Fangs and foliage: Unearthing the haemotoxic secrets of cannabis-dwelling rattlesnakes.

Despite a recent surge in high-throughput venom research that has enabled many species to be studied, some snake venoms remain understudied. The long-tailed rattlesnakes (Crotalus ericsmithi, C. lannomi, and C. stejnegeri) are one group where such research lags, largely owing to the rarity of these snakes and the hazardous areas, ripe with drug (marijuana and opium) production, they inhabit in Mexico. To fill this knowledge gap, we used multiple functional assays to examine the coagulotoxic (including across different plasma types), neurotoxic, and myotoxic activity of the venom of the long-tailed rattlesnakes. All crude venoms were shown to be potently anticoagulant on human plasma, which we discovered was not due to the destruction of fibrinogen, except for C. stejnegeri displaying minor fibrinogen destruction activity. All venoms exhibited anticoagulant activity on rat, avian, and amphibian plasmas, with C. ericsmithi being the most potent. We determined the mechanism of anticoagulant activity by C. ericsmithi and C. lannomi venoms to be phospholipid destruction and inhibition of multiple coagulation factors, leading to a net disruption of the clotting cascade. In the chick biventer assay, C. ericsmithi and C. lannomi did not exhibit neurotoxic activity but displayed potential weak myotoxic activity. BIRMEX® (Faboterápico Polivalente Antiviperino) antivenom was not effective in neutralising this venom effect. Overall, this study provides an in-depth investigation of venom function of understudied long-tailed rattlesnakes and provides a springboard for future venom and ecology research on the group.

Sugar-coated survival: N-glycosylation as a unique bearded dragon venom resistance trait within Australian agamid lizards.

In the ongoing evolutionary arms race between predators and prey, adaptive innovations often trigger a reciprocal response. For instance, the emergence of α-neurotoxins in snake venom has driven prey species targeted by these snakes to evolve sophisticated defense mechanisms. This study zeroes in on the particular motifs within the orthosteric sites of post-synaptic nicotinic acetylcholine receptors (nAChR) that confer resistance to α-neurotoxins, often through structural alterations of nAChR. This research examined Australian agamid lizards, a primary prey group for Australian elapid snakes, which are subject to predatory selection pressures. We previously showed that Pogona vitticeps (Central bearded dragon) was resistant to α-neurotoxic snake venoms through a steric hindrance form resistance evolving within the nAChR orthosteric, specifically through the 187-189NVT motif resulting in the presence of N-glycosylation, with the branching carbohydrate chains impeding the binding by the neurotoxins. This adaptive trait is thought to be a compensatory mechanism for the lizard's limited escape capabilities. Despite the significance of this novel adaptation, the prevalence and evolutionary roots of such venom resistance in Australian agamids have not been thoroughly investigated. To fill this knowledge gap, we undertook a comprehensive sequencing analysis of the nAChR ligand-binding domain across the full taxonomical diversity of Australian agamid species. Our findings reveal that the N-glycosylation resistance mechanism is a trait unique to the Pogona genus and absent in other Australian agamids. This aligns with Pogona's distinctive morphology, which likely increases vulnerability to neurotoxic elapid snakes, thereby increasing selective pressures for resistance. In contrast, biolayer interferometry experiments with death adder (Acanthophis species) venoms did not indicate any resistance-related binding patterns in other agamids, suggesting a lack of similar resistance adaptations, consistent with these lineages either being fast-moving, covered with large defensive spines, or being arboreal. This research not only uncovers a novel α-neurotoxin resistance mechanism in Australian agamids but also highlights the complex dynamics of the predator-prey chemical arms race. It provides a deeper understanding of how evolutionary pressures shape the interactions between venomous snakes and their prey.

High-Voltage Toxin'Roll: Electrostatic Charge Repulsion as a Dynamic Venom Resistance Trait in Pythonid Snakes.

