Many animals change traits over the course of their life history. It has been proposed that some of these developmental changes evolve in concert with shifts in ecology, but there are relatively few clear examples, and identifying the underlying molecular changes is challenging. Australian brown snakes (Pseudonaja spp.), one of Australia's most venomous snakes, may provide such an example, as previous work has shown that these species undergo a dietary shift from reptiles to mammals, accompanied by differences in venom activity. We show that the venom composition of adult brown snakes is distinguished by the collective expression of four procoagulant toxin families, which are absent in juvenile life stages, where venom composition is instead dominated by neurotoxins. The developmental onset of expression across all four procoagulant toxins correlates with a functional shift in venom activity to one that disrupts the blood-clotting system of mammalian prey. We further show that this developmental timing in both toxin expression and procoagulant venom activity coincides with a broader ecological transition in which Australian brown snakes begin to prey on mammals. In contrast, we found that the smallest brown snake species, a lizard specialist throughout its lifetime, only expresses neurotoxins and shows no comparable developmental shift in either procoagulant toxin expression or procoagulant activity. Our findings suggest that a coordinated shift in the expression of four procoagulant toxin gene families transitions the venom from a neurotoxic phenotype in lizard-feeding juveniles to a potent procoagulant phenotype in mammal-feeding adults. These results provide a clear example of how the concerted upregulation of multiple genes can drive a functional shift in a key ecological trait, linking changes in toxin gene expression to venom activity and, ultimately, to prey-capture strategy.

Cytotoxicity is a major pathological effect that can occur during snakebite envenoming. To better understand the underlying biochemical and molecular mechanisms behind snake venom-induced cytotoxicity, it is essential to use appropriate in vitro tools for bioassaying cytotoxicity evoked by snake venoms. Identifying the toxins causing cytotoxicity is also important in this regard, particularly in the context of developing more effective snakebite treatments. Cytotoxicity induced by venom toxins can result in local pathologies in snakebite victims, which can result in long-term morbidity, and is frequently observed after bites by medically important vipers. In the present study, we optimized and applied an analytical cytotoxicity profiling platform for in vitro cytotoxicity assessment of viper venoms. Using four cell lines (RPTEC/TERT1, HepaRG, iPSC-EC, HaCat), we applied an imaging analysis assay together with resazurin reduction to identify the mechanisms of cytotoxicity at the level of cell necrosis, extracellular matrix (ECM) degradation and/or cell apoptosis. Strong cytotoxic peaks are consistent with ECM-associated cytotoxic effects, as reflected by pronounced reductions in cell area and monolayer integrity. These cytotoxicity bioassays were integrated into nanofractionation analytics and high throughput venomics, which allowed for the identification of viper venom cytotoxins at the biological and chemical levels. Venom profiling showed ECM degradation as the main cytotoxic mechanism, except for Daboia russelii, which induced necrosis and apoptosis in three cell lines. Cytotoxicity largely disappeared after reversed-phase separation, prompting use of non-denaturing SEC in nanofractionation analytics, which revealed strong cytotoxic peaks for Bothrops jararaca and Calloselasma rhodostoma in RPTEC/TERT1 cells. The methodology presented here combined analytical and biochemical tools allowing rapid cytotoxicity profiling of viper venom toxins in parallel with toxin identification.

Snakebite envenoming is a neglected tropical disease that causes high mortality and morbidity. The current treatment, intravenous antivenom, comes with numerous disadvantages, making new therapeutics important. Optimized small molecules offer the possibility for oral use at the onset of envenoming, and the highly pathogenic, zinc-dependent snake venom metalloproteinase toxin family represents an attractive target for drug discovery. Through systematic chemical modification guided by molecular modeling, we describe the development of hydroxamic acid 23 (DC-174), a molecule that displays potent broad-spectrum metalloproteinase inhibition (IC50s < 10 nM) and neutralizes the procoagulant activities of multiple snake venoms. In oral dosing studies, 23 showed preclinical efficacy in a mouse model of severe envenoming, with efficacy boosted by a pharmacokinetically informed multiple dosing regimen. This rationally designed, orally bioavailable metalloproteinase inhibitor represents an excellent lead compound for the development of a small-molecule drug treatment for snakebite.

