Category: Diet

Snakebite venom inhibition

Snakebite venom inhibition

Subsequent studies led Vsnom Ferreira and colleagues to discover a set of nine peptides in the venom of vvenom that inhihition the Snakebote of bradykinin, Holistic physical therapy bradykinin potentiating Snakebite venom inhibition BPFs 9899 Heart J. Sousa, L. a Myotoxin I MT-Ia strongly myotoxic phospholipase A2 PLA2 from the venom of a terciopelo viper Bothrops asperattached to the sarcolemma. The distribution of the proportion of the most abundant protein families is shown in Supplementary Fig. Ainsworth S, Petras D, Engmark M, et al. Snakebite venom inhibition


Snakebite: How education and investment in anti venom treatments are a critical step in saving lives

Snakebite venom inhibition -

Neutralisation experiments with the two EAVs revealed marked inhibition of the coagulopathic toxins found in the majority of the coagulopathic venom immunogens Figs 4F and S6.

With the exception of C. subminiatus and E. carinatus Figs 4F and S6. atrox , EAV 2 also lacked inhibitory potency against the venom of E. Both venom immunogen mixtures were also found to be potently procoagulant, and this venom activity was effectively neutralised by both EAV 1 and EAV 2, although inhibition was greatest with EAV 1, irrespective of the venom immunogen mixture However, despite neither EAV by itself affecting normal coagulation, the addition of EAV 1 in the presence of the two venom immunogen mixtures resulted in a delay of clotting, and therefore net anticoagulant activity S2E Fig.

The reason for this remains unclear, but might indicate potent inhibition of procoagulant venom toxins, but ineffective inhibition of anticoagulant venom toxins, as seen elsewhere [ 43 ]. Contrastingly, EAV 2 effectively inhibited venom immunogen stimulated coagulation to control levels, though this same phenomenon was observed for both EAVs against many of the individual venom immunogens S6 Fig.

The SAIMR polyvalent antivenom was ineffective against all of the individual venoms and venom immunogen mixtures tested, likely as the result of no procoagulant snake venoms being used as immunogens during the production of this product Fig 4F. Following promising evidence of in vitro immunological cross-reactivity and venom neutralisation with the two EAVs, we next assessed their preclinical efficacy against a subset of haemotoxic venoms using a variation of the World Health Organization-recommended assay for assessing antivenom efficacy, the murine median effective dose [ 49 ].

We selected eight venoms for preclinical assessment; four that were included in both venom immunogen mixtures B. asper , C. rhodostoma , E. ocellatus and D. russelii , two that were only included in the immunogen mixture used to generate EAV 2 B.

arietans and E. carinatus and, to assess whether the breadth of antibodies generated by the diverse immunogen mixtures stimulated broad paraspecific efficacy, two haemotoxic venoms that were not used in either immunogen mixture V.

ammodytes and L. muta Table 2. Prior to performing antivenom efficacy experiments, the LD 50 of L. muta and C. rhodostoma venoms were determined, resulting in LD 50 s of 6.

The LD 50 s for the remaining species were sourced from previously published studies Table 2. Our dose-matched pilot in vivo findings demonstrated that, despite a reduction in the number of venoms used in the immunogen mixture 7 vs 12 , EAV 1 outperformed EAV 2 in terms of preclinical efficacy superior vs four venoms; equipotent vs two venoms; inferior vs one venom Fig 5.

Indeed, EAV 1 effectively prevented venom-induced lethality for the duration of the experiment against three of the four venoms used as immunogens B.

asper , E. russelii , and two of the four venoms tested that were not present in the immunising mixture B. arietans and V. ammodytes in this model Fig 5. Contrastingly, none of the venoms present in the immunogen mixture used to raise EAV 2 were fully neutralised i.

asper , B. carinatus venom Fig 5. The result with this latter venom proved to be the only example of superior preclinical efficacy exhibited by EAV 2 over EAV 1 four vs one survivor, respectively. russelii— arguably the two most medically-important species tested Fig 5.

Both EAVs exhibited comparable, limited protection against the lethal effects of C. rhodostoma venom two of five experimental animals survived the duration of the experiment , which correlates with our earlier observations of reduced immunological cross-reactivity to the toxins found in this venom see Fig 3 , and suggests that higher therapeutic doses than tested here are likely required to provide full protection.

Identical and contrasting efficacy observations were also observed in respect of the remaining two venoms tested, neither of which was used as an immunogen to generate either EAV; both antivenoms failed to provide any protection against the lethal effects of L.

muta venom, though both EAVs exhibited potent paraspecific preclinical efficacy against V. ammodytes Fig 5. To place these findings into context, we compared our preclinical efficacy data against that obtained using our antivenom control, SAIMR polyvalent, which is manufactured using venom from a variety of African vipers and elapids as immunogens.

As anticipated, both EAVs outperformed SAIMR polyvalent, with only EAV 1 proving inferior against the lethal effects of E. carinatus venom, while EAV 2 was only outperformed in experiments using E.

ocellatus and B. arietans venoms Fig 5. Groups of five mice were challenged intravenously with 5 x LD 50 doses of each venom purple lines or venom preincubated for 30 min at 37°C with EAV 1 blue or EAV 2 red , or the SAIMR polyvalent control antivenom green.

All experimental animals were monitored for 6 hours and survival times recorded. There are a number of major obstacles that need to be tackled to ensure the effective treatment of tropical snakebite victims. These include, but are not limited to, addressing the poor affordability, lack of availability and the often limited cross-species neutralising potency of commercial antivenom [ 19 , 50 ].

Currently, all existing antivenoms are manufactured for specific geographical regions, typically parts of a continent, or specific countries within a continent, leading to a highly fragmented drug market [ 3 , 50 ]. Ultimately, the venoms used as immunogens for antivenom production dictate the breadth of snake species efficacy of those products, and it has been suggested that simply adding additional venoms to the immunogen mixture to increase paraspecific efficacy could be potentially counter-productive by diluting those antibodies specific to the toxins delivered during the bite by any particular snake [ 11 , 19 , 32 ].

Despite the challenge posed by venom variation, in the current pilot study we explored the feasibility of generating a globally efficacious conventional antivenom against haemotoxic envenoming, based on encouraging recent findings [ 42 , 52 ]. To explore the potential trade-off between antivenom dose efficacy and the number of venoms included in the immunising mixture, we generated two different EAVs, each consisting of ovine polyclonal antibodies, but using either seven or twelve different venoms in the immunising mixture.

Time course analysis of the immunological responses of the sheep over the duration of immunisation 42 weeks , determined by ELISA analysis of serum samples, demonstrated highly comparable responses to the different immunogen mixtures, and high titres of cross-reactive antibodies resulting by the end of the immunising period.

Thereafter, IgG was purified from each week sample for use in in depth immunological and venom neutralisation experiments. Despite considerable differences in the two different immunogen mixtures, ELISA and immunoblotting experiments revealed that both EAVs exhibited high levels of cross-reactivity and detected the vast majority of the variable protein constituents found in each of the 12 snake venoms Figs 2 and 3A.

The most noticeable exceptions to this were i disparities in the general intensity of immunological cross-reactivity and ii differences in the recognition of lower molecular weight toxins i. EAV 2 outperformed EAV 1 on both counts, despite D. typus venom being included in both immunising mixtures, and these findings correlated with the binding levels observed in the end point ELISA experiments.

Contrastingly, the medium to high molecular weight toxins found across all of the various venoms e. the viperid venoms not used as venom immunogens.

The other major and, perhaps more surprising, observation from the immunological assays was the seemingly low levels of antibody binding, coupled with reduced binding avidities, of both EAVs against C. rhodostoma venom Fig 3. This was despite this venom being used as an immunogen to generate both antivenoms, and there being clear immunological cross-reactivity observed in the immunoblotting experiments Fig 2.

