Importance of the Cysteine-Rich Domain of Snake Venom Prothrombin Activators: Insights Gained from Synthetic Neutralizing Antibodies.

Snake venoms are cocktails of biologically active molecules that have evolved to immobilize prey, but can also induce a severe pathology in humans that are bitten. While animal-derived polyclonal antivenoms are the primary treatment for snakebites, they often have limitations in efficacy and can cause severe adverse side effects. Building on recent efforts to develop improved antivenoms, notably through monoclonal antibodies, requires a comprehensive understanding of venom toxins. Among these toxins, snake venom metalloproteinases (SVMPs) play a pivotal role, particularly in viper envenomation, causing tissue damage, hemorrhage and coagulation disruption. One of the current challenges in the development of neutralizing monoclonal antibodies against SVMPs is the large size of the protein and the lack of existing knowledge of neutralizing epitopes. Here, we screened a synthetic human antibody library to isolate monoclonal antibodies against an SVMP from saw-scaled viper (genus Echis) venom. Upon characterization, several antibodies were identified that effectively blocked SVMP-mediated prothrombin activation. Cryo-electron microscopy revealed the structural basis of antibody-mediated neutralization, pinpointing the non-catalytic cysteine-rich domain of SVMPs as a crucial target. These findings emphasize the importance of understanding the molecular mechanisms of SVMPs to counter their toxic effects, thus advancing the development of more effective antivenoms.

Toxic Peptides from the Mexican Scorpion Centruroides villegasi: Chemical Structure and Evaluation of Recognition by Human Single-Chain Antibodies.

Alternative recombinant sources of antivenoms have been successfully generated. The application of such strategies requires the characterization of the venoms for the development of specific neutralizing molecules against the toxic components. Five toxic peptides to mammals from the Mexican scorpion Centruroides villegasi were isolated by chromatographic procedures by means of gel filtration on Sephadex G-50, followed by ion-exchange columns on carboxy-methyl-cellulose (CMC) resins and finally purified by high-performance chromatography (HPLC) columns. Their primary structures were determined by Edman degradation. They contain 66 amino acids and are maintained well packed by four disulfide bridges, with molecular mass from 7511.3 to 7750.1 Da. They are all relatively toxic and deadly to mice and show high sequence identity with known peptides that are specific modifiers of the gating mechanisms of Na+ ion channels of type beta-toxin (ß-ScTx). They were named Cv1 to Cv5 and used to test their recognition by single-chain variable fragments (scFv) of antibodies, using surface plasmon resonance. Three different scFvs generated in our laboratory (10FG2, HV, LR) were tested for recognizing the various new peptides described here, paving the way for the development of a novel type of scorpion antivenom.

Mass spectrometric analysis of Odonthobuthus Doriae scorpion venom and its non-neutralized fractions after interaction with commercial antivenom.

It is believed that antivenoms play a crucial role in neutralizing venoms. However, uncontrolled clinical effects appear in patients stung by scorpions after the injection of antivenom. In this research, non-neutralized components of the venom of the Iranian scorpion Odonthobuthus doriae were analyzed after interacting with the commercial antivenom available in the market. The venom and antivenom interaction was performed, then centrifuged, and the supernatant was analyzed by high-performance liquid chromatography (HPLC). Two peaks of Odonthobuthus doriae venom were observed in the chromatogram of the supernatant. Two components were isolated by HPLC and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) instruments. Peptide sequencing was done by Liquid Chromatography Quadrupole Time-of-Flight Tandem Mass Spectrometry (LC-Q-TOF MS/MS). Results indicate that the components of scorpion venom mainly have a molecular weight below 10 kDa, consisting of toxic peptides that disrupt the function of sodium and potassium channels. The MALDI-TOF MS results show that two toxic peptides with molecular masses of 6941 Da and 6396 Da were not neutralized by the antivenom. According to the MS/MS sequencing data, the components have been related to peptides A0A5P8U2Q6_MESEU and A0A0U4FP89_ODODO, which belong to the sodium and potassium channels toxins family, respectively.

Evaluation of the Inhibitory Potential of Synthetic Peptides Homologous to CDR3 Regions of a Monoclonal Antibody against Bothropic Venom Serine Proteases.