Snake venom activity exhibits evolutionary patterns within genera and varies according to ecological niche. By examining venoms from species inhabiting distinct environments, niche-specific functional differences can be uncovered. Bothrops, a diverse and medically important Neotropical pit viper genus, shows known clade-specific differences in coagulotoxicity. Here, we expanded this framework by assessing intra-clade variation in cellular activity across twelve Bothrops species using a high-content fluorescence assay that simultaneously measures ion channel responses and membrane cytotoxicity. Most venoms induced rapid membrane damage and cell lysis, whereas arboreal species lacked both activities, suggesting reduced selection for these activities. In contrast, the terrestrial Bothrops mattogrossensis and Bothrops pauloensis, which are sister species inhabiting the Pantanal wetlands and outskirts, respectively, displayed unique intracellular calcium-modulatory effects in the absence of membrane disruption. High throughput venomics revealed candidate toxin families underlying these calcium responses. Our findings demonstrate niche-specific diversification of venom bioactivity and highlight Bothrops venoms as promising sources of pharmacological agents.

The variation in venom between and within snake species has significant implications for snakebite treatment. This highlights the critical importance of studying venom composition and its variations, not only for medical purposes but also from an evolutionary perspective. This study explores analytics for characterizing venom variability, focusing on venom toxin accurate masses, and emphasizes how the complexity of studying snake venom variability can be addressed by using liquid chromatography mass spectrometry (LC-MS) analysis with bioinformatics tools. This was demonstrated by investigating LC-MS data obtained from the venoms of 15 true cobras (Naja spp.), 5 mambas (Dendroaspis spp.) and 28 vipers (Crotalus and Bothrops spp.; total of 20 Elapidae and 28 Viperidae venoms), with newly developed bioinformatics tools. The measured LC-MS data was processed in an automated fashion and sorted based on the monoisotopic accurate masses of all toxins found, their peak intensities, and their retention times in LC. The data was then investigated using bioinformatic tools, before the toxin data available in open-source databases was used to predict the class of a toxin by means of its mass. This study highlights the importance of studying venom variability, which is performed by our combinatorial approach of intact-toxin analysis and toxin grouping by accurate mass.

Elapid snakebites cause severe toxicity, predominantly neurotoxicity and general cytotoxicity. However, the specific cellular impacts of individual venom toxins remain largely underexplored. This study developed a high-throughput platform for profiling cytotoxicity from elapid venoms, focusing on nanofractionation analytics to enhance selectivity and toxin identification. Elapid Venoms were tested on four human cell lines, representing kidney (RPTEC/TERT1), liver (HepaRG), endothelial (iPSC-EC), and skin (HaCaT) tissues. Cytotoxic effects were assessed through cell coverage, viability, and metabolic assays in both crude and nanofractionated venom samples. Nanofractionation revealed selective cytotoxicity in venom components, notably phospholipases A2 (PLA2s) and three-finger toxins (3FTxs), which impaired membrane integrity and cellular metabolism. Crude B. multicinctus venom displayed specific cytotoxicity toward liver and skin cells but not kidney or endothelial cells. Cytotoxicity of nanofractionated B. multicinctus venom was lost, likely due to denaturing conditions of the reversed-phase separation. Fractionation after size exclusion chromatography (SEC) for post-column bioassaying to avoid toxin denaturation yielded bioactive fractions, with 3FTxs, PLA2s, and Kunitz-type serine protease (KUNs) likely responsible for the observed cell permeability disruption, extracellular matrix (ECM) degradation, and metabolic loss. This integrated analytical workflow, combining nanofractionation with high-throughput cytotoxicity assays and venomics, enabled rapid identification of venom components with cell type-specific toxicity. Our findings contribute to understanding elapid venom toxicity and can aid in developing targeted snakebite treatments focusing on cytotoxicity responsible for tissue-specific damage.