Notwithstanding these immunological observations, in vitro neutralisation assays revealed that both EAVs inhibited C. rhodostoma SVMP and coagulopathic venom activities in a manner highly comparable to the other venoms tested Fig 4D and 4F. Indeed, broadly speaking, both antivenoms exhibited high levels of SVMP-inhibition against each of the venoms tested, and comparable findings were observed in terms of coagulotoxin inhibition, with the exception that EAV 2 provided little neutralisation effect against E.

ocellatus venom at the dose tested, while neither antivenom inhibited C. atrox venom-induced coagulation. The inhibitory effects of both antivenoms against SVSP toxin activity was far more variable Fig 4B , suggesting that both the titre and specificity of the resulting antibodies generated against these toxins may differ considerably between the two antivenoms, despite them sharing a number of snake venoms as immunogens.

One of the major challenges with interpreting the results described above in terms of predicted antivenom efficacy is that no single in vitro experiment accurately recapitulates the complex interaction of variable toxins on multiple physiological pathways following a snakebite. Indeed, some previous studies have demonstrated that high levels of immunological binding do not necessary result in effective neutralisation of venom toxicity in vivo [ 24 , 32 ].

Thus, to preliminarily assess the relative preclinical efficacies of the two EAVs described here, we employed an ethically-refined version of a previous described murine model of envenoming [ 49 ] and, to robustly investigate paraspecific neutralising capabilities, we used a diverse array of challenge venoms, including two that were not used as venom immunogens for generating either antivenom.

Despite highly comparable immunological and in vitro inhibitory characteristics, our in vivo studies demonstrated that EAV 1 exhibited increased in vivo preclinical efficacy than EAV 2, despite fewer venoms being used in the immunogen mixture.

In addition, EAV 1 provided some protection against lethality by C. rhodostoma and E. carinatus venoms. The former finding seems likely to reflect the low immunological binding observed against this venom, despite it being an immunogen, while the latter finding is more promising since this venom was not a venom immunogen, yet four of five experimental animals were protected in a paraspecific manner.

EAV 1 was, however, completely ineffective at protecting against the effects of L. muta venom, a venom that was not included in either immunising mixture. These findings, which were also observed for EAV 2, suggest that L. muta venom contains lethal toxins sufficiently distinct from, or in much greater abundances than, those found in the venoms used as immunogens to prevent any degree of paraspecific neutralisation.

Our in vivo findings revealed that EAV 2 failed to provide complete protection against venom-induced lethality stimulated by any of the six venoms used as immunogens at the therapeutic doses tested Fig 5.

A degree of protection was observed against five of these six venoms i. some experimental animals survived to the end of experiment , suggesting that the dose efficacy of EAV 2 is lower than that of EAV 1, and that perhaps with increased antivenom doses full protection might have been conveyed.

Crucially, these findings provide experimental evidence that increasing the number of venoms in an immunogen mixture could perhaps have detrimental effects on efficacy, likely by diluting the proportional amount of venom neutralising antibodies specific to any particular venom via the addition of numerous immunogens i.

those venom toxins unique to the additional venoms used for immunisation. However, it should be stressed that our in vitro immunological experiments did not predict reductions in antibody binding to the various crude venoms, and thus more detailed follow up experiments involving purified toxins or chromatographically separated toxin fractions i.

antivenomic-type approaches [ 58 , 59 ] are ultimately required to better understand the basis for these efficacy observations. Nonetheless, we suggest that a core set of venoms representing broad toxin diversity may be sufficient for the generation of antibodies that cross-react with and neutralise venoms in a paraspecific manner.

Experimentally identifying the optimal venom mixture for immunisation remains challenging though, particularly since using too few venoms seems likely to result in reduced paraspecific efficacy, whereas too many may cause reductions in dose efficacy, as observed here.

Despite these findings, there are a number of limitations with the described work. For example, due to financial constraints, only a single animal was immunised to generate each EAV, and thus differences between the efficacy of the two antivenoms could be partially due to the different immune responses of these animals during immunisation.

However, the highly comparable serological responses observed during the immunisation time course Fig 1 , and the comparable immunological cross-reactivity observed in ELISA and western blotting experiments Figs 2 and 3 , suggest that this is unlikely to be a major confounder of this study, though future iterations of attempts to generate pathology-focused experimental antivenoms should, where feasible, increase the number of immunisation animals to offset this risk.

Another limitation relates to the restricted preclinical efficacy testing performed, which was a result of both ethical and financial constraints imposed on the study. Furthermore, animal monitoring of acute envenoming was only undertaken for 6 hours due to ethical considerations.

Additional preclinical testing, involving those venoms omitted from this pilot work, and using varying antivenom doses to generate median effective dose data [ 48 ] for each venom, would provide a more complete picture of the efficacy and limitations of each EAV.

However, due to evidence of incomplete neutralisation observed in these pilot studies, and the apparent need to further optimise the immunisation process to increase the paraspecific efficacy of these pathology-specific antivenoms, such additional experiments in more robust preclinical efficacy models [ 60 ] cannot be justified at this time.

Despite these limitations, this pilot study provides at least some evidence that generating anti-haemotoxicity antivenoms with global efficacy may be a viable future strategy for tackling snakebite envenoming, analogous with recent findings exploring the potential of pan-continentally efficacious anti-neurotoxicity antivenoms [ 41 , 42 ].

Should further optimisation work address these current shortcomings, pathology-specific antivenoms may offer antivenom manufacturers with increased economies of scale to produce more sustainable conventional polyspecific antivenom products in the short to medium term to address the current therapeutic vacuum relating to tropical snakebite.

The EAVs EAV 1 and EAV 2 , commercial SAIMR polyvalent antivenom positive control and normal sheep IgG and normal horse IgG negative controls were serially diluted fivefold and tested by ELISA against each of the haemotoxic venoms used as immunogens. Venoms from B. jararaca , E. ocellatus , C.

rhodostoma , D. typus , D. russelii were used as immunogens for EAV 1, while all of the venoms shown were used as immunogens for EAV 2.

Error bars represent standard deviation SD of duplicate measurements. A End-point titration ELISA analysis of immunological binding between the EAVs and the two venom immunogen mixtures. B Chaotropic ELISA showing the relative avidity of the EAVs to the two venom immunogen mixtures in the presence of increasing molarities of the chaotropic agent ammonium thiocyanate NH 4 SCN.

For both panels A and B, data points represent the mean of duplicate readings and error bars represent standard deviations. C Quantification of the neutralisation of snake venom serine protease SVSP activity measured via chromogenic assay. Data points represent the rate of substrate cleavage measured kinetically.

D Quantification of the neutralisation of snake venom metalloproteinase SVMP activity measured via fluorescent assay. Data points represent area under the curve AUC of kinetic measurements.

E Quantification of the neutralisation of coagulopathic venom toxins via absorbance-based kinetic plasma clotting assay. negative control , with readings above the line promoting clotting i.

procoagulant , and those below inhibiting clotting i. The data represents percentage areas under the curve of the venom-only control area under the curve.

For C , D and E the data points represent the mean of triplicate readings and error bars represent SEM. Throughout, the antivenoms used consisted of the two EAVs EAV 1 and EAV 2 , the SAIMR polyvalent antivenom control positive control , and for the immunological assays, normal sheep and horse IgG were used as negative controls.

PBS was used as the negative control in the functional assays, and Bitis arietans and Echis ocellatus venom were used as the positive controls in the serine protease and metalloproteinase assays, respectively. EAVs EAV 1 and EAV 2 , commercial SAIMR polyvalent antivenom positive control and normal sheep IgG and normal horse IgG negative controls all at , dilutions were tested in the presence of increasing concentrations of ammonium thiocyanate M.

The SVSP venom activity 1 μg of each individual venom immunogen is displayed as the rate of substrate conversion kinetic readings at nm over 30 mins.

The antivenoms used consisted of the two EAVs EAV 1 and EAV 2 and the commercial SAIMR polyvalent antivenom as an antivenom comparator, and PBS was used as the negative control.

The positive control used across all experiments was Bitis arietans venom. Data points represent means of triplicate calculated rates, and error bars represent standard error of the mean SEM.

The SVMP venom activity 1 μg of each individual venom immunogen is displayed as area under the kinetic curve AUC of fluorescence nm excitation and nm emission over 40 mins.