Snakebite accidents, neglected tropical diseases per the WHO, pose a significant public health threat due to their severity and frequency. Envenomation by Bothrops genus snakes leads to severe manifestations due to proteolytic enzymes. While the antibothropic serum produced by the Butantan Institute saves lives, its efficacy is limited as it fails to neutralize certain serine proteases. Hence, developing new-generation antivenoms, like monoclonal antibodies, is crucial. This study aimed to explore the inhibitory potential of synthetic peptides homologous to the CDR3 regions of a monoclonal antibody targeting a snake venom thrombin-like enzyme (SVTLE) from B. atrox venom. Five synthetic peptides were studied, all stable against hydrolysis by venoms and serine proteases. Impressively, four peptides demonstrated uncompetitive SVTLE inhibition, with Ki values ranging from 10-6 to 10-7 M. These findings underscore the potential of short peptides homologous to CDR3 regions in blocking snake venom toxins, suggesting their promise as the basis for new-generation antivenoms. Thus, this study offers potential advancements in combatting snakebites, addressing a critical public health challenge in tropical and subtropical regions.

CTXP, The Major Cobra Toxin Peptide from Naja Naja Oxiana Venom; A Promising Target for Antivenom Agent Development.

BACKGROUND AND OBJECTIVE: Snakebite envenoming is a serious public health issue causing more than 135,000 annual deaths worldwide. Naja Naja Oxiana is one of the most clinically important venomous snakes in Iran and Central Asia. Conventional animal-derived polyclonal antibodies are the major treatment of snakebite envenoming. Characterization of venom components helps to pinpoint the toxic protein responsible for clinical manifestations in victims, which aids us in developing efficient antivenoms with minimal side effects. Therefore, the present study aimed to identify the major lethal protein of Naja Naja Oxiana by top-down proteomics. METHODS: Venom proteomic profiling was performed using gel filtration (GF), reversed-phase (RP) chromatography, and intact mass spectrometry. The toxicity of GF-, and RP-eluted fractions was analyzed in BALB/c mice. The rabbit polyclonal antisera were produced against crude venom, GF fraction V (FV), and RP peak 1 (CTXP) and applied in neutralization assays. RESULTS: Toxicity studies in BALB/c identified FV as the major toxic fraction of venom. Subsequently, RP separation of FV resulted in eight peaks, of which peak 1, referred to as "CTXP" (cobra toxin peptide), was identified as the major lethal protein. In vivo neutralization assays using rabbit antisera showed that polyclonal antibodies raised against FV and CTXP are capable of neutralizing at least 2-LD50s of crude venom, FV, and CTXP in all tested mice. CONCLUSION: Surprisingly, the Anti-CTXP antibody could neutralize 8-LD50 of the CTXP peptide. These results identified CTXP (a 7 kDa peptide) as a potential target for the development of novel efficient antivenom agents.

Identification of a common epitope in knottins and phospholipases D present in Loxosceles sp venom by a monoclonal antibody.

In the Americas and specially in Brazil, the Loxosceles intermedia, Loxosceles gaucho and Loxosceles laeta are the three most medically relevant brown spider species, and whose bites can lead to the condition known as loxoscelism. Here, we report the development of a tool capable of identifying a common epitope amongst Loxosceles sp. venom's toxins. A murine monoclonal antibody (LmAb12) and its recombinant fragments (scFv12P and diabody12P) have been produced and characterized. This antibody and its recombinant constructs were able to recognize proteins of Loxosceles spider venoms with specificity. The scFv12P variant was also able to detect low concentrations of Loxosceles venom in a competitive ELISA assay, displaying potential as a venom identification tool. The primary antigenic target of LmAb12 is a knottin, a venom neurotoxin, that has a shared identity of 100 % between the L. intermedia and L. gaucho species and high similarity to L. laeta. Furthermore, we observed LmAb12 was able to partially inhibit in vitro hemolysis, a cellular event typically induced by the Loxosceles sp. venoms. Such behavior might be due to LmAb12 cross-reactivity between the antigenic target of LmAb12 and the venom's dermonecrotic toxins, the PLDs, or even the existence of synergism between these two toxins.

Protective effects of novel diazepinone derivatives in snake venom induced sterile inflammation in experimental animals.