Snake venoms are complex bioactive mixtures designed to paralyse, kill, or digest prey. These venoms are of pharmacological interest due to their ability to modulate molecular targets such as ion channels and receptors with high specificity and potency. Traditional studies often focus on in vitro molecular analysis or in vivo behavioural effects, limiting comprehensive understanding. Here, we present a high-throughput screening platform that combines in vitro ion channel assays with in vivo zebrafish larval bioassays using nanofractionation analytics. This method integrates post-column calcium flux assays, zebrafish paralytic bioassays, toxin mass spectrometry, and proteomics to link bioactivity with toxin identification. Using elapid snake venoms (genus Dendroaspis, Naja, and Hemachatus) as a proof of concept, we identified several toxins modulating ion channels with paralytic effects on zebrafish larvae. Our approach enables parallel acquisition of in vitro and in vivo data, offering a robust guide for identifying and characterising ion channel modulators with defined molecular targets.

This study presents nanofractionation analytics coupled with in vivo profiling of zebrafish embryo paralysis and lethality in response to toxins in cone snail venoms. The focus of this study is on the development of this approach using venoms of Conus marmoreus, Conus ebraeus, and Conus bandanus. In brief, cone snail venoms were separated using reversed-phase chromatography following high-resolution nanofractionation on microplates with parallel mass spectrometry, enabled via a post-column flow split. All collected fractions were dried overnight, followed by assays on zebrafish embryos. For the paralysis assessment, we monitored swimming behavior and swimming distance and found that exposure to cone snail toxins led to paralysis and decreased movement and swim distance. To correlate the masses of eluted toxins with their paralyzing effects and potency, we compared the fractionation retention time versus normalized swimming distance. This allowed identification of the masses of toxins with paralyzing bioactivity, which were predominantly conopeptides. To assess lethality, zebrafish embryos were exposed to fractionated toxins for 24 h, after which they were inspected. The lethal doses and correlated toxins were identified by comparing retention times of fractionation versus the lethal dose values calculated for each fraction. We found that the most lethal venom was from C. bandanus, displaying the largest number of lethal peptides, followed by C. marmoreus and C. ebraeus. On the other hand, the most paralytic venom was from C. ebraeus, presenting a higher number of peptides with non-lethal paralytic effects, followed by C. bandanus and C. marmoreus. This study provides a pipeline to rapidly identify paralytic and lethal cone snail venom toxins using the zebrafish embryo model.

Snakebite envenoming is a persistent cause of mortality and morbidity worldwide due to the logistical challenges and costs of current antibody-based treatments. Their persistence motivates a broad interest in the discovery of inhibitors against multispecies venom phospholipase A2 (PLA2), which are underway as an alternative or supplemental treatment to improve health outcomes. Here, we present new computational strategies for improved inhibitor classification for challenging metalloenzyme targets across many species, including both a new method to utilize existing molecular docking, and subsequent data normalization. These methods were improved to support experimental screening efforts estimating the broader efficacy of candidate PLA2 inhibitors against diverse viper and elapid venoms.

Snakebite envenoming is a neglected tropical disease that causes high mortality and morbidity. The current treatment, intravenous antivenom, comes with numerous disadvantages making new therapeutics important. Optimised small molecules offer the possibility for oral use at the onset of envenoming, and the highly pathogenic, zinc-dependent, snake venom metalloproteinase toxin family represents an attractive target for drug discovery. Through systematic chemical modification guided by molecular modelling, we describe the development of hydroxamic acid DC-174, a molecule that displays potent broad spectrum metalloproteinase inhibition and neutralises the procoagulant activities of multiple snake venoms. In oral-dosing studies, DC-174 showed preclinical efficacy in a mouse model of severe envenoming, with efficacy boosted by a pharmacokinetically-informed multiple dosing regimen. This rationally designed metalloproteinase inhibitor offers a potential paradigm shift from delayed treatment with antivenom in tertiary hospitals to a contemporary approach using oral drugs amenable for rapid use in snakebite-affected communities. ### Competing Interest Statement The authors have declared no competing interest. Wellcome Trust, 221712/Z/20/Z