The evolutionary interplay between predator and prey has significantly shaped the development of snake venom, a critical adaptation for subduing prey. This arms race has spurred the diversification of the components of venom and the corresponding emergence of resistance mechanisms in the prey and predators of venomous snakes. Our study investigates the molecular basis of venom resistance in pythons, focusing on electrostatic charge repulsion as a defense against α-neurotoxins binding to the alpha-1 subunit of the postsynaptic nicotinic acetylcholine receptor. Through phylogenetic and bioactivity analyses of orthosteric site sequences from various python species, we explore the prevalence and evolution of amino acid substitutions that confer resistance by electrostatic repulsion, which initially evolved in response to predatory pressure by Naja (cobra) species (which occurs across Africa and Asia). The small African species Python regius retains the two resistance-conferring lysines (positions 189 and 191) of the ancestral Python genus, conferring resistance to sympatric Naja venoms. This differed from the giant African species Python sebae, which has secondarily lost one of these lysines, potentially due to its rapid growth out of the prey size range of sympatric Naja species. In contrast, the two Asian species Python brongersmai (small) and Python bivittatus (giant) share an identical orthosteric site, which exhibits the highest degree of resistance, attributed to three lysine residues in the orthosteric sites. One of these lysines (at orthosteric position 195) evolved in the last common ancestor of these two species, which may reflect an adaptive response to increased predation pressures from the sympatric α-neurotoxic snake-eating genus Ophiophagus (King Cobras) in Asia. All these terrestrial Python species, however, were less neurotoxin-susceptible than pythons in other genera which have evolved under different predatory pressure as: the Asian species Malayopython reticulatus which is arboreal as neonates and juveniles before rapidly reaching sizes as terrestrial adults too large for sympatric Ophiophagus species to consider as prey; and the terrestrial Australian species Aspidites melanocephalus which occupies a niche, devoid of selection pressure from α-neurotoxic predatory snakes. Our findings underline the importance of positive selection in the evolution of venom resistance and suggest a complex evolutionary history involving both conserved traits and secondary evolution. This study enhances our understanding of the molecular adaptations that enable pythons to survive in environments laden with venomous threats and offers insights into the ongoing co-evolution between venomous snakes and their prey.

A Russian Doll of Resistance: Nested Gains and Losses of Venom Immunity in Varanid Lizards.

The interplay between predator and prey has catalyzed the evolution of venom systems, with predators honing their venoms in response to the evolving resistance of prey. A previous study showed that the African varanid species Varanus exanthematicus has heightened resistance to snake venoms compared to the Australian species V. giganteus, V. komodoensis, and V. mertensi, likely due to increased predation by sympatric venomous snakes on V. exanthematicus. To understand venom resistance among varanid lizards, we analyzed the receptor site targeted by venoms in 27 varanid lizards, including 25 Australian varanids. The results indicate an active evolutionary arms race between Australian varanid lizards and sympatric neurotoxic elapid snakes. Large species preying on venomous snakes exhibit inherited neurotoxin resistance, a trait potentially linked to their predatory habits. Consistent with the 'use it or lose it' aspect of venom resistance, this trait was secondarily reduced in two lineages that had convergently evolved gigantism (V. giganteus and the V. komodoensis/V. varius clade), suggestive of increased predatory success accompanying extreme size and also increased mechanical protection against envenomation due to larger scale osteoderms. Resistance was completely lost in the mangrove monitor V. indicus, consistent with venomous snakes not being common in their arboreal and aquatic niche. Conversely, dwarf varanids demonstrate a secondary loss at the base of the clade, with resistance subsequently re-evolving in the burrowing V. acanthurus/V. storri clade, suggesting an ongoing battle with neurotoxic predators. Intriguingly, within the V. acanthurus/V. storri clade, resistance was lost again in V. kingorum, which is morphologically and ecologically distinct from other members of this clade. Resistance was also re-evolved in V. glebopalma which is terrestrial in contrast to the arboreal/cliff dwelling niches occupied by the other members of its clade (V. glebopalma, V. mitchelli, V. scalaris, V. tristis). This 'Russian doll' pattern of venom resistance underscores the dynamic interaction between dwarf varanids and Australian neurotoxic elapid snakes. Our research, which included testing Acanthophis (death adder) venoms against varanid receptors as models for alpha-neurotoxic interactions, uncovered a fascinating instance of the Red Queen Hypothesis: some death adders have developed more potent toxins specifically targeting resistant varanids, a clear sign of the relentless predator-prey arms race. These results offer new insight into the complex dynamics of venom resistance and highlight the intricate ecological interactions that shape the natural world.

High-content fluorescence bioassay investigates pore formation, ion channel modulation and cell membrane lysis induced by venoms.

