of Cbtx to Lymnea acetylcholine-binding protein (AChBP); (ii) shows that most of the structures of AChBP obtained in the presence of antagonists are compatible with Cbtx binding; and (iii) reveals a complex between Cbtx and muscle nAChR that corresponds to the basal "resting" state conformation. The structures are made available for further understanding of the allosteric transitions of the nAChR as well as for drug design.

Alpha-cobratoxin as a possible therapy for multiple sclerosis: a review of the literature leading to its development for this application.

The use of snake venom in the treatment of multiple sclerosis has been, at best, controversial. The anecdotal reports for snake venom's beneficial effects in this condition may be supportable now by recent scientific evidence. Cobratoxin, a neurotoxin obtained from the venom of the Thailand cobra, has demonstrated several pharmacological activities that strongly support its use in this application. By employing a chemical detoxification step, the neurotoxin can be rendered safe for administration to humans with minimal side effects. This modified neurotoxin has demonstrated neuromodulatory, antiviral, and analgesic activity, elements associated with the multiple sclerosis condition. Modified cobratoxin has demonstrated potent immunosuppressive activity in acute and chronic animal models of the disease. The drug is under investigation for use in adrenomyeloneuropathy and clinical trials in Multiple sclerosis are planned.

Cobrotoxin inhibits NF-kappa B activation and target gene expression through reaction with NF-kappa B signal molecules.

Cobrotoxin is known to bind with cysteine residues of biological molecules such as nicotine acetylcholine receptor. Cobrotoxin may modify IKKs and p50 through protein-protein interaction since cysteine residues are present in the kinase domains of IKKalpha and IKKbeta and in the p50 of NF-kappaB. Our surface plasmon resonance analysis showed that cobrotoxin directly binds to p50 (K(d) = 1.54 x 10(-)(5) M), IKKalpha (K(d) = 3.94 x 10(-)(9) M) and IKKbeta (K(d) = 3.4 x 10(-)(8) M) with high binding affinity. Moreover, these protein-protein interactions suppressed the lipopolysaccharide (LPS, 1 microg/mL)- and the sodium nitroprusside (SNP, 200 microM)-induced DNA binding activity of NF-kappaB and NF-kappaB-dependent luciferase activity in astrocytes and Raw 264.7 macrophages. These inhibitory effects were correlated with the inhibition of IkappaB release and p50 translocation. Inhibition of NF-kappaB by cobrotoxin resulted in reductions in the LPS-induced expressions of COX-2, iNOS, cPLA(2), IL-4, and TNF-alpha in astrocytes and in COX-2 expression induced by SNP, LPS, and TNF-alpha in astrocytes. Moreover, these inhibitory effects of cobrotoxin were reversed by adding reducing agents, dithiothreitol and glutathione. In addition, cobrotoxin did not have any inhibitory effect on NF-kappaB activity in cells carrying mutant p50 (C62S), IKKalpha (C178A), and IKKbeta (C179A), with the exception of IKKbeta (K44A) mutant plasmid. Confocal microscopic analysis showed that cobrotoxin is uptaken into the nucleus of cells. These results demonstrate that cobrotoxin directly binds to the sulfhydryl groups of p50 and IKKs, and that this results in reduced IkappaB release and the translocation of p50, thereby inhibiting the activation of NF-kappaB.