Snake envenomation leads to the formation of damage-associated molecular patterns (DAMPs), which are mediated by endogenous intracellular molecules. These are recognized by pattern-recognition receptors (PRRs) and can induce sterile inflammation. AIMS: In the present study, we aim at understanding the mechanisms involved in DAMPs induced sterile inflammation to unravel the novel therapeutic strategies for treating snake bites. The potential of benzodiazepinone derivatives to act against snake venom induced inflammation has been explored in the present investigation. MAIN METHODS: Three compounds VA 17, VA 43 and PA 03 were taken from our library of synthetic compounds. Oxidative stress markers such as lipid peroxidation, superoxide and nitric oxide were measured along with the analysis of DAMPs (IL6, HMGB1, vWF, S100b and HSP70). These compounds have been docked using molecular docking against the snake venom PLA2 structure (PDB code: 1OXL). KEY FINDINGS: The compounds have been found to effectively neutralize viper and cobra venoms induced lethal activity both ex vivo and in vivo. The compounds have also neutralized the viper venom induced hemorrhagic, coagulant, anticoagulant reactions as well as inflammation. The fold of protection have always been found to be higher in case of ex vivo than in in vivo. These compounds have neutralized the venom induced DAMPs as exhibited by IL6, HMGB1, vWF, S100b and HSP70. The fold of neutralization is found to be higher in VA 43. SIGNIFICANCE: The identified compounds could be used as potential candidates for developing treatment of snakebites in areas where antiserums are not yet available.

The molecular basis of venom resistance in the non-venomous snake Sinonatrix annularis.

Various snake species and snake predators have natural neutralization against snake toxins, which their antidotal abilities are commonly attributed to the intrinsic inhibitors produced by the liver, e.g., phospholipase A2 inhibitor (PLI) and metalloproteinase inhibitor (SVMPI). Sinonatrix annularis was found to possess broad-spectrum neutralization to different snake venoms in our lab. Although the anti-venom compound PLIγ has been previously characterized in our laboratory, the mechanism of resistance of S. annularis to snake venoms remains obscure. In this research, a venom affinity chromatography was constructed by immobilizing D. acutus venom to NHS-agarose beads and applied for antitoxins mining from S. annularis. The binding capacity of the venom column was validated using a self-prepared rabbit antivenom against D. acutus. Serum and liver homogenate of S. annularis were then applied to the column, the bound components were profiled using SDS-PAGE and mass spectrometry. PLIs, snake venom metalloproteins inhibitor (SVMPI), small serum protein (SSP), heat shock proteins, etc were identified. To identify their toxin targets in D. acutus venom, a reverse separation was conducted by coupling the fractionated S. annularis serum proteins to NHS-agarose beads. Fifteen toxins of five families were captured and identified as follows: PLA2s, metalloproteinases, cysteine-rich secretory proteins, snake venom serine proteinases, and C-type lectins. These discoveries increased our understanding of the capacity and mechanism of the natural neutralization of S. annularis to snake venom. These natural inhibitors are medically significant due to their powerful and broad antidotal activities, which may provide alternative and promising drug candidates for snakebite treatment.

A Strategy for Efficient Preparation of Genus-Specific Diagnostic Antibodies for Snakebites.

As said by former United Nations Secretary-General Kofi Annan, "Snakebite is the most important tropical disease you've never heard of." Listed as a priority neglected tropical disease by the World Health Organization, snakebite envenoming (SBE) kills in excess of 125,000 people per year. However, due to the complexity and overlap of snake venom compositions, few reliable venom diagnostic methods for genus-/species-specific identification, which is crucial for successful SBE therapy, are available. Here, we develop a strategy to select and prepare genus-specific snake venom antibodies, which allows rapid and efficient clinical diagnosis of snakebite. Multi-omics approaches are used to choose candidate antigens from snake venoms and identify genus-specific antigenic epitope peptide fragments (GSAEPs) with ideal immunogenicity, specificity, and spatial accessibility. Double-antibody sandwich ELISA kit was established by matching a polyclonal antibody against a natural antigen and a monoclonal antibody that was prepared by natural protein as antigen and can specifically target the GSAEPs. The kit shows the ability to accurately identify venoms from similar genera of Trimeresurus and Protobothrops with a detection limit of 6.25 ng/ml on the snake venoms and a little cross-reaction, thus proving high feasibility and applicability.

Effects of photobiomodulation therapy on the local experimental envenoming by Bothrops leucurus snake.