Venoms comprise highly sophisticated bioactive molecules modulating ion channels, receptors, coagulation factors, and the cellular membranes. This array of targets and bioactivities requires advanced high-content bioassays to facilitate the development of novel envenomation treatments and biotechnological and pharmacological agents. In response to the existing gap in venom research, we developed a cutting-edge fluorescence-based high-throughput and high-content cellular assay. This assay enables the simultaneous identification of prevalent cellular activities induced by venoms such as membrane lysis, pore formation, and ion channel modulation. By integrating intracellular calcium with extracellular nucleic acid measurements, we have successfully distinguished these venom mechanisms within a single cellular assay. Our high-content bioassay was applied across three cell types exposed to venom components representing lytic, ion pore-forming or ion channel modulator toxins. Beyond unveiling distinct profiles for these action mechanisms, we found that the pore-forming latrotoxin α-Lt1a prefers human neuroblastoma to kidney cells and cardiomyocytes, while the lytic bee peptide melittin is not selective. Furthermore, evaluation of snake venoms showed that Elapid species induced rapid membrane lysis, while Viper species showed variable to no activity on neuroblastoma cells. These findings underscore the ability of our high-content bioassay to discriminate between clades and interspecific traits, aligning with clinical observations at venom level, beyond discriminating among ion pore-forming, membrane lysis and ion channel modulation. We hope our research will expedite the comprehension of venom biology and the diversity of toxins that elicit cytotoxic, cardiotoxic and neurotoxic effects, and assist in identifying venom components that hold the potential to benefit humankind.

Malaysian and Chinese King Cobra Venom Cytotoxicity in Melanoma and Neonatal Foreskin Fibroblasts Is Mediated by Age and Geography.

Snake venoms constitute a complex, rapidly evolving trait, whose composition varies between and within populations depending on geographical location, age and preys (diets). These factors have determined the adaptive evolution for predatory success and link venom heterogeneity with prey specificity. Moreover, understanding the evolutionary drivers of animal venoms has streamlined the biodiscovery of venom-derived compounds as drug candidates in biomedicine and biotechnology. The king cobra (Ophiophagus hannah; Cantor, 1836) is distributed in diverse habitats, forming independent populations, which confer differing scale markings, including between hatchlings and adults. Furthermore, king cobra venoms possess unique cytotoxic properties that are used as a defensive trait, but their toxins may also have utility as promising anticancer-agent candidates. However, the impact of geographical distribution and age on these potential venom applications has been typically neglected. In this study, we hypothesised that ontogenetic venom variation accompanies the morphological distinction between hatchlings and adults. We used non-transformed neonatal foreskin (NFF) fibroblasts to examine and compare the variability of venom cytotoxicity between adult captive breeding pairs from Malaysian and Chinese lineages, along with that of their progeny upon hatching. In parallel, we assessed the anticancer potential of these venoms in human-melanoma-patient-derived cells (MM96L). We found that in a geographical distribution and gender-independent manner, venoms from hatchlings were significantly less cytotoxic than those from adults (NFF; ~Log EC50: 0.5-0.6 vs. 0.2-0.35 mg/mL). This is consistent with neonates occupying a semifossorial habitat, while adults inhabit more above-ground habitats and are therefore more conspicuous to potential predators. We also observed that Malaysian venoms exhibited a slightly higher cytotoxicity than those from the Chinese cobra cohorts (NFF; Log EC50: 0.1-0.3 vs. 0.3-0.4 mg/mL), which is consistent with Malaysian king cobras being more strongly aposematically marked. These variations are therefore suggestive of differential anti-predator strategies associated with the occupation of distinct niches. However, all cobra venoms were similarly cytotoxic in both melanoma cells and fibroblasts, limiting their potential medical applications in their native forms.

Comparative Analysis of Alpha-1 Orthosteric-Site Binding by a Clade of Central American Pit Vipers (Genera Atropoides, Cerrophidion, Metlapilcoatlus, and Porthidium).