Snakebite envenoming remains a major global health issue, particularly in underserved regions. To better understand venom composition and toxic effects, crude snake venoms can be fractionated into distinct toxin groups using high-throughput nanofractionation analytics. These fractions can then be assessed using venomics to identify specific toxins, in conjunction with bioassays to evaluate their bioactivity. Traditional in vivo testing in rodents is limited by legal and ethical concerns, the high number of fractions, and the small quantities of toxins in each. In this study, zebrafish larvae were introduced as an efficient alternative model for in vivo analysis of toxins that impair locomotion. A behavioral bioassay was developed using a light-dark challenge to assess locomotion in 5-day post-fertilization (dpf) larvae. A Locomotion Index was created to compare movement during the dark phase between venom-exposed and control groups. Using venom from the spitting cobra Naja nigricollis, two fractions were found to reduce locomotor activity significantly. Microscopic screening also revealed three tissue-toxic fractions, one of which caused bradycardia. Dose-response testing was performed for each toxic fraction. Furthermore, oxygen consumption assays were conducted with venom fractions from N. nigricollis, N.haje, N. siamensis, and N. subfulva, identifying two fractions that elevated oxygen consumption. High-throughput venomics linked these toxic effects to three-finger toxins and phospholipases A2. This integrative approach offers a scalable, efficient method for in vivo toxicity screening of venom components and shows promise for broader applications in the discovery and profiling of diverse bioactive compounds.

Background Synthetic cathinones (SCs) are a large category of new psychoactive substances (NPS), which pose a serious threat to public health due to limited information about their toxicology and pharmacology. Many SCs are closely related in their chemical structures, with some substances being positional isomers. In this study, we propose a new workflow for the identification of SC isomers using liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS2) combined with electron activated dissociation (EAD) and chemometrics. Differentiation between isomeric SCs is essential for both legislative and public safety reasons, since minor differences in their molecular structures may change their legal status and pharmacological profiles. Results The workflow was optimized using ring-substituted isomers of methylmethcathinones, methylethcathinones, and chloromethcathinones. The kinetic energy in the EAD cell was investigated at three levels (i.e., 15, 18, and 20 eV) for each group. Two data analysis methods (i.e., t-distributed stochastic neighbor embedding [t-SNE] and a Random Forest [RF] algorithm) were applied using the obtained EAD mass spectral data. The three sets of ring-substituted SCs were clearly distinguished using t-SNE and an RF algorithm. Moreover, the RF approach resulted in a 97 % classification accuracy for isomer identification using various combinations of compounds, isomers, and electron kinetic energies. This workflow was subsequentially applied to the analysis of 26 blind street samples, resulting in a 92 % classification accuracy for isomer identification. However, the accuracy varied based on the kinetic electron energy. A subset of the original data set, focusing on 15-eV data only, was used, resulting in a classification accuracy of 100 %. Significance This study presents the first LC-HRMS2 workflow based on EAD and chemometrics, which resulted in a classification accuracy of 100 % of authentic street samples. The developed LC-HRMS2 workflow demonstrates that EAD product ions and their characteristic ion ratios can be successfully used to identify ring-substituted positional isomers of SCs.

Envenoming resulting from snakebites is recognized as a priority neglected tropical disease by The World Health Organization. The Bothrops genus, consisting of different pitviper species, is considered the most medically significant taxa in Central and South America. Further research into Bothrops venom composition is important to aid in the development of safer and more effective snakebite treatments. In addition, the discovery of Bothrops toxins that could potentially be used for medical or diagnostic purposes is of interest to the pharmaceutical industry. This study aimed to employ high-throughput (HT) venomics to qualitatively analyze venom composition while utilizing coagulation bioassays for identifying coagulopathic toxins and characterizing coagulopathic activity in various Bothrops venoms. Using the recently demonstrated HT venomics workflow in combination with post-column coagulopathic bioassaying, focus was placed at anticoagulant toxins. Well-known procoagulant toxins were also investigated, taking into account that using the HT venomics workflow, procoagulant toxins are especially prone to denaturation during the reversed-phase chromatographic separations performed in the workflow. The findings revealed that the venoms of B. atrox and B. jararaca harbored procoagulant toxins, whereas those of B. alternatus and B. neuwiedi contained both procoagulant and anticoagulant toxins. In general, anticoagulation was associated with phospholipases A2s, while procoagulation was associated with snake venom metalloproteinases and snake venom serine proteases. These results showed the identification of coagulopathic venom toxins in the Bothrops venoms analyzed using multiple analytical methods that complement each other. Additionally, each venom underwent qualitative characterization of its composition.