Bothrops leucurus is the major causative agent of snakebites in Brazil's Northeast. The systemic effects of its venom are effectively neutralized by antivenom therapy, preventing bitten patients' death. However, antivenom fails in neutralizing local effects that include intense pain, edema, bleeding, and myonecrosis. Such effects can lead to irreversible sequels, representing a clinically relevant issue for which there is no current effective treatment. Herein, the effects of photobiomodulation therapy (PBMT) were tested in the local actions induced by B. leucurus venom (BLV) in mice (n = 123 animals in 20 experimental groups). A continuous emission AlGaAs semiconductor diode laser was used in two wavelengths (660 or 780 nm). Mechanical nociceptive thresholds were assessed with the electronic von Frey apparatus. Local edema was determined by measuring the increase in paw thickness. Hemorrhage was quantified by digital measurement of the bleeding area. Myotoxicity was evaluated by serum creatine kinase (CK) activity and histopathological analysis. PBMT promoted anti-hypernociception in BLV-injected mice; irradiation with the 660 nm laser resulted in faster effect onset than the 780 nm laser. Both laser protocols reduced paw edema formation, whether irradiation was performed immediately or half an hour after venom injection. BLV-induced hemorrhage was not altered by PBMT. Laser irradiation delayed, but did not prevent myotoxicity caused by BLV, as shown by a late increase in CK activity and histopathological alterations. PBMT was effective in the control of some of the major local effects of BLV refractory to antivenom. It is a potential complementary therapy that could be used in bothropic envenoming, minimizing the morbidity of these snakebite accidents.

Repurposing Cancer Drugs Batimastat and Marimastat to Inhibit the Activity of a Group I Metalloprotease from the Venom of the Western Diamondback Rattlesnake, Crotalus atrox.

Snakebite envenomation causes over 140,000 deaths every year, predominantly in developing countries. As a result, it is one of the most lethal neglected tropical diseases. It is associated with incredibly complex pathophysiology due to the vast number of unique toxins/proteins present in the venoms of diverse snake species found worldwide. Here, we report the purification and functional characteristics of a Group I (PI) metalloprotease (CAMP-2) from the venom of the western diamondback rattlesnake, Crotalus atrox. Its sensitivity to matrix metalloprotease inhibitors (batimastat and marimastat) was established using specific in vitro experiments and in silico molecular docking analysis. CAMP-2 shows high sequence homology to atroxase from the venom of Crotalus atrox and exhibits collagenolytic, fibrinogenolytic and mild haemolytic activities. It exerts a mild inhibitory effect on agonist-induced platelet aggregation in the absence of plasma proteins. Its collagenolytic activity is completely inhibited by batimastat and marimastat. Zinc chloride also inhibits the collagenolytic activity of CAMP-2 by around 75% at 50 µM, while it is partially potentiated by calcium chloride. Molecular docking studies have demonstrated that batimastat and marimastat are able to bind strongly to the active site residues of CAMP-2. This study demonstrates the impact of matrix metalloprotease inhibitors in the modulation of a purified, Group I metalloprotease activities in comparison to the whole venom. By improving our understanding of snake venom metalloproteases and their sensitivity to small molecule inhibitors, we can begin to develop novel and improved treatment strategies for snakebites.

Delineating the venom toxin arsenal of Malabar pit viper (Trimeresurus malabaricus) from the Western Ghats of India and evaluating its immunological cross-reactivity and in vitro cytotoxicity.

The venom protein components of Malabar pit viper (Trimeresurus malabaricus) were identified by combining SDS-PAGE and ion-exchange chromatography pre-fractionation techniques with LC-MS/MS incorporating Novor and PEAKS-assisted de novo sequencing strategies. Total 97 proteins that belong to 16 protein families such as L-amino acid oxidase, metalloprotease, serine protease, phospholipase A2, 5'-nucleotidase, C-type lectins/snaclecs and disintegrin were recognized from the venom of a single exemplar species. Of the 97 proteins, eighteen were identified through de novo approaches. Immunological cross-reactivity assessed through ELISA and western blot indicate that the Indian antivenoms binds less effectively to Malabar pit viper venom components compared to that of Russell's viper venom. The in vitro cell viability assays suggest that compared to the normal cells, MPV venom induces concentration dependent cell death in various cancer cells. Moreover, crude venom resulted in chromatin condensation and apoptotic bodies implying the induction of apoptosis. Taken together, the present study enabled in dissecting the venom proteome of Trimeresurus malabaricus and revealed the immuno-cross-reactivity profiles of commercially available Indian polyvalent antivenoms that, in turn, is expected to provide valuable insights on the need in improving antivenom preparations against its bite.