The distribution and relative potency of post-synaptic neurotoxic activity within Crotalinae venoms has been the subject of less investigation in comparison with Elapidae snake venoms. No previous studies have investigated post-synaptic neurotoxic activity within the Atropoides, Metlapilcoatlus, Cerrophidion, and Porthidium clade. Given the specificity of neurotoxins to relevant prey types, we aimed to uncover any activity present within this clade of snakes that may have been overlooked due to lower potency upon humans and thus not appearing as a clinical feature. Using biolayer interferometry, we assessed the relative binding of crude venoms to amphibian, lizard, bird, rodent and human α-1 nAChR orthosteric sites. We report potent alpha-1 orthosteric site binding in venoms from Atropoides picadoi, Metlapilcoatlus occiduus, M. olmec, M. mexicanus, M. nummifer. Lower levels of binding, but still notable, were evident for Cerrophidion godmani, C. tzotzilorum and C. wilsoni venoms. No activity was observed for Porthidium venoms, which is consistent with significant alpha-1 orthosteric site neurotoxicity being a trait that was amplified in the last common ancestor of Atropoides/Cerrophidion/Metlapilcoatlus subsequent to the split by Porthidium. We also observed potent taxon-selective activity, with strong selection for non-mammalian targets (amphibian, lizard, and bird). As these are poorly studied snakes, much of what is known about them is from clinical reports. The lack of affinity towards mammalian targets may explain the knowledge gap in neurotoxic activity within these species, since symptoms would not appear in bite reports. This study reports novel venom activity, which was previously unreported, indicating toxins that bind to post-synaptic receptors may be more widespread in pit vipers than previously considered. While these effects appear to not be clinically significant due to lineage-specific effects, they are of significant evolutionary novelty and of biodiscovery interest. This work sets the stage for future research directions, such as the use of in vitro and in vivo models to determine whether the alpha-1 orthosteric site binding observed within this study confers neurotoxic venom activity.

Resistance Is Not Futile: Widespread Convergent Evolution of Resistance to Alpha-Neurotoxic Snake Venoms in Caecilians (Amphibia: Gymnophiona).

Predatory innovations impose reciprocal selection pressures upon prey. The evolution of snake venom alpha-neurotoxins has triggered the corresponding evolution of resistance in the post-synaptic nicotinic acetylcholine receptors of prey in a complex chemical arms race. All other things being equal, animals like caecilians (an Order of legless amphibians) are quite vulnerable to predation by fossorial elapid snakes and their powerful alpha-neurotoxic venoms; thus, they are under strong selective pressure. Here, we sequenced the nicotinic acetylcholine receptor alpha-1 subunit of 37 caecilian species, representing all currently known families of caecilians from across the Americas, Africa, and Asia, including species endemic to the Seychelles. Three types of resistance were identified: (1) steric hindrance from N-glycosylated asparagines; (2) secondary structural changes due to the replacement of proline by another amino acid; and (3) electrostatic charge repulsion of the positively charged neurotoxins, through the introduction of a positively charged amino acid into the toxin-binding site. We demonstrated that resistance to alpha-neurotoxins convergently evolved at least fifteen times across the caecilian tree (three times in Africa, seven times in the Americas, and five times in Asia). Additionally, as several species were shown to possess multiple resistance modifications acting synergistically, caecilians must have undergone at least 20 separate events involving the origin of toxin resistance. On the other hand, resistance in non-caecilian amphibians was found to be limited to five origins. Together, the mutations underlying resistance in caecilians constitute a robust signature of positive selection which strongly correlates with elapid presence through both space (sympatry with caecilian-eating elapids) and time (Cenozoic radiation of elapids). Our study demonstrates the extent of convergent evolution that can be expected when a single widespread predatory adaptation triggers parallel evolutionary arms races at a global scale.

Horizontal gene transfer underlies the painful stings of asp caterpillars (Lepidoptera: Megalopygidae).

Larvae of the genus Megalopyge (Lepidoptera: Zygaenoidea: Megalopygidae), known as asp or puss caterpillars, produce defensive venoms that cause severe pain. Here, we present the anatomy, chemistry, and mode of action of the venom systems of caterpillars of two megalopygid species, the Southern flannel moth Megalopyge opercularis and the black-waved flannel moth Megalopyge crispata. We show that megalopygid venom is produced in secretory cells that lie beneath the cuticle and are connected to the venom spines by canals. Megalopygid venoms consist of large aerolysin-like pore-forming toxins, which we have named megalysins, and a small number of peptides. The venom system differs markedly from those of previously studied venomous zygaenoids of the family Limacodidae, suggestive of an independent origin. Megalopygid venom potently activates mammalian sensory neurons via membrane permeabilization and induces sustained spontaneous pain behavior and paw swelling in mice. These bioactivities are ablated by treatment with heat, organic solvents, or proteases, indicating that they are mediated by larger proteins such as the megalysins. We show that the megalysins were recruited as venom toxins in the Megalopygidae following horizontal transfer of genes from bacteria to the ancestors of ditrysian Lepidoptera. Megalopygids have recruited aerolysin-like proteins as venom toxins convergently with centipedes, cnidarians, and fish. This study highlights the role of horizontal gene transfer in venom evolution.

Venom biotechnology: casting light on nature's deadliest weapons using synthetic biology.

