The analysis and detection of snake venom toxins are a matter of great importance in clinical diagnosis for fast treatment and the discovery of new pharmaceutical products. Current detection methods have high associated costs and require the use of sophisticated bioreceptors, which in some cases are difficult to obtain. Herein, we report the synthesis of template-based molecularly imprinted micromotors for dynamic detection of α-bungarotoxin as a model toxin present in the venom of many-banded krait (Bungarus multicinctus). The specific recognition sites are built-in in the micromotors by incubation of the membrane template with the target toxin, followed by a controlled electrodeposition of a poly(3,4-ethylenedioxythiophene)/poly(sodium 4-styrenesulfonate) polymeric layer, a magnetic Ni layer to promote magnetic guidance and facilitate washing steps, and a Pt layer for autonomous propulsion in the presence of hydrogen peroxide. The enhanced fluid mixing and autonomous propulsion increase the likelihood of interactions with the target analyte as compared with static counterparts, retaining the tetramethylrhodamine-labeled α-bungarotoxin on the micromotor surface with extremely fast dynamic sensor response (after just 20 s navigation) in only 3 μL of water, urine, or serum samples. The sensitivity achieved meets the clinically relevant concentration postsnakebite (from 0.1 to 100 μg/mL), illustrating the feasibility of the approach for practical applications. The selectivity of the protocol is very high, as illustrated by the absence of fluorescence in the micromotor surface in the presence of α-cobratoxin as a representative toxin with a size and structure similar to those of α-bungarotoxin. Recoveries higher than 95% are obtained in the analysis of urine- and serum-fortified samples. The new strategy holds considerable promise for fast, inexpensive, and even onsite detection of several toxins using multiple molecularly imprinted micromotors with tailored recognition abilities.

Nicotinic acetylcholine receptors (nAChRs) are a family of ligand-gated ion channel receptors that contribute to cognition, memory, and motor control in many organisms. The pharmacological targeting of these receptors, using small molecules or peptides, presents an important strategy for the development of drugs that can treat important human diseases, including neurodegenerative disorders. The Aplysia californica acetylcholine binding protein (Ac-AChBP) is a structural surrogate of the nAChR with high homology to the extracellular ligand binding domain of homopentameric nAChRs. In this study, we optimized protein-painting-based mass spectrometry to identify regions of interaction between the Ac-AChBP and several nAChR ligands. Using molecular dyes that adhere to the surface of a solubilized Ac-AChBP complex, we identified amino acid residues that constitute a contact site within the Ac-AChBP for α-bungarotoxin, choline, nicotine, and amyloid-β 1-42. By integrating innovation in protein painting mass spectrometry with computational structural modeling, we present a new experimental tool for analyzing protein interactions of the nAChR.

Current treatment of snakebite relies on immunoglobulin-rich antivenoms. However, production of these antivenoms is complicated and costly. Aptamers - single-stranded DNAs or RNAs with specific folding structures that bind to specific target molecules - represent excellent alternatives or complements to antibody-based therapeutics. However, no studies have systematically assessed the feasibility of using aptamers to mitigate venom-induced toxicity in vivo. β-bungarotoxin is the predominant protein responsible for the toxicity of the venom of Bungarus multicinctus, a prominent venomous snake inhabiting Taiwan. In this study, we reported the screening and optimization of a DNA aptamer against β-bungarotoxin and tested its utility in a mouse model. After 14 rounds of directed evolution of ligands by exponential enrichment, an aptamer, called BB3, displaying remarkable binding affinity and specificity for β-bungarotoxin was obtained. Following structural prediction and point-modification experiments, BB3 underwent truncation and was modified with 2\'-O-methylation and a 3\'-inverted dT. This optimized aptamer showed sustained, high-affinity binding for β-bungarotoxin and exhibited remarkable nuclease resistance in plasma. Importantly, administration of this optimized aptamer extended the survival time of mice treated with a lethal dose of β-bungarotoxin. Collectively, our data provide a compelling illustration of the potential of aptamers as promising candidates for development of recombinant antivenom therapies.