Edematic and coagulant effects caused by the venom of Bothrops rhombeatus neutralized by the ethanolic extract of Piper auritum.

ETHNOPHARMACOLOGICAL RELEVANCE: In Colombia, the only authorized treatment to cure snakebite envenomation is with the use of antivenom. The antivenom neutralizes the systemic effects properly, but is not very effective at neutralizing local effects, thus several cases have lead to complications. On the other hand, rural communities turn to the use of plants that are easily accessible and available for basic health care. One of these plants is named Piper auritum (PA), which is traditionally highlighted in some indigenous communities of Antioquia and Chocó. AIM OF THE STUDY: The main objective of this work was to characterize the venom's toxicity by determining the Minimum Edema Dose (MED), the Minimum Coagulant Dose-Plasma (MCD-P), the Minimum Hemorrhagic Dose (MHD) and to determine the neutralizing power of the Total Ethanolic Extract (TEE) from leaves of PA on the localized and systemic effects caused by the Bothrops rhombeatus venom. MATERIALS AND METHODS: To begin, the minimum dose that causes edema-forming, coagulant and hemorrhagic activities was determined. The protocols investigated include coagulant and edematic activities caused by the venom of Bothrops rhombeatus which were neutralized by the TEE of PA. RESULTS: The MCD-P was found to be 0.206 ±â€¯0.026 µg, the MED is the same at 0.768 ±â€¯0.065 µg, and the MHD is 3.553 ±â€¯0.292 µg, which are different from the reports for Bothrops asper and Bothrops ayerbei. Next, a phytochemical screening was done to the TEE where mainly triterpenes, steroids, coumarins, saponins, and lignans were identified. Also present were 43,733 ±â€¯2106 mg AG/g ES of phenols, which are secondary metabolites that are probably responsible for the neutralization of coagulant and edematic activities at rates of 2363.870 µL and 1787.708 µL of extract/mg of venom, respectively. As a comparative parameter, the National Institute Health's (NHI) effective dose of the antivenom was used as a comparative parameter. In addition, we determined the toxicity of the TEE of PA on to Artemia salina, being moderately toxic at 6 and 24 h, while the essential oil of PA at the same observation hours is in the extremely toxic range. CONCLUSIONS: The results reflect that the extract of P. auritum has an anti-inflammatory effect similar to that of the NIH serum. It could be used as a complement of NIH antivenom, using them together so it contributes to effectively reduce inflammation and the socio-economic impact generated by the permanence of a patient victim of snakebite in health centers. CLASSIFICATIONS: Immunological products and vaccines.

Comprehensive Study of the Proteome and Transcriptome of the Venom of the Most Venomous European Viper: Discovery of a New Subclass of Ancestral Snake Venom Metalloproteinase Precursor-Derived Proteins.

The nose-horned viper, its nominotypical subspecies Vipera ammodytes ammodytes ( Vaa), in particular, is, medically, one of the most relevant snakes in Europe. The local and systemic clinical manifestations of poisoning by the venom of this snake are the result of the pathophysiological effects inflicted by enzymatic and nonenzymatic venom components acting, most prominently, on the blood, cardiovascular, and nerve systems. This venom is a very complex mixture of pharmacologically active proteins and peptides. To help improve the current antivenom therapy toward higher specificity and efficiency and to assist drug discovery, we have constructed, by combining transcriptomic and proteomic analyses, the most comprehensive library yet of the Vaa venom proteins and peptides. Sequence analysis of the venom gland cDNA library has revealed the presence of messages encoding 12 types of polypeptide precursors. The most abundant are those for metalloproteinase inhibitors (MPis), bradykinin-potentiating peptides (BPPs), and natriuretic peptides (NPs) (all three on a single precursor), snake C-type lectin-like proteins (snaclecs), serine proteases (SVSPs), P-II and P-III metalloproteinases (SVMPs), secreted phospholipases A2 (sPLA2s), and disintegrins (Dis). These constitute >88% of the venom transcriptome. At the protein level, 57 venom proteins belonging to 16 different protein families have been identified and, with SVSPs, sPLA2s, snaclecs, and SVMPs, comprise ∼80% of all venom proteins. Peptides detected in the venom include NPs, BPPs, and inhibitors of SVSPs and SVMPs. Of particular interest, a transcript coding for a protein similar to P-III SVMPs but lacking the MP domain was also found at the protein level in the venom. The existence of such proteins, also supported by finding similar venom gland transcripts in related snake species, has been demonstrated for the first time, justifying the proposal of a new P-IIIe subclass of ancestral SVMP precursor-derived proteins.