Venoms are complex chemical arsenals that have evolved independently many times in the animal kingdom. Venoms have attracted the interest of researchers because they are an important innovation that has contributed greatly to the evolutionary success of many animals, and their medical relevance offers significant potential for drug discovery. During the last decade, venom research has been revolutionized by the application of systems biology, giving rise to a novel field known as venomics. More recently, biotechnology has also made an increasing impact in this field. Its methods provide the means to disentangle and study venom systems across all levels of biological organization and, given their tremendous impact on the life sciences, these pivotal tools greatly facilitate the coherent understanding of venom system organization, development, biochemistry, and therapeutic activity. Even so, we lack a comprehensive overview of major advances achieved by applying biotechnology to venom systems. This review therefore considers the methods, insights, and potential future developments of biotechnological applications in the field of venom research. We follow the levels of biological organization and structure, starting with the methods used to study the genomic blueprint and genetic machinery of venoms, followed gene products and their functional phenotypes. We argue that biotechnology can answer some of the most urgent questions in venom research, particularly when multiple approaches are combined together, and with other venomics technologies.

Functional and Proteomic Insights into Aculeata Venoms.

Aculeate hymenopterans use their venom for a variety of different purposes. The venom of solitary aculeates paralyze and preserve prey without killing it, whereas social aculeates utilize their venom in defence of their colony. These distinct applications of venom suggest that its components and their functions are also likely to differ. This study investigates a range of solitary and social species across Aculeata. We combined electrophoretic, mass spectrometric, and transcriptomic techniques to characterize the compositions of venoms from an incredibly diverse taxon. In addition, in vitro assays shed light on their biological activities. Although there were many common components identified in the venoms of species with different social behavior, there were also significant variations in the presence and activity of enzymes such as phospholipase A2s and serine proteases and the cytotoxicity of the venoms. Social aculeate venom showed higher presence of peptides that cause damage and pain in victims. The venom-gland transcriptome from the European honeybee (Apis mellifera) contained highly conserved toxins which match those identified by previous investigations. In contrast, venoms from less-studied taxa returned limited results from our proteomic databases, suggesting that they contain unique toxins.

Children and Snakebite: Snake Venom Effects on Adult and Paediatric Plasma.

Snakebite is a globally neglected tropical disease, with coagulation disturbances being the primary pathology of many deadly snake venoms. Age-related differences in human plasma have been abundantly reported, yet the effect that these differences pose regarding snakebite is largely unknown. We tested for differences in coagulotoxic effects (via clotting time) of multiple snake venoms upon healthy human adult (18+) and paediatric (median 3.3 years old) plasma in vivo and compared these effects to the time it takes the plasmas to clot without the addition of venom (the spontaneous clotting time). We tested venoms from 15 medically significant snake species (from 13 genera) from around the world with various mechanisms of coagulotoxic actions, across the three broad categories of procoagulant, pseudo-procoagulant, and anticoagulant, to identify any differences between the two plasmas in their relative pathophysiological vulnerability to snakebite. One procoagulant venom (Daboia russelii, Russell's Viper) produced significantly greater potency on paediatric plasma compared with adult plasma. In contrast, the two anticoagulant venoms (Pseudechis australis, Mulga Snake; and Bitis cornuta, Many-horned Adder) were significantly more potent on adult plasma. All other procoagulant venoms and all pseudo-procoagulant venoms displayed similar potency across both plasmas. Our preliminary results may inform future studies on the effect of snake venoms upon plasmas from different age demographics and hope to reduce the burden of snakebite upon society.

Children and snakebite: snake venom effects on adult and paediatric plasma

Snakebite is a globally neglected tropical disease, with coagulation disturbances being the primary pathology of many deadly snake venoms. Age-related differences in human plasma have been abundantly reported, yet the effect that these differences pose regarding snakebite is largely unknown. We tested for differences in coagulotoxic effects (via clotting time) of multiple snake venoms upon healthy human adult (18+) and paediatric (median 3.3 years old) plasma in vivo and compared these effects to the time it takes the plasmas to clot without the addition of venom (the spontaneous clotting time). We tested venoms from 15 medically significant snake species (from 13 genera) from around the world with various mechanisms of coagulotoxic actions, across the three broad categories of procoagulant, pseudo-procoagulant, and anticoagulant, to identify any differences between the two plasmas in their relative pathophysiological vulnerability to snakebite. One procoagulant venom (Daboia russelii, Russell’s Viper) produced significantly greater potency on paediatric plasma compared with adult plasma. In contrast, the two anticoagulant venoms (Pseudechis australis, Mulga Snake; and Bitis cornuta, Many-horned Adder) were significantly more potent on adult plasma. All other procoagulant venoms and all pseudo-procoagulant venoms displayed similar potency across both plasmas. Our preliminary results may inform future studies on the effect of snake venoms upon plasmas from different age demographics and hope to reduce the burden of snakebite upon society.