The antivenoms used consisted of the two EAVs EAV 1 and EAV 2 and the SAIMR polyvalent antivenom as an antivenom comparator, and PBS was used as the negative control. The positive control used across all experiments was Echis ocellatus venom. The data displayed represents the mean AUC of triplicate measurements and error bars represent standard error of the mean SEM.

The data displayed shows the percentage of plasma clotting normalised to venom only readings of the means of triplicate area under the clotting curve measurements absorbance plotted over time , with error bars represent the standard error of the mean SEM.

The dashed line represents normal clotting i. negative control [PBS] readings. The antivenoms used consisted of the two EAVs EAV 1 and EAV 2 and the SAIMR polyvalent antivenom as an antivenom comparator.

The authors wish to thank Paul Rowley for expert snake husbandry at LSTM, and Ig-Innovations Ltd, UK for the commercial generation of ovine antibodies. Article Authors Metrics Comments Media Coverage Reader Comments Figures.

Correction 2 Jun Alomran N, Alsolaiss J, Albulescu LO, Crittenden E, Harrison RA, et al. Abstract Background Snakebite is a neglected tropical disease that causes high global rates of mortality and morbidity.

Author summary Snakebite is a major public health concern that causes extensive death and disability in the tropical world. Introduction Snakebite envenoming is classified as a neglected tropical disease NTD , and as such is most prevalent in tropical and subtropical regions of the world, particularly rural regions [ 1 , 2 ].

Materials and methods Ethics statement All animal experiments were conducted using protocols approved by the Animal Welfare and Ethical Review Boards of the Liverpool School of Tropical Medicine and the University of Liverpool. Antivenom generation Venom immunogens.

Download: PPT. Table 1. The venoms used in the immunisation mixtures for the generation of experimental antivenoms EAVs. Ovine immunisation. Immunoglobulin purification. In vitro immunological assays SDS-PAGE gel electrophoresis and immunoblotting.

End-point titration by enzyme-linked immunosorbent assay ELISA. Relative avidity by ELISA. In vitro functional assays Serine protease assay. Metalloproteinase assay. Plasma coagulation assay. In vivo efficacy assays In vivo venom median lethal dose.

Table 2. The murine intravenous median lethal dose LD 50 of the haemotoxic snake venoms used in this study. In vivo antivenom efficacy. Results Monitoring seroconversion during immunisation To assess the specificity of the immune response towards the venom immunogens over the course of the immunisation period, serum samples were collected every four weeks and assessed via ELISA to quantify antibody binding levels.

Fig 1. Time course analysis of the immunological cross-reactivity of ovine serum to the immunogen mixtures over 42 weeks of immunisation. Immunological cross-reactivity of the experimental antivenoms To visualise the immunological recognition of the two EAVs against the venom proteins found in the 12 venoms used in the immunisation process, we subjected each venom and each venom immunogen mixture to reduced SDS-PAGE gel electrophoresis and western blotting.

Fig 2. SDS-PAGE gel electrophoretic profiles of the individual venoms and the venom immunogen mixtures and their immunological recognition by experimental antivenoms.

Fig 3. A comparative summary of the immunological binding observed between the two experimental antivenoms and the individual venoms and venom immunogen mixtures as measured by end-point and avidity ELISA. In vitro venom neutralisation by the EAVs While high levels of immunological binding appear to be a prerequisite for antivenom efficacy, immunological binding does not necessarily result in toxin neutralisation.

Fig 4. The in vitro venom activity of the venom immunogens and quantification of venom neutralisation by the experimental antivenoms. In vivo neutralisation of venom lethality Following promising evidence of in vitro immunological cross-reactivity and venom neutralisation with the two EAVs, we next assessed their preclinical efficacy against a subset of haemotoxic venoms using a variation of the World Health Organization-recommended assay for assessing antivenom efficacy, the murine median effective dose [ 49 ].

Fig 5. Kaplan—Meier survival curves for murine preclinical efficacy experiments using representative, geographically diverse, haemotoxic snake venoms and the experimental antivenoms. Discussion There are a number of major obstacles that need to be tackled to ensure the effective treatment of tropical snakebite victims.

Supporting information. S1 Fig. End point titration ELISA analyses of immunological binding between the two experimental antivenoms EAVs and each of the individual venoms used as immunogens. s TIF. S2 Fig. In vitro immunological and neutralisation results for EAVs against each of the venom immunogen mixtures.

S3 Fig. The relative avidity of the EAVs to the venoms used as immunogens measured by immunological binding in the presence of increasing molarities of the chaotropic agent ammonium thiocyanate NH 4 SCN.

S4 Fig. The serine protease activity of the individual venom immunogens and their inhibition by the EAVs as measured by kinetic chromogenic assay. S5 Fig. The metalloproteinase activity of the individual venom immunogens and their inhibition by the EAVs as measured by kinetic fluorescent assay.

S6 Fig. The coagulopathic activity of the individual venom immunogens and their inhibition by the EAVs as measured by plasma coagulation assay. S1 Data. A multi-tabbed excel file containing the raw data presented in the various figures of the manuscript.

s XLSX. Acknowledgments The authors wish to thank Paul Rowley for expert snake husbandry at LSTM, and Ig-Innovations Ltd, UK for the commercial generation of ovine antibodies. Nevertheless, the in vitro efficacy of nafamostat demonstrated here justified its evaluation in in vivo models of envenoming to select the most efficacious mixture of inhibitors.

b Neutralization of SVSP venom activity by the serine protease-inhibitor nafamostat. We used an established in vivo model of envenoming 41 , 42 to test the efficacy of small molecule toxin inhibitors. We first tested the ability of marimastat, varespladib and nafamostat as solo therapies to prevent venom-induced lethality in mice challenged with a 2.

We selected this snake venom and venom dose as our initial model based upon its medical importance and results from our recent work exploring the preclinical venom-neutralizing efficacy of metal chelators All five of the experimental animals receiving only E.

Drug-only controls are presented as black dashed lines at the top of each graph none of the drugs exhibited any observable toxicity at the given doses. ocellatus venom 2. c Quantified thrombin-antithrombin TAT levels in the envenomed animals from a and b.

Where the time of death was the same within experimental groups e. The data displayed represents means of the duplicate technical repeats plus SDs. carinatus h Quantified TAT levels in the envenomed animals from d to g , with data presented as described for c. We next tested the preclinical efficacy of two different inhibitor combinations against the lethal effects of E.

Both toxin inhibitor mixtures resulted in survival of all experimental animals until the end of the experiment Fig. We next assessed markers of venom-induced coagulopathy in the envenomed animals via the quantification of thrombin-antithrombin TAT levels, a proxy for thrombin generation, in plasma collected following euthanasia.

In line with previous reports 25 , 28 , TAT levels correlated well with treatment efficacy. While animals in the venom-only group displayed very high TAT levels mean of TAT levels in the marimastat solo therapy group were also reduced In combination, these findings suggest that marimastat is likely responsible for much of the observed efficacy against the lethal effects of E.

ocellatus venom, but that small molecule combinations with additional toxin inhibitors provide superior preclinical efficacy than treatment with marimastat alone. We next investigated whether the two inhibitor combination therapies were equally effective against the other viper venoms tested in vitro, as these venoms exhibit highly variable toxin compositions in comparison with the SVMP-rich toxin profile of E.

ocellatus Fig. We adopted the same approach as described above, and intravenously challenged groups of experimental animals with 2. These results of these studies demonstrate the therapeutic potential of small molecule inhibitors, as despite extensive venom differences, we found that the dual mixture of marimastat and varespladib protected mice from the lethal effects of all four venoms for the duration of the experiment Fig.

russelii venom Fig. TAT levels increased in all venom-only groups Fig. arietans venom—a finding in line with our in vitro data suggesting that this venom has little coagulopathic activity Fig.

TAT levels were consistently reduced in the experimental animals treated with the two inhibitor mixtures, resulting in Comparable preclinical efficacy between the MV and MVN inhibitor combinations against a variety of medically important viper venoms suggests that the SVSP-inhibitor nafamostat does not contribute substantially to venom neutralization.