Intact protein mass spectrometry reveals intraspecies variations in venom composition of a local population of Vipera kaznakovi in Northeastern Turkey.

We report on the variable venom composition of a population of the Caucasus viper (Vipera kaznakovi) in Northeastern Turkey. We applied a combination of venom gland transcriptomics, de-complexing bottom-up and top-down venomics. In contrast to sole bottom-up venomics approaches and gel or chromatography based venom comparison, our combined approach enables a faster and more detailed comparison of venom proteomes from multiple individuals. In total, we identified peptides and proteins from 15 toxin families, including snake venom metalloproteinases (svMP; 37.8%), phospholipases A2 (PLA2; 19.0%), snake venom serine proteinases (svSP; 11.5%), C-type lectins (CTL; 6.9%) and cysteine-rich secretory proteins (CRISP; 5.0%), in addition to several low abundant toxin families. Furthermore, we identified intraspecies variations of the venom composition of V. kaznakovi, and find these were mainly driven by the age of the animals, with lower svSP abundance detected in juveniles. On the proteoform level, several small molecular weight toxins between 5 and 8 kDa in size, as well as PLA2s, drove the differences observed between juvenile and adult individuals. This study provides novel insights into the venom variability of V. kaznakovi and highlights the utility of intact mass profiling for fast and detailed comparison of snake venom. BIOLOGICAL SIGNIFICANCE: Population level and ontogenetic venom variation (e.g. diet, habitat, sex or age) can result in a loss of antivenom efficacy against snakebites from wide ranging snake populations. The current state of the art for the analysis of snake venoms are de-complexing bottom-up proteomics approaches. While useful, these have the significant drawback of being time-consuming and following costly protocols, and consequently are often applied to pooled venom samples. To overcome these shortcomings and to enable rapid and detailed profiling of large numbers of individual venom samples, we integrated an intact protein analysis workflow into a transcriptomics-guided bottom-up approach. The application of this workflow to snake individuals of a local population of V. kaznakovi revealed intraspecies variations in venom composition, which are primarily explained by the age of the animals, and highlighted svSP abundance to be one of the molecular drivers for the compositional differences observed.

Vipera berus berus Venom from Russia: Venomics, Bioactivities and Preclinical Assessment of Microgen Antivenom.

The common European adder, Vipera berus berus, is a medically relevant species, which is widely distributed in Russia and thus, is responsible for most snakebite accidents in Russia. We have investigated the toxic and enzymatic activities and have determined the proteomic composition of its venom. Phospholipases A2 (PLA2, 25.3% of the venom proteome), serine proteinases (SVSP, 16.2%), metalloproteinases (SVMP, 17.2%), vasoactive peptides (bradykinin-potentiating peptides (BPPs), 9.5% and C-type natriuretic peptides (C-NAP, 7.8%), cysteine-rich secretory protein (CRISP, 8%) and L-amino acid oxidase (LAO, 7.3%) represent the major toxin classes found in V. b. berus (Russia) venom. This study was also designed to assess the in vivo and in vitro preclinical efficacy of the Russian Microgen antivenom in neutralizing the main effects of V. b. berus venom. The results show that this antivenom is capable of neutralizing the lethal, hemorrhagic and PLA2 activities. Third-generation antivenomics was applied to quantify the toxin-recognition landscape and the maximal binding capacity of the antivenom for each component of the venom. The antivenomics analysis revealed that 6.24% of the anti-V. b. berus F(ab')2 molecules fraction are toxin-binding antibodies, 60% of which represent clinically relevant antivenom molecules.

