Elda Eliza Sanchez

Snakebite maims or kills several hundred thousand people each year. For more than a century, treatment has relied on antivenoms derived from animals immunized with whole venoms, but their efficacy, safety, and availability are highly variable, and it is often not well understood which specific venom components must be inhibited to prevent mortality and major morbidities. New therapeutic approaches are needed. Here, we take an evolutionary approach to antivenom design inspired by the longstanding observation that vipers have evolved serum-borne toxin inhibitors that confer resistance to their own venoms. We have investigated the abilities of a family of four rattlesnake metalloproteinase (MP) inhibitors derived from the ancestral serum glycoprotein Fetuin-A (FETUAs) to neutralize the enzymatic, hemorrhagic, and lethal activities of viper venoms. We find that while certain individual FETUA proteins are able to inhibit enzymatic or hemorrhagic activity, they are unable or only partially able to inhibit venom lethality. However, we show that specific combinations of FETUA proteins complement one another’s activities and are sufficient to fully neutralize rattlesnake venom lethality with approximately 10 times greater potency than commercial antivenom. Moreover, we demonstrate that FETUA proteins are well-conserved among viper subfamilies and that rattlesnake FETUAs are able to inhibit the MPs and neutralize the lethality of several evolutionarily distant pit viper or true viper venoms. Our results highlight the critical importance of inhibiting MPs in hemorrhagic venoms and the potential general utility of combinations of naturally evolved, recombinant MP inhibitors in the treatment of viper snakebite. Significance Statement: Snakebite remains an undertreated tropical disease that affects over one million people annually, and new therapeutic approaches are needed. Here, we develop a novel approach to viper antivenoms inspired by the observations that these snakes are typically resistant to the hemorrhagic and lethal effects of their own venoms and that this resistance can be transferred to experimental animals through snake sera. We show that a small set of serum metalloproteinase inhibitors from the Western Diamondback rattlesnake are potent inhibitors of the lethality of its own venom, and able to block the lethality of other rattlesnakes and evolutionarily distant vipers. Recombinant natural antitoxins offer a promising approach for formulating more potent viper antivenoms.

The Mojave rattlesnake venom shows significant geographical variability. The venom of Type A animals primarily contains β-neurotoxin referred to as Mojave Toxin (MTX), which makes bites from this snake particularly feared. We performed a genome-wide transcriptomic analysis of the neurocellular response to Mojave Type A rattlesnake venom using induced pluripotent stem cell-derived neural stem cells to unveil the molecular mechanisms underlying the damage caused by this snake’s envenomation. Our results suggest that snake venom metalloproteases, although having a limited repertoire in Type A venom, facilitate venom spread by digesting the tissue’s extracellular matrix. The MTX, which is composed of heterodimers of basic and acidic phospholipase-A2, co-opts the host arachidonic acid and Ca²⁺ second messenger mechanisms and triggers multiple signaling cascades, such as the activation of MAPKs and NF-κB-regulated proinflammatory genes; the neurotransmitter overload in excitatory synapses leading to a presynaptic blockade of nerve signals; and the upregulation of unfolded protein response (UPR) due to the depletion of Ca²⁺ from the endoplasmic reticulum. The upregulated UPR and the oxidative stress caused by reactive oxygen species generated in cytochromeP4501A1-mediated hydroxylation of arachidonic acid contribute to mitochondrial toxicity. The activation of UPR, mitochondrial toxicity, and oxidative stress synergistically contributed to apoptotic and ferroptotic cell death.

Synopsis: Venoms are complex bioactive mixtures that have independently evolved across diverse animal lineages, including snails, insects, sea anemones, spiders, scorpions, and snakes. Despite the growing interest in venom research, data is fragmented across disparate databases which lack standardization and interoperability. A vision for the proposed VenomsBase platform presented here seeks to address these challenges by using the best practices approach in creating a centralized, open-access platform adhering to FAIR principles (Findable, Accessible, Interoperable, and Reproducible). VenomsBase will unify venom datasets, standardize terminology, and enable comparative analyses across species, facilitating novel toxin discovery and functional annotation. Key features of VenomsBase include user-friendly data submission modules with built-in validation, advanced cross-species analysis tools, and integration of multidisciplinary datasets spanning genomics, transcriptomics, proteomics, functional assays, and ecological metadata. A modular, cloud-based design will ensure scalability, while heuristic scoring systems will guide users toward high-confidence data entries. To promote accessibility, the envisioned VenomsBase will provide tutorials, regular training sessions, case studies, and feedback loops, supporting researchers at all levels. By harmonizing venom research and addressing the limitations of outdated or nonstandardized methods, VenomsBase aims to revolutionize the field, while being continuously improved and refined by venom experts. This initiative will unlock venoms’ potential to make groundbreaking discoveries, address global health challenges, and foster collaboration and innovation across the scientific community.