Because i SVSP-inhibitors such as nafamostat can induce off-target effects by interacting with serine proteases found in the coagulation cascade, ii the inclusion of every additional molecule in an inhibitory therapeutic combination substantially increases regulatory hurdles for future translation, and iii the inhibition of SVMP and PLA 2 toxins appears sufficient to protect against lethality caused by a diverse array of viper venoms, we decided to proceed with the marimastat and varespladib combination as our lead candidate for testing in more therapeutically challenging preclinical models of envenoming.

To this end, we injected venom from each of the five viper species in doses equivalent to at least 5× the intravenous i. For E.

arietans venoms we challenged mice with 5× i. LD 50 46 and D. russelii 13× i. Survival of mice receiving: a E. The drug-only control is presented as a black dashed line at the top of each graph no toxicity was observed at the given dose. f Quantified thrombin-antithrombin TAT levels in the envenomed animals from a to e.

Quantified TAT levels from the envenomed animals correlated with survival, with those receiving the inhibitor mixture exhibiting Contrastingly, quantification of soluble thrombomodulin, a marker of endothelial cell damage, was only elevated in B.

These findings suggest that, in addition to protecting against the lethal effects of the various viper venoms, the marimastat and varespladib therapeutic combination is capable of preventing coagulopathy, and in the case of B.

asper , inhibiting toxins acting to disrupt certain components of the endothelium. Although conventional polyclonal immunoglobulin-based antivenoms save thousands of lives each year, their lack of specificity, poor cross-species efficacy, reliance on delivery in clinical settings and low affordability severely hamper their accessibility and utility for treating tropical snakebite victims 1 , Consequently, new strategies capable of circumventing variation in snake venom composition to deliver broad neutralization across snake species, while simultaneously improving the safety, affordability and storage logistics of treatment, are urgently needed 3 , Approaches showing signs of promise include the rational design of immunogens to improve the neutralizing breadth of conventional products 48 , the selection of human or humanized toxin-specific monoclonal or oligoclonal antibodies 49 , 50 , and the use of small molecule inhibitors specific to certain toxin families 12 , such as the PLA 2 -inhibitor varespladib 22 , 26 , 27 and the metal chelator DMPS Small molecule toxin inhibitors offer a number of desirable characteristics over existing snakebite therapies, including desirable specificity, potent dose-efficacy, higher tolerability, greater stability and superior affordability These characteristics, combined with their oral formulation, provide an opportunity to explore their utility as prehospital treatments for snakebite, thereby circumventing one of the major challenges faced by impoverished snakebite victims, who have great difficulty in rapidly accessing the secondary and tertiary healthcare facilities where current treatments are held.

Here, we show that a small molecule mixture consisting of the inhibitors marimastat and varespladib, which are directed against the hemotoxicity-inducing SVMP and PLA 2 toxin families, provides preclinical protection against lethality caused by a geographically diverse array of medically important viper venoms that differ considerably in their toxin compositions.

Due to their importance in many snake venoms, we first rationally selected an inhibitory molecule capable of abrogating the activity of SVMP toxins. In vitro SVMP and coagulation assays convincingly demonstrated that the Phase 2-approved peptidomimetic hydroxamate inhibitors batimastat and marimastat provided superior venom neutralization over the metal chelators DMPS and dimercaprol Figs.

Despite previous reports of batimastat exhibiting increased efficacy over marimastat in preventing venom-induced local hemorrhage 23 , we found both drugs to be equipotent in vitro.

We selected marimastat as our candidate for in vivo efficacy experiments due to a number of desirable characteristics that make it amenable for a future field intervention for snakebite, specifically its oral vs intraperitoneal route of administration, and its increased solubility and tolerability compared to batimastat Indeed, these characteristics seemingly contributed to the demise of batimastat during development, although both drugs were ultimately discontinued following lack of efficacy in Phase 3 clinical trials 38 , despite showing early promise as cancer therapeutics Marimastat displays particularly good oral bioavailability.

However, murine bridging studies incorporating pharmacokinetic PK profiling coupled with pharmacodynamic PD assessments of venom neutralization are required in the future to enable accurate simulations of predicted human doses.

The second drug in our mixture, varespladib, is a secretory PLA 2 -inhibitor previously investigated for use in the treatment of various acute coronary syndromes Both varespladib and varespladib methyl its oral prodrug, which is rapidly converted in vivo to varespladib have been used clinically in Phase 1 and 2 trials 55 , 56 , 57 , although a lack of efficacy at Phase 3 ultimately resulted in discontinuation More recently, varespladib has been explored for repurposing as a potential therapeutic for the treatment of snakebite.

Both varespladib and its oral prodrug have been shown to exhibit promising neutralizing capabilities against a variety of different snake venoms 22 , 29 , but have proven to be particularly effective at mitigating the life-threatening effects of neurotoxicity caused by certain elapid venoms in animal models of envenoming 26 , 27 , Given that the dose of varespladib used in these clinical studies is ~fold higher than the facilely-extrapolated human equivalent dose used intraperitoneally in our animal model 0.

The addition of the serine protease-inhibitor nafamostat to the therapeutic mixture resulted in no additional protection to the marimastat and varespladib dual combination Fig. These findings, alongside evidence that nafamostat provides no protection against the lethal effects of E.

ocellatus venom when used as a solo therapy Fig. For those various reasons, our lead candidate therapeutic mixture remained restricted to the marimastat and varespladib combination. In our previous work, we demonstrated that the licensed metal chelator DMPS, which shows much promise as an early intervention therapeutic against snakes with SVMP-rich venoms e.

While DMPS remains a promising future therapeutic for snakebite, not least because of its oral formulation, licensed drug status and decades of therapeutic use for other indications 61 , 62 , it seems unlikely to be highly efficacious as a solo therapy against a wide variety of different snake species due to only targeting SVMP toxins russelii having substantially different abundances of distinct venom toxins to that of E.

ocellatus 4 , 63 , 64 , 65 Fig. Given that all existing antivenoms are geographically-restricted in terms of their snake species efficacy e. ocellatus , for example , these findings suggest that this therapeutic combination of small molecule toxin inhibitors may represent a highly specific yet generic future treatment for viperid snakebite.

Notwithstanding the apparent therapeutic promise of this small molecule toxin inhibitor combination, a considerable amount of future research is required to facilitate its translation. Despite the combination of animal models used here providing confidence of broad anti-envenoming efficacy, these models remain limited in terms of accurately recapitulating cases of human envenoming e.

Thus, additional preclinical studies are needed to further explore the neutralizing efficacy of this drug combination, including the use of oral dosing regimens, and repeat dosing experiments combined with pharmacokinetic analyses, to effectively model the oral dose required to maintain effective concentrations of the drugs sufficient to provide prolonged protection from envenoming.

This may be particularly challenging for cases where envenoming may result in prolonged treatment times, for example, as the result of recurrence of coagulopathy or acute kidney injury, and thus additional model development addressing this point is needed. The in vitro and in vivo venom neutralization data presented here should also be extended to additional medically important snake species, and the efficacy of this combination therapy against the local, morbidity-inducing, effects of snake venoms should be explored.

Finally, the successful delivery and uptake of any prehospital snakebite treatment comes with a number of long-term implementation challenges that require careful consideration, including ensuring i acceptable safety profiles across the target population e.

While these findings hold much promise, we propose that the future translation of this inhibitor combination should occur in parallel with other small molecule toxin inhibitor lead candidates, such as DMPS and varespladib 26 , 27 , 28 , 59 , to increase the breadth of new molecules being added to the snakebite treatment toolbox and, most importantly, to help offset the risk of potential drug failures during clinical trials.

Venoms were sourced from either wild-caught specimens maintained in, or historical venom samples stored in, the Herpetarium of the Liverpool School of Tropical Medicine. This facility and its protocols for the expert husbandry of snakes are approved and inspected by the UK Home Office and the LSTM and University of Liverpool Animal Welfare and Ethical Review Boards.