Venom proteome of Bungarus sindanus (Sind krait) from Pakistan and in vivo cross-neutralization of toxicity using an Indian polyvalent antivenom.

The proteome of the Pakistani B. sindanus venom was investigated with reverse-phase HPLC and nano-ESI-LCMS/MS analysis. At least 36 distinct proteins belonging to 8 toxin protein families were identified. Three-finger toxin (3FTx), phospholipase A2 (including ß-bungarotoxin A-chains) and Kunitz-type serine protease inhibitor (KSPI) were the most abundant, constituting ~95% of total venom proteins. The other toxin proteins of low abundance are snake venom metalloproteinase (SVMP), L-amino acid oxidase (LAAO), acetylcholinesterase (AChE), vespryn and cysteine-rich secretory protein (CRiSP). The venom was highly lethal to mice with LD50 values of 0.04 µg/g (intravenous) and 0.15 µg/g (subcutaneous). The 3FTx proteins are diverse, comprising kappa-neurotoxins, neurotoxin-like protein, non-conventional toxins and muscarinic toxin-like proteins. Kappa-neurotoxins and ß-bungarotoxins represent the major toxins that mediate neurotoxicity in B. sindanus envenoming. Alpha-bungarotoxin, commonly present in the Southeast Asian krait venoms, was undetected. The Indian VINS Polyvalent Antivenom (VPAV) was immunoreactive toward the venom, and it moderately cross-neutralized the venom lethality (potency = 0.25 mg/ml). VPAV was able to reverse the neurotoxicity and prevent death in experimentally envenomed mice, but the recovery time was long. The unique toxin composition of B. sindanus venom may be considered in the formulation of a more effective pan-regional, polyspecific antivenom. BIOLOGICAL SIGNIFICANCE: Bungarus sindanus, an endemic krait species distributed mainly in the Sindh Province of Pakistan is a cause of snake envenomation. Its specific antivenom is, however, lacking. The proteomic study of its venom revealed a substantial presence of κ-bungarotoxins and ß-bungarotoxins. The toxin profile corroborates the potent neurotoxicity and lethality of the venom tested in vivo. The heterologous Indian VINS polyvalent antivenom (VPAV) cross-reacted with B. sindanus venom and cross-neutralized the venom neurotoxicity and lethality in mice, albeit the efficacy was moderate. The findings imply that B. sindanus and the phylogenetically related B. caeruleus of India share certain venom epitopes. Research should be advanced to improve the efficacy spectrum of a pan-regional polyspecific antivenom.

Proteomics, functional characterization and antivenom neutralization of the venom of Pakistani Russell's viper (Daboia russelii) from the wild.

The venom proteome of wild Pakistani Russell's viper (Daboia russelii) was investigated through nano-ESI-LCMS/MS of the reverse-phase HPLC fractions. A total of 54 venom proteins were identified and clustered into 11 protein families. Phospholipase A2 (PLA2, 63.8%) and Kunitz-type serine protease inhibitor (KSPI, 16.0%) were most abundant, followed by snake venom serine protease (SVSP, 5.5%, mainly Factor V activating enzyme), vascular endothelial growth factor (VEGF, 4.3%), snake venom metalloproteinase (SVMP, 2.5%, mainly Factor X activating enzyme) and phosphodiesterase (PDE, 2.5%). Other minor proteins include cysteine-rich secretory protein (CRiSP), snake venom C-type lectin/lectin-like protein (snaclec), nerve growth factor, L-amino acid oxidase and 5'-nucleotidase. PLA2, KSPI, SVSP, snaclec and SVMP are hemotoxic proteins in the venom. The study indicated substantial venom variation in D. russelii venoms of different locales, including 3 Pakistani specimens kept in the USA. The venom exhibited potent procoagulant activity on human plasma (minimum clotting dose = 14.5 ng/ml) and high lethality (rodent LD50 = 0.19 µg/g) but lacked hemorrhagic effect locally. The Indian VINS Polyvalent Antivenom bound the venom immunologically in a concentration-dependent manner. It moderately neutralized the venom procoagulant and lethal effects (normalized potency against lethality = 2.7 mg venom neutralized per g antivenom). BIOLOGICAL SIGNIFICANCE: Comprehensive venom proteomes of D. russelii from different locales will facilitate better understanding of the geographical variability of the venom in both qualitative and quantitative terms. This is essential to provide scientific basis for the interpretation of differences in the clinical presentation of Russell's viper envenomation. The study revealed a unique venom proteome of the Pakistani D. russelii from the wild (Indus Delta), in which PLA2 predominated (~60% of total venom proteins). The finding unveiled remarkable differences in the venom compositions between the wild (present study) and the captive specimens reported previously. The integration of toxicity tests enabled the correlation of the venom proteome with the envenoming pathophysiology, where the venom showed potent lethality mediated through coagulopathic activity. The Indian VINS Polyvalent Antivenom (VPAV) showed binding activity toward the venom protein antigens; however the immunorecognition of small proteins and PLA2-dominating fractions was low to moderate. Consistently, the antivenom neutralized the toxicity of the wild Pakistani Russell's viper venom at moderate efficacies. Our results suggest that it may be possible to enhance the Indian antivenom potency against the Pakistani viper venom by the inclusion of venoms from a wider geographical range including that from Pakistan into the immunogen formulation.