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Animal testing has been the primary approach to assess the neutralization potency of antivenom for decades. However, the necessity to sacrifice large numbers of experimental animals during this process has recently raised substantial welfare concerns. Furthermore, the laborious and expensive nature of animal testing highlights the critical need to develop alternative in vitro assays. Here, we developed an antibody-detection enzyme-linked immunosorbent assay (ELISA) technique as an alternative approach to evaluate the neutralization potency of hyperimmunized equine plasma against B. multicinctus, a medically important venomous snake in Taiwan. Firstly, five major protein components of B. multicinctus venom, specifically, α-BTX, β-BTX, γ-BTX, MTX, and NTL, were isolated. To rank their relative medical significance, a toxicity score system was utilized. Among the proteins tested, β-BTX presenting the highest score was regarded as the major toxic component. Subsequently, antibody-detection ELISA was established based on the five major proteins and used to evaluate 55 hyperimmunized equine plasma samples with known neutralization potency. ELISA based on β-BTX, the most lethal protein according to the toxicity score, exhibited the best sensitivity (75.6 %) and specificity (100 %) in discriminating between high-potency and low-potency plasma, supporting the hypothesis that highly toxic proteins offer better discriminatory power for potency evaluation. Additionally, a phospholipase A2 (PLA2) competition process was implemented to eliminate the antibodies targeting toxicologically irrelevant domains. This optimization greatly enhanced the performance of our assay, resulting in sensitivity of 97.6 % and specificity of 92.9 %. The newly developed antibody-detection ELISA presents a promising alternative to in vivo assays to determine the neutralization potency of antisera against B. multicinctus during the process of antivenom production.

SLURP-1 is a three-finger human protein targeting nicotinic acetylcholine receptors (nAChRs). The recombinant forms of SLURP-1 produced in E. coli differ in added fusion fragments and in activity. The closest in sequence to the naturally occurring SLURP-1 is the recombinant rSLURP-1, differing by only one additional N-terminal Met residue. sSLURP-1 can be prepared by peptide synthesis and its amino acid sequence is identical to that of the natural protein. In view of recent NMR analysis of the conformational mobility of rSLURP-1 and cryo-electron microscopy structures of complexes of α-bungarotoxin (a three-finger snake venom protein) with Torpedo californica and α7 nAChRs, we compared conformations of sSLURP-1 and rSLURP-1 by Raman spectroscopy and CD-controlled thermal denaturation, analyzed their competition with α-bungarotoxin for binding to the above-mentioned nAChRs, compared the respective receptor complexes with computer modeling and compared their inhibitory potency on the α9α10 nAChR. The CD revealed a higher thermostability of sSLURP-1; some differences between sSLURP-1 and rSLURP-1 were observed in the regions of disulfides and tyrosine residues by Raman spectroscopy, but in binding, computer modeling and electrophysiology, the proteins were similar. Thus, sSLURP-1 and rSLURP-1 with only one additional Met residue appear close in structure and functional characteristics, being appropriate for research on nAChRs.

This review covers briefly the work carried out at our institute (IBCh), in many cases in collaboration with other Russian and foreign laboratories, for the last 50 years. It discusses the discoveries and studies of various animal toxins, including protein and peptide neurotoxins acting on the nicotinic acetylcholine receptors (nAChRs) and on other ion channels. Among the achievements are the determination of the primary structures of the α-bungarotoxin-like three-finger toxins (TFTs), covalently bound dimeric TFTs, glycosylated cytotoxin, inhibitory cystine knot toxins (ICK), modular ICKs, and such giant molecules as latrotoxins and peptide neurotoxins from the snake, as well as from other animal venoms. For a number of toxins, spatial structures were determined, mostly by 1H-NMR spectroscopy. Using this method in combination with molecular modeling, the molecular mechanisms of the interactions of several toxins with lipid membranes were established. In more detail are presented the results of recent years, among which are the discovery of α-bungarotoxin analogs distinguishing the two binding sites in the muscle-type nAChR, long-chain α-neurotoxins interacting with α9α10 nAChRs and with GABA-A receptors, and the strong antiviral effects of dimeric phospholipases A2. A summary of the toxins obtained from arthropod venoms includes only highly cited works describing the molecules\' success story, which is associated with IBCh. In marine animals, versatile toxins in terms of structure and molecular targets were discovered, and careful work on α-conotoxins differing in specificity for individual nAChR subtypes gave information about their binding sites.