Worldwide, it is estimated that there are 1.8 to 2.7 million cases of envenoming caused by snakebites. Snake venom is a complex mixture of protein toxins, lipids, small molecules, and salts, with the proteins typically responsible for causing pathology in snakebite victims. For their chemical characterization and identification, analytical methods are required. Reversed-phase liquid chromatography coupled with electrospray ionization mass spectrometry (RP-LC-ESI-MS) is a widely used technique due to its ease of use, sensitivity, and ability to be directly coupled after LC separation. This method allows for the efficient separation of complex mixtures and sensitive detection of analytes. On the other hand, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is also sometimes used, and though it typically requires additional sample preparation steps, it offers desirable suitability for the analysis of larger biomolecules. In this study, seven medically important viperid snake venoms were separated into their respective venom toxins and measured by ESI-MS. In parallel, using nanofractionation analytics, post-column high-resolution fractionation was used to collect the eluting toxins for further processing for MALDI-MS analysis. Our comparative results showed that the deconvoluted snake venom toxin masses were observed with good sensitivity from both ESI-MS and MALDI-MS approaches and presented overlap in the toxin masses recovered (between 25% and 57%, depending on the venom analyzed). The mass range of the toxins detected in high abundance was between 4 and 28 kDa. In total, 39 masses were found in both the ESI-MS and/or MALDI-MS analyses, with most being between 5 and 9 kDa (46%), 13 and 15 kDa (38%), and 24 and 28 kDa (13%) in size. Next to the post-column MS analyses, additional coagulation bioassaying was performed to demonstrate the parallel post-column assessment of venom activity in the workflow. Most nanofractionated venoms exhibited anticoagulant activity, with three venoms additionally exhibiting toxins with clear procoagulant activity (Bothrops asper, Crotalus atrox, and Daboia russelii) observed post-column. The results of this study highlight the complementarity of ESI-MS and MALDI-MS approaches for characterizing snake venom toxins and provide a complementary overview of defined toxin masses found in a diversity of viper snake venoms.

Snakebite envenoming results in ∼100,000 deaths per year, with close to four times as many victims left with life-long sequelae. Current antivenom therapies have several limitations including high cost, variable cross-snake species efficacy and a requirement for intravenous administration in a clinical setting. Next-generation snakebite therapies are being widely investigated with the aim to improve cost, efficacy, and safety. In recent years several small molecule drugs have shown considerable promise for snakebite indication, with oral bioavailability particularly promising for community delivery rapidly after a snakebite. However, only two such drugs have entered clinical development for snakebite. To offset the risk of attrition during clinical trials and to better explore the chemical space for small molecule venom toxin inhibitors, here we describe the first high throughput drug screen against snake venom metalloproteinases (SVMPs)—a pathogenic toxin family responsible for causing haemorrhage and coagulopathy. Following validation of a 384-well fluorescent enzymatic assay, we screened a repurposed drug library of 3,547 compounds against five geographically distinct and toxin variable snake venoms. Our drug screen resulted in the identification of 14 compounds with pan-species inhibitory activity. Following secondary potency testing, four SVMP inhibitors were identified with nanomolar EC50s comparable to the previously identified matrix metalloproteinase inhibitor marimastat and superior to the metal chelator dimercaprol, doubling the current global portfolio of SVMP inhibitors. Following analysis of their chemical structure and ADME properties, two hit-to-lead compounds were identified. These clear starting points for the initiation of medicinal chemistry campaigns provide the basis for the first ever designer snakebite specific small molecules.