First report of the characterization of a snake venom apyrase (Ruviapyrase) from Indian Russell's viper (Daboia russelii) venom.

A novel apyrase from Russell's viper venom (RVV) was purified and characterized, and it was named Ruviapyrase (Russell's viper apyrase). It is a high molecular weight (79.4 kDa) monomeric glycoprotein that contains 2.4% neutral sugars and 58.4% N-linked oligosaccharides and strongly binds to Concanavalin A. The LC-MS/MS analysis did not identify any protein in NCBI protein database, nevertheless some de novo sequences of Ruviapyrase showed putative conserved domain of apyrase superfamily. Ruviapyrase hydrolysed adenosine triphosphate (ATP) to a significantly greater extent (p < .05) as compared to adenosine diphosphate (ADP); however, it was devoid of 5'-nucleotidase and phosphodiesterase activities. The Km and Vmax values for Ruviapyrase towards ATP were 2.54 µM and 615 µM of Pi released min-1, respectively with a turnover number (Kcat) of 24,600 min-1. Spectrofluorometric analysis demonstrated interaction of Ruviapyrase with ATP and ADP at Kd values of 0.92 nM and 1.25 nM, respectively. Ruviapyrase did not show cytotoxicity against breast cancer (MCF-7) cells and haemolytic activity, it exhibited marginal anticoagulant and strong antiplatelet activity, and dose-dependently reversed the ADP-induced platelet aggregation. The catalytic activity and platelet deaggregation property of Ruviapyrase was significantly inhibited by EDTA, DTT and IAA, and neutralized by commercial monovalent and polyvalent antivenom.

In vitro cleavage of bioactive peptides by peptidases from Bothrops jararaca venom and its neutralization by bothropic antivenom produced by Butantan Institute: Major contribution of serine peptidases.

In Brazil, envenomation by Bothrops pitvipers is responsible for over 73% of snakebites, and their venom is a rich source of proteolytic enzymes. Most studies have demonstrated that Bothrops jararaca venom acts on macromolecular substrates, causing an imbalance in the victim's hemostatic system. In contrast, fewer studies have examined the proteolytic activity on small molecules such as peptides. In this study, we used a set of bioactive peptides (insulin B chain, Met-enkephalin, Leu-enkephalin, neuropeptide Y, peptide YY, pancreatic polypeptide, substance P and somatostatin) to identify new peptide substrates for the metallopeptidases and serine peptidases from the B. jararaca venom. The majority of these peptides were substrates for the venom, but neuropeptide Y and pancreatic polypeptide presented higher hydrolyses rates. Although most of the peptides were simultaneously substrates for both classes of proteases, serine peptidases were the most active. Substance P was an exclusive substrate for metallopeptidases, while somatostatin was a selective substrate for serine peptidases. The neutralizing efficacy of the bothropic antivenom produced by the Butantan Institute was also assessed and found to totally prevent substance P hydrolysis, whereas somatostatin cleavage was not inhibited. Thus, the antivenom effectively inhibited metallopeptidase activity, but did not neutralize some of the serine peptidases. These results indicate that, in addition to cleaving proteins, the proteolytic enzymes from this venom also hydrolyze bioactive peptides, and this peptidase activity could effectively contribute to some of the many dire manifestations of envenomation.