The venom pools were from vipers with diverse geographical localities, namely: E. ocellatus Nigeria , E. carinatus sochureki India, referred to throughout as E.

carinatus , B. arietans Nigeria , B. asper Atlantic coast of Costa Rica and D. russelii Sri Lanka. Note that the Indian E. carinatus venom was collected from a single specimen that was inadvertently imported to the UK via a boat shipment of stone, and then rehoused at LSTM on the request of the UK Royal Society for the Prevention of Cruelty to Animals RSPCA.

The SVMP assay measuring metalloproteinase activity and the plasma assay measuring coagulation in the presence or absence of venoms and inhibitors were performed as previously described The negative control PBS-only was also expressed relative to the venom, and the variation in background was presented as an interval delineated by the lowest and highest values in the PBS-only samples across concentrations and inhibitors for a specific venom.

However, only the latter two concentrations were sufficiently depleted of DMSO for viable use in the assay. We used a previously developed plasma clotting assay 35 to measure venom-induced coagulation.

We calculated the maximum clotting velocity of each of the curves as per clot waveform analysis 67 , by calculating the maximum of the first derivative. The means of at least three independent experimental runs for each condition were plotted at each inhibitor concentration with SEMs.

SVSP activity was measured using a chromogenic substrate S, Cambridge Bioscience. Fifty microlitres of venom solution D.

The total flow rate of the mobile phase solution was 0. Neutralization of coagulopathic venom toxins by marimastat and varespladib was assessed by assaying the D.

russelii venom nanofractionated plates in the plasma coagulation assay, as recently described The final assay concentrations of the inhibitors tested in the assay were 20, 4, 0.

The obtained results were normalized by dividing the slope measured in each well by the median of all slope values across the plate, and the processed coagulation chromatograms were plotted to visualize very fast coagulation, medium increased coagulation and anticoagulation, as previously described All animal experiments were conducted using protocols approved by the Animal Welfare and Ethical Review Boards of the Liverpool School of Tropical Medicine and the University of Liverpool, and performed in specific pathogen-free conditions under licensed approval PPL and PF90 of the UK Home Office and in accordance with the Animal [Scientific Procedures] Act and institutional guidance on animal care.

The experimental design was based upon 3R-refined WHO-recommended protocols 28 , 41 , with animals randomized and observers being blinded to the experimental condition. The median lethal doses venom LD 50 used for E.

carinatus India , B. asper Costa Rica , D. russelii Sri Lanka and B. arietans Nigeria venoms were previously determined 25 , 41 , 44 , For our initial in vivo experiments, we used 2. Groups of five mice received experimental doses that consisted of either: a venom only 2. For all experimental animals described above, blood samples were collected via cardiac puncture immediately post-euthanasia.

All available plasma samples some were unobtainable via cardiac puncture due to extensive internal hemorrhage were assessed if the time of death within the group varied, whereas three samples were randomly chosen if the time of death was the same e.

The resulting data was plotted as the median of duplicate measurements for each animal and is presented with standard deviations SDs. Further information on research design is available in the Nature Research Reporting Summary linked to this article.

The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request. The raw data supporting the findings of this study and that are displayed in Figs.

Data used to construct the species distributions displayed in Fig. Source data are provided with this paper. Gutiérrez, J. et al. Snakebite envenoming. PubMed Google Scholar. Harrison, R. The time is now: a call for action to translate recent momentum on tackling tropical snakebite into sustained benefit for victims.

PubMed PubMed Central Google Scholar. Williams, D. Strategy for a globally coordinated response to a priority neglected tropical disease: Snakebite envenoming. PLoS Negl. Casewell, N. Medically important differences in snake venom composition are dictated by distinct postgenomic mechanisms.

Natl Acad. USA , — ADS CAS PubMed PubMed Central Google Scholar. Tasoulis, T. A review and database of snake venom proteomes. Toxins 9 , PubMed Central Google Scholar.

Ending the drought: New strategies for improving the flow of affordable, effective antivenoms in Asia and Africa. CAS Google Scholar. Arnold, C. Vipers, mambas and taipans: the escalating health crisis over snakebites.

Nature , 26—28 ADS CAS PubMed Google Scholar. Global availability of antivenoms: The relevance of public manufacturing laboratories.

Toxins 11 , 5 Google Scholar. Pre-clinical assays predict pan-African Echis viper efficacy for a species-specific antivenom. de Silva, H. Low-dose adrenaline, promethazine, and hydrocortisone in the prevention of acute adverse reactions to antivenom following snakebite: A randomised, double-blind, placebo-controlled trial.

PLoS Med. Mohapatra, B. Snakebite mortality in India: a nationally representative mortality survey. Bulfone, T. Developing small molecule therapeutics for the initial and adjunctive treatment of snakebite. Knudsen, C. Recent advances in next generation snakebite antivenoms. Dis 3 , 42 Habib, A. Snake bite in Nigeria.

Otero-Patiño, R. Epidemiological, clinical and therapeutic aspects of Bothrops asper bites. Toxicon 54 , — Kumar, K. Clinical and epidemiologic profile and predictors of outcome of poisonous snake bites — an analysis of 1, cases from a tertiary care center in Malabar, North Kerala, India.

Slagboom, J. Haemotoxic snake venoms: their functional activity, impact on snakebite victims and pharmaceutical promise. Snake venom metalloproteinases: their role in the pathogenesis of local tissue damage.

Biochimie 82 , — Hemorrhage caused by snake venom metalloproteinases: a journey of discovery and understanding. Toxins Basel. Ferraz, C. Multifunctional toxins in snake venoms and therapeutic implications: from pain to hemorrhage and necrosis.

Howes, J. Furthermore, SVSPs and SVMPs induce hemostatic and cardiovascular effects as coagulopathy, hypotension and hemorrhage Slagboom et al.

Interestingly, some PLA2s, SVSPs, and SVMPs are also capable of triggering severe pain by modulating pain pathways through activation of ion channels, such as transient receptor potential vanilloid type 1 TRPV1 and acid-sensing ion channel ASIC Bohlen et al.

The inflammation induced by the elapid and viper venoms is widely reported to produce pain or hyperalgesia in human and in experimental models Hifumi et al. Unfortunately, these are not completely reversed by antivenom and anti-inflammatory therapies Picolo et al.

Although sufficient in most cases, snakebite treatments have been challenged by the continuous high numbers of clinical illness and mortality associated with snakebites worldwide WHO, Furthermore, chronic morbidity following snakebites have been underestimated, with many victims reporting chronic symptoms in the bitten region, including complex regional pain syndrome CRPA Seo et al.

Available snakebite treatments face challenges associated with limited para-specificity, poor antibody specificity, high incidences of adverse reactions, low availability and poor affordability to those who need them, along with poor efficacy against local tissue effects Williams et al. Therefore, current research efforts are directed to the development of more effective snakebite therapies able to generically fully inhibit the major toxic components of snake venoms in order to better overcome severe acute and chronic effects caused by snakebite.

In light of the public health importance and the complexity of snake venoms, in this review we highlight the multifunctionality, structure-activity relationships and evolution of proteins and peptides in snake venoms.

We aim to provide a better understanding of their action mechanisms and effects, and to bring attention to their undetermined targets and a host of potential novel therapeutic targets that might have implications for improving the treatments of snakebites.

Phospholipases A 2 play an important role in the neurotoxic and myotoxic effects of snakebites Harris and Scott-Davey, These proteins have molecular masses of 13—15 kDa and are classified into groups I and II, which are found as major components in the venoms of Elapidae and Viperidae, respectively Six and Dennis, ; Harris and Scott-Davey, In addition, a third group of PLA2s, termed IIE, have been predominately recovered from the venoms of non-front fanged snakes, although their importance in the venom arsenal remains unclear Fry et al.

Studies reconstructing the evolutionary history of this multi-locus gene family have demonstrated that each of these PLA2s types I, II, and IIE have been independently recruited into snake venom systems Fry et al.

Although PLA2s from vipers and elapids share similar enzymatic properties, both types have undergone extensive gene duplication over evolutionary time, seemingly facilitating the evolution of new toxic functions, and resulting in different patterns of residue conservation Lynch, ; Vonk et al.