Extracellular vesicles (EVs) are nanoparticle-sized vesicles secreted by nearly all cell types under normal physiological conditions. In toxicological research, EVs have emerged as a crucial link between public health and multi-omics approaches, offering insights into cellular responses to disease-causing injury agents such as environmental and biological toxins, contaminants, and drugs. Notably, EVs present a unique opportunity to deepen our understanding of the pathophysiology of envenomation by natural toxins. Recent advancements in isolating and purifying EV cargo, mass spectrometry techniques, and bioinformatics have positioned EVs as potential biomarkers that could elucidate biological signaling pathways and provide valuable information on the relationship between venomous toxins, their mechanisms of action, and the effectiveness of antivenoms. Additionally, EVs hold promise as proxies for various aspects of envenomation, including the toxin dosage, biological characterization, injury progression, and prognosis during therapeutic interventions. These aspects can be explored through multi-omics technology applied to EV contents from the plasma, saliva, or urine samples of envenomated individuals, offering a comprehensive integrative approach to understanding and managing envenomation cases.

King cobra (Ophiophagus hannah) venom comprises a diverse array of proteins and peptides. However, the roles and properties of these individual components are still not fully understood. Among these, Cysteine-rich secretory proteins (CRiSPs) are recognized but not fully characterized. This study investigates the biological effects of Opharin, the CRiSP from king cobra venom (KCV). The effects of Opharin on cytokine production, specifically on IL-1β, IL-6, IL-8, TNF-α, and IL-10 release, were evaluated over 24 h in monocyte-derived macrophage (MDM) cells. Notably, the levels of these inflammatory cytokines were significantly increased over 24 h, with values higher than those observed in cells treated with crude KCV at most time points. Additionally, the in vivo Miles assay in mice revealed that Opharin increased vascular permeability by 26% compared to the negative control group. These findings highlight the Opharin’s role in severe inflammatory and vascular responses observed in king cobra envenomation. Still, further research is essential to elucidate the pharmacological and toxicological effects of venom components, ultimately enhancing the clinical management of envenomation.

This study investigated the intraspecific and interspecific variability in the venom effects of Agkistrodon viperid snake species and subspecies (eleven venoms total) on plasma clotting times, fibrinogen levels, and fibrin clot strength. Significant delays in plasma clotting time were observed for A. conanti, A. contortrix mokasen, A. contortrix phaeogaster, A. howardgloydi, A. piscivorus leucostoma, and A. piscivorus piscivorus. Notably, the phylogenetically disjunct lineages A. conanti, A. contortrix mokasen, and A. howardgloydi exhibited the most potent anticoagulant effects, indicating the independent amplification of a basal trait. Inhibition assays with the activated clotting enzymes Factors XIa, IXa, Xa, and IIa (thrombin) revealed that FXa inhibition is another basal trait amplified independently on multiple occasions within the genus, but with A. howardgloydi, notably more potent than all others. Phospholipid degradation and zymogen destruction were identified as mechanisms underlying the variability in venom effects observed experimentally and in previous clinical reports. Thromboelastography demonstrated that the venoms did not clot fibrinogen directly but affected fibrin clot strength by damaging fibrinogen and that thrombin was subsequently only able to cleave into weak, unstable clots. The ability to activate Protein C, an endogenous anticoagulant enzyme, varied across species, with some venoms exceeding that of A. contortrix contortrix, which previously yielded the protein diagnostic agent Protac^®. Phylogenetic analysis suggested that both fibrinogen degradation and Protein C activation were each amplified multiple times within the genus, albeit with negative correlation between these two modes of action. This study highlights the evolutionary, clinical, and biodiscovery implications of venom variability in the Agkistrodon species, underscoring their dynamic evolution, emphasising the need for tailored clinical approaches, and highlighting the potential for novel diagnostic and therapeutic developments inspired by the unique properties of snake venoms.