γ-bungarotoxin (γ-BGT) is an RGD motif-containing protein, derived from the venom of Bungarus multicinctus, leading to acute death in mice. These RGD motif-containing proteins from snake venom belonging to the disintegrin family can interfere with vascular endothelial homeostasis by directly binding cell surface integrins. Targeting integrins that generate vascular endothelial dysfunction may contribute to γ-BGT poisoning, however, the underlying mechanisms have not been investigated in detail. In this study, the results showed that γ-BGT played a role in -promoting the permeability of the vascular endothelial barrier. Depending on its selective binding to integrin α5 in vascular endothelium (VE), γ-BGT initiated downstream events, including focal adhesion kinase dephosphorylation and cytoskeleton remodeling, resulting in the intercellular junction interruption. Those alternations facilitated paracellular permeability of VE and barrier dysfunction. Proteomics profiling identified that as a downstream effector of the integrin α5 / FAK signaling pathway cyclin D1 partially mediated the cellular structural changes and barrier dysfunction. Furthermore, VE-released plasminogen activator urokinase and platelet-derived growth factor D could serve as potential diagnostic biomarkers for γ-BGT-induced vascular endothelial dysfunction. Our results indicate the mechanisms through which γ-BGT as a novel disintegrin directly interacts with the VE, with consequences for barrier dysfunction.

Nicotinic acetylcholine receptor of α7 type (α7-nAChR) presented in the nervous and immune systems and epithelium is a promising therapeutic target for cognitive disfunctions and cancer treatment. Weak toxin from Naja kaouthia venom (WTX) is a non-conventional three-finger neurotoxin, targeting α7-nAChR with weak affinity. There are no data on interaction mode of non-conventional neurotoxins with nAChRs. Using α-bungarotoxin (classical three-finger neurotoxin with high affinity to α7-nAChR), we showed applicability of cryo-EM to study complexes of α7-nAChR extracellular ligand-binding domain (α7-ECD) with toxins. Using cryo-EM structure of the α7-ECD/WTX complex, together with NMR data on membrane active site in the WTX molecule and mutagenesis data, we reconstruct the structure of α7-nAChR/WTX complex in the membrane environment. WTX interacts at the entrance to the orthosteric site located at the receptor intersubunit interface and simultaneously forms the contacts with the membrane surface. WTX interaction mode with α7-nAChR significantly differs from α-bungarotoxin\'s one, which does not contact the membrane. Our study reveals the important role of the membrane for interaction of non-conventional neurotoxins with the nicotinic receptors.

The pathophysiological mechanisms underlying the constellation of symptoms that characterize COVID-19 are only incompletely understood. In an effort to fill these gaps, a \"nicotinic hypothesis,\" which posits that nicotinic acetylcholine receptors (AChRs) act as additional severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptors, has recently been put forth. A key feature of the proposal (with potential clinical ramifications) is the suggested competition between the virus\' spike protein and small-molecule cholinergic ligands for the receptor\'s orthosteric binding sites. This notion is reminiscent of the well-established role of the muscle AChR during rabies virus infection. To address this hypothesis directly, we performed equilibrium-type ligand-binding competition assays using the homomeric human α7-AChR (expressed on intact cells) as the receptor, and radio-labeled α-bungarotoxin (α-BgTx) as the orthosteric-site competing ligand. We tested different SARS-CoV-2 spike protein peptides, the S1 domain, and the entire S1-S2 ectodomain, and found that none of them appreciably outcompete [125I]-α-BgTx in a specific manner. Furthermore, patch-clamp recordings showed no clear effect of the S1 domain on α7-AChR-mediated currents. We conclude that the binding of the SARS-CoV-2 spike protein to the human α7-AChR\'s orthosteric sites-and thus, its competition with ACh, choline, or nicotine-is unlikely to be a relevant aspect of this complex disease.