In addition, the venoms of viper snakes contain isoforms of group II PLA2s that are catalytically-active e. Figure 1. Structure of PLA2s from snake venoms. A Alignment of the primary structure of PLA2s from snakes belong to Elapidae Class I and Viparinae Class II.

β-Bungarotoxin β-BTX from Bungarus multicinctus Class I, Basic Uniprot P , OS2 from Oxyuranus scutellatus Class I, Basic Uniprot Q45Z47 , Myotoxin II from Bothrops asper Class II, Basic Uniprot P , APP-D49 from A. piscivorus Class II, Basic Uniprot P , BpirPLA2-I from Bothrops pirajai Class II, Acid Uniprot C9DPL5 , Bothropstoxin-1 BThTx-I from Bothrops jararacussu Class II, Basic Uniprot Q , and Crotoxin B from Crotalus durissus terrificus Class II, Basic Uniprot P Green: N-terminal region critical for enzymatic and neurotoxic properties, C-terminal region essential for enzymatic activity and central Histidine in the catalytic site Rouault et al.

B—E Cartoon representation of the three-dimensional structure of B β-Bungarotoxin with the PLA2 domain in red and knutiz domain in green PDB 1BUN , C MitTx1 and ASIC1a channel complex with PLA2 domain in red, knutiz domain in green and ASIC1a channel in blue PDB 4NTY , D Crotoxin B PDB 2QOG and E BThrTx1 PDB 3HZD.

D,E Highlighted in blue are the amino acids positions involved in the enzymatic, toxic and pharmacological properties of Crotoxin B Soares et al. The central histidine in the catalytic site of Crotoxin B is highlighted in red.

A number of PLA2s exert strong myotoxic effects which often lead to severe necrosis Harris and Maltin, ; Gutierrez and Ownby, , and many of these toxins also promote inflammation, including edema formation, cytokine production and leukocyte recruitment, pain by inducing thermal allodynia and mechanical hyperalgesia, paralysis through block of neuromuscular transmission and intensify hemorrhage by inhibiting coagulation Table 1 Camara et al.

Neurotoxic effects caused by these toxins, as well as some of their proinflammatory effects, occurs via the modulation of pre-synaptic terminals as well as sensory nerve-endings Camara et al.

The PLA2s pre-synaptic effects are characteristic of β-neurotoxins and target the motor nerve terminals at the neuromuscular junction Sribar et al. Overall, these pre-synaptic effects induce robust exocytosis of the neurotransmitters vesicles reserves which consequently lead to the depletion of neurotransmitter release in the neuromuscular junction to promote muscle paralysis Harris et al.

Table 1. Snake toxins and their multifunctional roles in the toxicity induced by snakebites. The inflammation induced by PLA2s has non-neurogenic and neurogenic substance-P dependent components Camara et al.

The non-neurogenic component is mostly caused by the hydrolysis of membrane lipids that generate potent pro-inflammatory lipid mediators Costa et al. Additional non-neurogenic and neurogenic inflammations induced by PLA2s use more complex mechanisms still not fully understood.

For example, leukocyte recruitment De Castro et al. Furthermore, substance-P mediated neurogenic inflammation has been described to be induced by PLA2s from Crotalus durissus cascavella Camara et al.

Interestingly, the C-terminal of Myotoxin-II a LysPLA2 isolated from Bothrops asper was able to activate macrophages, showing this region maybe be crucial for the observed enzymatic-independent inflammation Giannotti et al.

The pain induced by PLA2s is driven by inflammatory processes and sensory neuronal activation. Bradykinin is an important mediator of the inflammatory pain induced by PLA2s Moreira et al. It induces mechanical hyperalgesia dependent on the production of TNF-α, IL-1β, and prostaglandins Cunha et al.

This suggests that PLA2s contribute to an increase in arachidonic acid release from cell membranes and its availability to be processed by cyclooxygenase resulting in prostaglandin production Verri et al. Direct activation of sensory neurons was demonstrated by MitTx from Micrurus tener tener , a heteromeric complex between a PLA2 and a kunitz peptide Bohlen et al.

MitTx activates somatosensory neurons and was found to be a potent and selective agonist of ASIC channels Figure 1C.

This agonistic effect induces robust pain behavior in mice via activation of ASIC1 channels on capsaicin-sensitive nerve fibers Bohlen et al. BomoTx also activated a cohort of sensory neurons to induce ATP release followed by activation of purinergic receptors Zhang et al.

Unfortunately, the primary target of this neuronal activation is still unknown. The multifunctionality of PLA2s is evidenced by their myotoxic, neurotoxic and enzymatic functions, as well as by their inflammatory properties.

There is evidence that separate domains and regions of the PLA2s structure participate in these various activities Figures 1A,B. For example, for the LysPLA2 from Bothrops asper and Agkistrodon piscivorus piscivorus , the C-terminal region of these toxins residues — were identified as the active sites responsible for their myotoxic effects Lomonte et al.

Interestingly, the same C-terminal region in BpirPLA2-I isolated from Bothrops pirajai had anticoagulant activity through inhibition of platelet aggregation Teixeira et al.

Crotoxin B, an AspPLA2, and a major component of the venom of Crotalus durissus terrificus , has toxic active sites fully independent of its enzymatic activity Soares et al.

A detailed mutational study using the PLA2 OS2 from the Australian Taipan snake Oxyuranus scutellatus scutellatus revealed that a fold loss in enzymatic activity had only a minor effect on its neurotoxicity Rouault et al.

Furthermore, the enzymatic activity of OS2 was dependent of the N- and C-terminal regions, and the N-terminal region had a major role in the central nervous system neurotoxicity. An alanine scan of the LysPLA2 from Bothrops jararacussu BThTx-I demonstrated distinct regions involved in the hyperalgesia and edema Zambelli et al.

In this study, the mutant ArgAla lost both nociceptive and edematogenic properties, LysAla and LysAla lost the nociceptive effects without interfering with the edema formation and LysAla lost the nociceptive properties and had weak inflammatory effects Figure 1E.

Similarly, an independent study showed the LysAla substitution led to reduced membrane damaging and myotoxic activities Ward et al. This C-terminal region is characterized by the presence of basic and hydrophobic residues which have been strongly associated with the ability of PLA2s to interact and penetrate the lipid bilayer Delatorre et al.

Many snake venom toxins are known to be encoded by multi-locus gene families Casewell et al. The process of gene duplication and loss underpins the evolution of many snake venom toxin families, including the PLA 2 s Lynch, ; Vonk et al.

Indeed, studies have demonstrated that extremely divergent venom phenotypes e. haemorrhagic observed within populations of the same snake species, or between closely related species, are at least partially the result of major genomic differences in PLA 2 toxin loci, with variation at different gene complexes resulting in markedly different haplotypes Dowell et al.

It remains unclear as to the specific processes that underpin such diversity, although natural selection driven by environmental factors and hybridization events have both been proposed Dowell et al.

Snake Venom MetalloProteinases SVMPs are zinc-dependent proteinases ranging from 20 to kDa in size and are categorized into P-I, P-II, and P-III classes according to their structural domains Hite et al.

These toxins are major components of viper venoms and play a key role in the toxicity of these snake venoms Table 1 ; Tasoulis and Isbister, Venom SVMPs have evolved from ADAM a disintegrin and metalloproteinase proteins, specifically ADAM28 Casewell, , with the P-III being the most basal structural variant consisting of metalloproteinase, disintegrin-like, and cysteine-rich domains Moura-Da-Silva et al.

Subsequently, P-II SVMPs diverged from P-IIIs and consist of a metalloproteinase and disintegrin domain, with the latter typically detected in venom as a proteolytically processed product Fox and Serrano, ; Casewell et al.

The final class, P-I SVMPs which consist only of the metalloproteinase domain, appeared to have evolved on multiple independent occasions in specific lineages as a result of loss of the P-II disintegrin-encoding domain Casewell et al.