Increased vascular permeability is a frequent outcome of viperid snakebite envenomation, leading to local and systemic complications. We reported that snake venom cysteine-rich secretory proteins (svCRiSPs) from North American pit vipers increase vascular permeability both in vitro and in vivo. They also induce acute activation of several adhesion and signaling molecules that may play a critical role in the pathophysiology of snakebites. Extracellular vesicles (EVs) have gained interest for their diverse functions in intercellular communication, regulating cellular processes, blood-endothelium interactions, vascular permeability, and immune modulation. They also hold potential as valuable biomarkers for diagnosing, predicting, and monitoring therapeutic responses in different diseases. This study aimed to identify proteins in peritoneal exudate and plasma EVs isolated from BALB/c mice following a 30 min post-injection of Crotalus scutulatus scutulatus venom and its purified CRiSP (Css-CRiSP). EVs were isolated from these biofluids using the EVtrap method. Proteomic analysis of exudate- and plasma-derived EVs was performed using LC-MS/MS. We observed significant upregulation or downregulation of proteins involved in cell adhesion, cytoskeleton rearrangement, signal transduction, immune responses, and vesicle-mediated transports. These findings suggest that svCRiSPs play a crucial role in the acute effects of venom and contribute to the local and systemic toxicity of snakebites.

The global exploration of snakebites requires the use of quantitative omics approaches to characterize snake venom as it enters into the systemic circulation. These omics approaches give insights into the venom proteome, but a further exploration is warranted to analyze the venom-reactome for the identification of snake venom biomarkers. The recent discovery of extracellular vesicles (EVs), and their critical cellular functions, has presented them as intriguing sources for biomarker discovery and disease diagnosis. Herein, we purified EV’s from the snake venom (svEVs) of Crotalus atrox and C. oreganus helleri, and from plasma of BALB/c mice injected with venom from each snake using EVtrap in conjunction with quantitative mass spectrometry for the proteomic identification and quantification of svEVs and plasma biomarkers. Snake venom EVs from C. atrox and C. o. helleri were highly enriched in 5′ nucleosidase, L-amino acid oxidase, and metalloproteinases. In mouse plasma EVs, a bioinformatic analysis for revealed upregulated responses involved with cytochrome P450, lipid metabolism, acute phase inflammation immune, and heat shock responses, while downregulated proteins were associated with mitochondrial electron transport, NADH, TCA, cortical cytoskeleton, reticulum stress, and oxidative reduction. Altogether, this analysis will provide direct evidence for svEVs composition and observation of the physiological changes of an envenomated organism.

Cysteine-Rich Secretory Proteins (CRiSPs) are typically found in many snake venoms; however, the role that these toxins play in the pathophysiology of snakebites is still unclear. Herein, we compared the effects of snake venom CRiSPs (svCRiSPs) from the most medically important species of North American snakes on endothelial cell permeability and vascular permeability. We used reverse phase protein array (RPPA) to identify key signaling molecules on human dermal lymphatic (HDLECs) and blood (HDBECs) endothelial cells treated with svCRiSPs. The results showed that Css-CRiSP isolated from Crotalus scutulatus scutulatus and App-CRiSP from Agkistrodon piscivorus piscivorus are the most potent causes of increase vascular and endothelial permeability in comparison with other svCRiSPs used in this study. We examined the protein expression levels and their activated phosphorylation states in HDLECs and HDBECs induced by App-CRiSP and Css-CRiSP using RPPA. Interestingly, both App-CRiSP and Css-CRiSP induced caveolin-1 expression in HDBECs. We also found that stimulating HDBECs with Css-CRiSP and App-CRiSP significantly induced the phosphorylation of mTOR and Src, respectively. In HDLECs, Css-CRiSP significantly downregulated the expression of N-Cadherin and phospholipase C-gamma, while App-CRiSP significantly enhanced Akt and JNK phosphorylation. These results suggest that the increased endothelial permeability in HDLECs and HDBECs by Css-CRiSP and App-CRiSP may occur through different pathways.

Snake envenomation can result in hemorrhage, local necrosis, swelling, and if not treated properly can lead to adverse systemic effects such as coagulopathy, nephrotoxicity, neurotoxicity, and cardiotoxicity, which can result in death. As such, snake venom metalloproteinases (SVMPs) and disintegrins are two toxic components that contribute to hemorrhage and interfere with the hemostatic system. Administration of a commercial antivenom is the common antidote to treat snake envenomation, but the high-cost, lack of efficacy, side effects, and limited availability, necessitates the development of new strategies and approaches for therapeutic treatments. Herein, we describe the neutralization ability of anti-disintegrin polyclonal antibody on the activities of isolated disintegrins, P-II/P-III SVMPs, and crude venoms. Our results show disintegrin activity on platelet aggregation in whole blood and the migration of the SK-Mel-28 cells that can be neutralized with anti-disintegrin polyclonal antibody. We characterized a SVMP and found that anti-disintegrin was also able to inhibit its activity in an in vitro proteolytic assay. Moreover, we found that anti-disintegrin could neutralize the proteolytic and hemorrhagic activities from crude Crotalus atrox venom. Our results suggest that anti-disintegrin polyclonal antibodies have the potential for a targeted approach to neutralize SVMPs in the treatment of snakebite envenomations.