Extreme Procoagulant Potency in Human Plasma of Venoms from the African Viperid Genera Atheris, Cerastes, and Proatheris and the Relative Efficacy of Antivenoms and Synthetic Enzyme-Inhibitors.

The African viperid snake genera Atheris, Cerastes, and Proatheris are closely related, similar in size, but occupy extremely divergent ecological niches (arboreal in tropical rainforests, fossorial in deserts, and swamp-dwelling, respectively). Their venoms have not previously been subjected to comparative analyses for their action upon the coagulation of blood, most notably with significant data deficiencies from Atheris and Proatheris. In contrast, the closely related genus Echis is well-documented as capable of producing potent procoagulant effects. In light of this, we set out to compare the coagulotoxic actions of Atheris ceratophora, A. chlorechis, A. desaixi, A. nitschei, A. squamigera, C. cerastes, C. cerastes gasperettii, C. vipera, and Proatheris superciliaris and explore potential pharmacological interventions to reestablish normal blood coagulation. All venoms displayed extremely potent procoagulant effects, over twice as fast as the most potent Echis reported to date. Although Cerastes is used in the immunising mixture of two different regionally available antivenoms (Inoserp-MENA with C. cerastes, C. cerastes gasperettii, C. vipera and Saudi Arabian polyvalent with C. cerastes), none of the other species in this study are included in the immunising mixture of any antivenom. Notably, all the Cerastes species were only neutralised by the Inoserp-MENA antivenom. C. cerastes venom was not neutralised well by the Saudi Arabian antivenom, with the low levels of recognition for any of the Cerastes venoms suggesting a strong regional variation in the venom of this species, as the C. cerastes venom tested was of African (Tunisian) origin versus Saudi locality used in that antivenom's production. The other antivenoms (Micropharm EchiTAbG, ICP EchiTAb-Plus-ICP, Inosan Inoserp Pan-Africa, Premium Serums PANAF Sub-Sahara Africa, South African Vaccine Producers Echis, South African Vaccine Producers Polyvalent) all displayed trivial-to-no ability to neutralise the procoagulant toxicity of any of the Atheris, Cerastes, or Proatheris venoms. Comparative testing of the enzyme inhibitors DMPS, marimastat, and prinomastat, revealed a very potent neutralising capacity of marimastat, with prinomastat showing lower but still significant potency at the same molar concentration, while a 5× molar concentration of DMPS had no apparent effect on procoagulant venom effects normalized by the other inhibitors. These results and methods contribute to the body of knowledge of potential clinical effects and data necessary for evidence-based advancement of clinical management strategies.

Varespladib in the Treatment of Snakebite Envenoming: Development History and Preclinical Evidence Supporting Advancement to Clinical Trials in Patients Bitten by Venomous Snakes.

The availability of effective, reliably accessible, and affordable treatments for snakebite envenoming is a critical and long unmet medical need. Recently, small, synthetic toxin-specific inhibitors with oral bioavailability used in conjunction with antivenom have been identified as having the potential to greatly improve outcomes after snakebite. Varespladib, a small, synthetic molecule that broadly and potently inhibits secreted phospholipase A2 (sPLA2s) venom toxins has renewed interest in this class of inhibitors due to its potential utility in the treatment of snakebite envenoming. The development of varespladib and its oral dosage form, varespladib-methyl, has been accelerated by previous clinical development campaigns to treat non-envenoming conditions related to ulcerative colitis, rheumatoid arthritis, asthma, sepsis, and acute coronary syndrome. To date, twenty-nine clinical studies evaluating the safety, pharmacokinetics (PK), and efficacy of varespladib for non-snakebite envenoming conditions have been completed in more than 4600 human subjects, and the drugs were generally well-tolerated and considered safe for use in humans. Since 2016, more than 30 publications describing the structure, function, and efficacy of varespladib have directly addressed its potential for the treatment of snakebite. This review summarizes preclinical findings and outlines the scientific support, the potential limitations, and the next steps in the development of varespladib's use as a snakebite treatment, which is now in Phase 2 human clinical trials in the United States and India.