Throughout this diverse evolutionary history, SVMPs show evidence of extensive gene duplication events, coupled with bursts of accelerated molecular evolution Casewell et al.

While these P-III SVMPs are typically relatively lowly abundant venom components in elapid snakes e. These abundance differences likely underpin the distinct pathologies observed following envenomings by snakes found in these families.

SVMPs contribute extensively to the hemorrhagic and coagulopathic venom activities following bites by viperid snakes, and the diversity of SVMPs isoforms often present in their venom likely facilitate synergistic effects, such as simultaneous action on multiple steps of the blood clotting cascade Kini and Koh, ; Slagboom et al.

However, it is relatively uncommon for elapid snakebites to cause systemic hemotoxicity Slagboom et al. Figure 2. Structure of metalloproteinaises from snake venoms.

A Alignment of the primary structure of the SVMPs Jararhagin from Bothrops jararaca Uniprot P and VAPB2 from Crotalus atrox Uniprot Q belonging to the class P-III, BlatH1 from Bothriechis lateralis Uniprot U5PZ28 belonging to the class P-II, and Ba-PI from Bothrops asper Uniprot P and Adamalysin from Crotalus adamanteus Uniprot P belonging to classes P-I.

Cysteines are colored in red, the disintegrin-like domain is highlighted in green and the cysteine-rich domain is highlighted in blue. B Cartoon representation of the three-dimensional structure of the class P-III metalloproteinase VAPB2 from Crotalus atrox PDB 2DW0. The metalloproteinase domain is colored in orange, the disintegrin-like domain D-like is colored in green and the cysteine-rich domain Cys-rich is colored in blue.

The disulphide bridges are colored in yellow. Research has revealed that the effects of SVMP-induced hemorrhage relies on a mechanism that occurs in two steps Gutierrez et al.

First, SVMPs cleave the basement membrane and adhesion proteins of endothelial cells-matrix complex to weaken the capillary vessels. During the second stage, the endothelial cells detach from the basement membrane and become extremely thin, resulting in disruption of the capillary walls and effusion of blood from the fragile capillary walls.

In addition to the proteinase activity, SVMPs impact on homeostasis by altering coagulation, which contributes to their toxic hemorrhagic effects Markland, ; Takeda et al. This occurs through modulation of factors such as fibrinogenase and fibrolase that mediate the coagulation cascade, depletion of pro-coagulation factors through consumption processes e.

Some SVMPs also induce inflammation, including edema, and pain by triggering hyperalgesia Dale et al. The inflammation induced by jararhagin, a class P-IIIb metalloproteinase with potent hemorrhagic and dermonecrotic activity isolated from Bothrops jararaca , produced TNF-α and IL-1β in vivo Laing et al.

Curiously, the edema formation induced by jararhagin was independent of pro-inflammatory mediators such as TNF, IL-1β, and IL-6 Laing et al. Neurogenic inflammation was also implicated in the local hemorrhage induced by Bothrops jararaca which was shown to be dependent on serotonin and other neuronal factors Goncalves and Mariano, The mechanisms on how neurogenic inflammation is triggered by the snake venom components and how it participates in the hemorrhagic process are still not understood.

Pain induced by SVMPs is characterized by hyperalgesia and inflammatory pain, which is dependent on the production of cytokines, nitric oxide, prostaglandins, histamine, leukotrienes, and migration of leukocytes, mast cell degranulation and NFkB activation Fernandes et al.

However, the mechanisms underlying SVMP-induced pain are still poorly understood, with neurogenic inflammation and neuronal excitatory properties still underexplored.

The multifunctional properties of SMVPs are also well-described. Class P-III SVMPs tend to display stronger hemorrhagic activity compared to P-I and P-II SVMPs, possibly due to the disintegrin-like and cysteine-rich domains enabling binding to relevant targets in the extracellular matrix of capillary vessels.

The functions of these domains have been investigated in inflammation, revealing that these domains are sufficient to induce pro-inflammatory responses through production of TNF-α, IL-1β and IL-6, and leukocyte migration, in which mechanisms and primary targets are still unknown Clissa et al.

These observations suggest that these domains are involved in the inflammatory hyperalgesia induced by SVMPs.

Furthermore, the pronounced hemorrhagic and necrotic activities are strongly dependent on biological effects driven by the disintegrin-like and cysteine-rich domains, as observed for BJ-PI2 da Silva et al.

The hemorrhagic activity of Bothrops jararaca venom was also shown dependent on neurogenic inflammation Goncalves and Mariano, Snake Venom Serine Proteinases SVSPs belong to the S1 family of serine proteinases and display molecular masses ranging from 26 to 67 kDa and two distinct structural domains Figure 3.

These venom toxins have evolved from kallikrein-like serine proteases and, following their recruitment for use in the venom gland, have undergone gene duplication events giving rise to multiple isoforms Fry et al.

SVSPs catalyze the cleavage of polypeptide chains on the C-terminal side of positively charged or hydrophobic amino acid residues Page and Di Cera, ; Serrano, Whilst the SVMPs are well-known for their ability to rupture capillary vessels, SVSPs execute their primary toxicity by altering the hemostatic system of their victims, and by inducing edema and hyperalgesia through mechanisms still poorly understood Table 1.

Hemotoxic effects caused by SVSPs include perturbations of blood coagulation pro-coagulant or anti-coagulant , fibrinolysis, platelet aggregation and blood pressure, with potential deadly consequences for snakebite victims Murakami and Arni, ; Kang et al.

Figure 3. Structure of Serine proteinases from snake venoms. A Alignment of the primary structure of the SVSPs Dav-PA from Deinagkistrodon acutus Uniprot Q9I8X1 , BPA from Bothrops jararaca Uniprot Q9PTU8 , and ACC-C from Agkistrodon contortrix Uniprot P In Dav-PA, the N- and C-terminal domains are highlighted in blue and green, respectively.

B Cartoon representation of the three-dimensional structure of the SVSP Dav-PA from Deinagkistrodon acutus PDB 1OP0 , with N- and C-terminal domains colored in blue and green, respectively.

Pro-coagulant SVSPs have been described to activate multiple coagulation factors, including prothrombin and factors V, VII, and X Kini, ; Serrano, For example, the activation of prothrombin produces thrombin which in turn produces fibrin polymers that are cross-linked.

Thrombin also activates aggregation of platelets which, together with the formation of fibrin clots, results in coagulation Murakami and Arni, In addition, platelet-aggregating SVSPs will activate the platelet-receptors to promote binding to fibrinogen and clot formation Yip et al.

These procoagulant and platelet-aggregating activities will lead to the rapid consumption of key factors in the coagulation cascade and clot formation.

On the other hand, anticoagulant SVSPs effects involve the activation of Protein C, which subsequently inactivates the coagulant factors Va and VIIIa Kini, Furthermore, fibrinolytic SVSPs play an important role in the elimination of blood clots by acting as thrombin-like enzymes or plasminogen activators, which eliminates the fibrin in the clots and contributes significantly to the establishment of the coagulopathy Kang et al.

Little is known about inflammatory responses and hyperalgesia induced by SVSPs. Studies suggest SVMPs and PLA2s have a pivotal role in the inflammatory responses and pain induced by snake venoms, while SVSPs have an important role in inflammation and a minor role in pain Zychar et al.

SVSPs in the venoms of Bothrops jararaca and Bothrops pirajai induce inflammation through edema formation, leucocyte migration mainly neutrophils and mild mechanical hyperalgesia, however, the mediators involved in these effects are still unknown Zychar et al.

Three-fingers toxins 3FTXs are non-enzymatic neurotoxins ranging from 58 to 81 residues that contain a three-finger fold structure stabilized by disulfide bridges Osipov and Utki, ; Kessler et al. They are present mostly in the venoms of elapid and colubrid snakes, and exert their neurotoxic effects by binding postsynaptically at the neuromuscular junctions to induce flaccid paralysis in snakebite victims Barber et al.

Three-finger toxins differ in length, with short-chain 3FTXs including α-neurotoxins, β-cardiotoxins, cytotoxins, fasciculins and mambalgins, which comprise of 57—62 residues and 4 disulfide bridges, and long-chain 3FTXs which include α-neurotoxins and γ-neurotoxins, hannalgesin and κ-neurotoxins, and comprise 66—74 residues and five disulfide bridges.