Engineering the Cyclization Loop of MCoTI-II Generates Targeted Cyclotides that Potently Inhibit Factor XIIa.

Factor XIIa (FXIIa) is a promising target for developing new drugs that prevent thrombosis without causing bleeding complications. A native cyclotide (MCoTI-II) is gaining interest for engineering FXIIa-targeted anticoagulants as this peptide inhibits FXIIa but not other coagulation proteases. Here, we engineered the native biosynthetic cyclization loop of MCoTI-II (loop 6) to generate improved FXIIa inhibitors. Decreasing the loop length led to gains in potency up to 7.7-fold, with the most potent variant having five residues in loop 6 (Ki = 25 nM). We subsequently examined sequence changes within loop 6 and an adjacent loop, with substitutions at P4 and P2' producing a potent FXIIa inhibitor (Ki = 2 nM) that displayed more than 700-fold selectivity, was stable in human serum, and blocked the intrinsic coagulation pathway in human plasma. These findings demonstrate that engineering the biosynthetic cyclization loop can generate improved cyclotide variants, expanding their potential for drug discovery.

Diverse and Dynamic Alpha-Neurotoxicity Within Venoms from the Palearctic Viperid Snake Clade of Daboia, Macrovipera, Montivipera, and Vipera.

The targeting of specific prey by snake venom toxins is a fascinating aspect of molecular and ecological evolution. Neurotoxic targeting by elapid snakes dominates the literature in this regard; however, recent studies have revealed viper toxins also induce neurotoxic effect. While this effect is thought to primarily be driven by prey selectivity, no study has quantified the taxonomically specific neurotoxicity of the viper clade consisting of Daboia, Macrovipera, Montivipera, and Vipera genera. Here, we tested venom toxin binding from 28 species of vipers from the four genera on the alpha 1 neuronal nicotinic acetylcholine receptors (nAChRs) orthosteric sites of amphibian, avian, lizard, rodent, and human mimotopes (synthetic peptides) using the Octet HTX biolayer interferometry platform. Daboia siamensis and D. russelii had broad binding affinity towards all mimotopes, while D. palestinae had selectivity toward lizard. Macrovipera species, on the other hand, were observed to have a higher affinity for amphibian mimotopes except for M. schweizeri, which inclined more toward lizard mimotopes. All Montivipera and most Vipera species also had higher affinity toward lizard mimotopes. Vipera a. montandoni, V. latastei, V. nikolski, and V. transcaucasina had the least binding to any of the mimotopes of the study. While a wide range of affinity binding towards various mimotopes were observed within the clade, the lowest affinity occurred towards the human target. Daboia siamensis and Macrovipera lebetina exhibited the greatest affinity toward the human mimotope, albeit still the least targeted of the mimotopes within those species. Overlaying this toxin-targeting trait over phylogeny of this clade revealed multiple cases of amplification of this trait and several cases of secondary loss. Overall, our results reveal dynamic variation, amplification, and some secondary loss of the prey targeting trait by alpha-neurotoxins within the venoms of this clade, indicating evolutionary selection pressure shaping the basic biochemistry of these venoms. Our work illustrates the successful use of this biophysical assay to further research snake venom neurotoxins and emphasizes the risk of generalizing venom effects observed on laboratory animals to have similar effects on humans.

Proteomic and toxicological characterization of the venoms of the most enigmatic group of rattlesnakes: The long-tailed rattlesnakes.

The most enigmatic group of rattlesnakes is the long-tailed rattlesnake group, consisting of three species: Crotalus ericsmithi, Crotalus lannomi and Crotalus stejnegeri. These species have been the least studied rattlesnakes in all aspects, and no study on the characterization of their venoms has been carried out to date. Our main objective was to investigate the proteomic composition, as well as some of the biochemical and toxic activities of these venoms, and their neutralization by commercial antivenom. The venom proteome of C. ericsmithi mainly contains metalloproteinases (SVMP; 49.3%), phospholipases A2 (PLA2; 26.2%), disintegrins (Dis; 12.6%), and snake venom serine proteases (SVSP; 6.8%), while C. lannomi venom mainly consists of SVMP (47.1%), PLA2 (19.3%), Dis (18.9%), SVSP (6%) and l-amino acid oxidase (LAAO; 2.6%). For these venoms high lethality was recorded in mice, the most potent being that of C. lannomi (LD50 of 0.99 µg/g body weight), followed by C. ericsmithi (1.30 µg/g) and finally C. stejnegeri (1.79 µg/g). The antivenoms Antivipmyn® from SILANES and Fabotherapic polyvalent antiviperin® from BIRMEX neutralized the lethal activity of the three venoms. Although this group of snakes is phylogenetically related to the C. viridis group, no neurotoxic components (crotoxin or crotoxin-like proteins) common in rattlesnakes were found in their venoms. This study expands current knowledge on the venoms of understudied snake species of the Mexican herpetofauna.

Keel venom: Rhabdophis subminiatus (Red-Necked Keelback) venom pathophysiologically affects diverse blood clotting pathways.

Venoms are evolutionary novelties that have real-world implications due to their impact upon human health. However, relative to the abundant studies of elapid and viperid snake venoms, fewer investigations have been undertaken on those of rear-fanged snakes as they are more problematic for obtaining venom. While most rear-fanged venomous snakes are not considered to be of great medical importance, several species are capable of producing fatalities. Most notable among these are snakes from the genus Rhabdophis, the Asian "keelback" snakes. Prior work have described potent procoagulant toxicity suggesting Factor X and prothrombin activation, but did not investigate the ability to activate other clotting factors. Here we show that in addition to activating both Factor X and prothrombin (with prothrombin twice that of FX), the venom of Rhabdophis subminiatus is able to more potently activate Factor VII (ten times that of prothrombin), while also activating FXII and FIX equipotently to prothrombin, and with FXI also activated but at a much lower level. The ability to activate FVII represents a third convergent evolution of this trait. The Australian elapid clade of [Oxyuranus (taipans) + Pseudonaja (brown snakes)] was the first identified to have evolved this trait. and only recently was it shown to be independently present in another lineage (the Central American viperid species Porthidium volcanicum). In addition, the abilities to activate FXI and FXII are also convergent between R. subminiatus and P. volcanicum, but with R. subminiatus being much more potent. By testing across amphibian, avian, and mammalian plasmas we demonstrate that the venom is potently procoagulant across diverse plasma types. However, consistent with dietary preference, R. subminiatus venom was most potent upon amphibian plasma. While a Rhabdophis antivenom is produced in Japan to treat R. tigrinus envenomings, it is scarce even within Japan and is not exported. As this genus is very wide-ranging in Asia, alternate treatment options are in need of development. Hence we tested the ability of candidate, broad-spectrum enzyme inhibitors to neutralize R. subminiatus venom: marimastat was more effective than prinomastat but both marimastat and prinomastat were significantly more effective than DMPS (2,3-Dimercapto-1-propanesulfonic acid). The findings of this study shed light on the evolution of these fascinating rear-fanged snakes as well as explored their systemic effects upon blood coagulation and point to potential treatment options for the rare, but potentially lethal encounters.

The Target Selects the Toxin: Specific Amino Acids in Snake-Prey Nicotinic Acetylcholine Receptors That Are Selectively Bound by King Cobra Venoms.

Snake venom is an adaptive ecological trait that has evolved primarily as a form of prey subjugation. Thus, the selection pressure for toxin diversification is exerted by the prey's physiological targets, with this pressure being particularly acute for specialist feeders, such as the King Cobra species, all of which are snake-prey specialists. However, while extensive research has been undertaken to elucidate key amino acids that guide toxin structure-activity relationships, reciprocal investigations into the specific sites guiding prey-lineage selective effects have been lacking. This has largely been due to the lack of assay systems amenable to systematic amino acid replacements of targeted proteins in the prey's physiological pathways. To fill this knowledge gap, we used a recently described approach based upon mimotope peptides corresponding to the orthosteric site of nicotinic acetylcholine receptor alpha-1 subunits, a major binding site for snake venom neurotoxins that cause flaccid paralysis. We investigated the venoms of four different types of King Cobra (Cambodian, Javan, Malaysian, and Thai). This approach allowed for the determination of the key amino acid positions in King Cobra snake prey that are selectively bound by the toxins, whereby replacing these amino acids in the snake-prey orthosteric site with those from lizards or rats resulted in a significantly lower level of binding by the venoms, while conversely replacing the lizard or rat amino acids with those from the snake at that position increased the binding. By doing such, we identified three negatively charged amino acids in the snake orthosteric site that are strongly bound by the positively charged neurotoxic three-finger toxins found in King Cobra venom. This study, thus, sheds light on the selection pressures exerted by a specialist prey item for the evolution of lineage-selective toxins.