Furthermore, they can exist as monomers and as covalent or non-covalent homo or heterodimers. The diversity of 3FTX isoforms described above are a direct result of a diverse evolutionary history, whereby ancestral 3FTXs have diversified by frequent gene duplication and accelerated rates of molecular evolution.

These processes, which are broadly similar to those underpinning the evolution of the other toxin families described above, are particularly associated with the evolution of a high-pressure hollow-fanged venom delivery system observed in elapid snakes Sunagar et al.

For example, gene duplication events have resulted in the expansion of 3FTX loci from one in non-venomous snakes like pythons, to 19 in the elapid Ophiophagus hannah king cobra Vonk et al.

The consequences of this evolutionary history are the differential production of numerous 3FTX isoforms that often exhibit considerable structural differences and distinct biological functions Figures 4B—E.

Although many elapid snakes exhibit broad diversity of these functionally varied toxins in their venom e.

Figure 4. Structure of three-finger toxins from snake venoms. A Alignment of the primary structure of α-cobratoxin from Naja kaouthia Uniprot P , α-bungarotoxin from Bungarus multicinctus Uniprot P , fasciculin from Dendroaspis angusticeps Uniprot P0C1Y9 , Calciseptin from Dendroaspis polylepis polylepis Uniprot P , mambin from Dendroaspis jamesoni kaimosae Uniprot P , mambalgin-1 from Dendroaspis polylepis polylepis Uniprot P0DKR6 , cytotoxin 1 from Naja atra Uniprot P , and calliotoxin from Calliophis bivirgatus Uniprot P0DL B—E Cartoon representation of the three-dimensional structure of the 3FTXs short-chain mambalgin-1 from Dendroaspis polylepis polylepis PDB 5DU1 B , long-chain α-bungarotoxin from Bungarus multicinctus PDB 1ABT C , non-covalent homodimer α-cobratoxin from Naja kaouthia PDB 4AEA D , and covalent heterodimer irditoxin from Boiga irregularis PDB 2H7Z E.

F Cartoon representation of the three-dimensional structure of Fascilulin-2 bound to the AChE PDB 4BDT. The residues Arg24, Lys25, Pro31 and Leu35 which form hydrogen bonds with AChE are shown in orange. G—J Cartoon representation of the three-dimensional structure of the muscarinotoxin 1 MT1, PDB 4DO8 G and muscarinotoxin 7 MT7, PDB 2VLW H , and respective analogs displaying the modified loop 1 PDB 3FEV I and loop 3 PDB 3NEQ J in light orange color.

K Neurotoxin II from N. oxiana PDB 1NOR. The residues Ser29, His31, Gly33 and Thr34 which form hydrogen bonds with the α-subunit of nAChR are shown in orange, and the residue Arg32 forming ionic interactions with the α-subunit of nAChR is shown in yellow.

L Neurotoxin b NTb from O. Hannah PDB 1TXA. The residues Lys24, Trp26, and Asp28 that form hydrogen bonds with the α-subunit nAChR are shown in orange. Despite the shared three-finger fold, the 3FTXs have diverse targets and biological activities.

For example, α-neurotoxins inhibit muscle acetylcholine receptors nAChR Changeux, , κ-neurotoxins inhibits neuronal AChR Grant and Chiappinelli, , muscarinic toxins inhibit muscarinic receptors Marquer et al. Their toxic biological effects include flaccid or spastic paralysis due to the inhibition of AChE and ACh receptors Grant and Chiappinelli, ; Changeux, ; Marchot et al.

In addition to their multitude of bio-activities, 3FTXs can remarkably display toxicities that target distinct classes of organisms as demonstrated in non-front fanged snake venoms that produce 3FTX isoforms which are non-toxic to mice but highly toxic to lizards, and vice-versa Modahl et al.

Some 3FTXs are able to induce analgesia through inhibition of ASIC channels Salinas et al. Furthermore, 3FTXs are relatively small compared to the other snake toxins discussed herein, and do not exhibit multiple domains to produce their multiple toxic functions.

Nevertheless, the number of receptors, ion channels, and enzymes targeted by snake 3FTXs highlights the unique capacity of this fold to modulate diverse biological functions and the arsenal of toxic effects that are induced by 3FTXs.

The unique multifunctionality of the 3FTX scafold occurs because of their resistance to degradation and tolerance to mutations and large deletions Kini and Doley, Therefore, the structure-activity relationship of the 3FTXs is complex and yet to be fully understood.

Their functional sites are located on various segments of the molecule surface. Conserved regions determine structural integrity and correct folding of 3FTXs to form the three loops, including eight conserved cysteine residues found in the core region.

Aromatic residues Tyr25 or Phe27 are conserved in most 3FTXs and essential to their folding. Another conserved features are the antiparallel β-sheet structure and charged amino acid residues also essential to stabilize the native conformation of the protein by forming hydrogen and ionic interactions, respectively Torres et al.

Additional disulfide bonds can be observed either in the loop I or loop II which can potentially change the activity of the 3FTX in some cases. Specific amino acid residues in critical segments of the 3FTXs have been identified to be important for binding to their targets.

For example, the interactions between fasciculin and AChE enzyme has been studied. The first loop or finger of fasciculin reaches down the outer surface of the enzyme, while the second loop inserts into the active site and exhibit hydrogen bonds and hydrophobic interaction Harel et al.

Several basic residues in fasciculin make key contacts with AChE. From docking studies, hydrogen bonds, and hydrophobic interactions where shown to establish receptor-toxin assembly.

Six amino acid residues Lys25, Arg24, Asn47, Pro31, Leu35 and Ala12 of fasciculin interact with the AChE residues by forming hydrogen bonds at its active site. Hydrophobic interactions are also observed between eight amino acid residues Lys32, Cys59, Val34, Leu48, Ser26, Gly36, Thr15, Asn20 from fasciculin and the enzyme active site Waqar and Batool, These interactions involve charged residues but lacks intermolecular salt linkages.

Muscarinic toxins from mamba venoms, such as MT1 and MT7 Figures 4G,H , act as highly potent and selective antagonists of M1 receptor subtype through allosteric interactions with the M1 receptor. Fruchart-Gaillard et al. In this study, substitution within loop 1 and loop 3 weaken the toxin interactions with the M1 receptor, resulting in a 2-fold decrease in affinity Figures 4I,J.

Furthermore, modifications in loop 2 of the MT1 and MT7 significantly reduce the affinity for the M1 receptor. Interestingly, a significant increase in affinity was achieved on the α1 A -adrenoceptor by combined modifications in loops 1 and 3, where loop 1 forms a critical interaction with the receptor Fruchart-Gaillard et al.

Another muscarinic toxin named MTβ was designed based on ρ-Da1a protein from Black Mamba which is known to have affinity for the muscarinic receptor. These two residues were not located at the tip of the toxin loop, however, they played a critical role in the interactions with their molecular targets Bourne et al.

Neurotoxin II NTII from Naja oxiana is a potent blocker of nAChR. NTII is a short chain α-neurotoxin which consists of 61 amino acid residues and four disulfide bridges Figure 4K.

A computational model for examining the interactions of NTII with the Torpedo californica nAChR has been studied Mordvintsev et al.

The model showed that the binding of the short α-neurotoxin occurs by rearranging the aromatic residues in the binding pocket. The insertion of the loop II into the binding pocket of a nAChR induces the neurotoxin activity and significantly determines the toxin-receptor interactions, while loop I and III contact the receptor residues by their tips only and determine the immunogenicity of the short neurotoxins.

In the model, the Arg32 NTII residue forms an ionic pair with Trp from the nAChR and is observed as the strongest interaction. Hydrogen bond interactions such as Asp30 from loop II with Tyr of the nAChR and Lys46 from loop III with Thr of the nAChR complement the ionic interaction between NTII and its target receptor.

Aside from hydrogen bonds, van-der-Waals interactions were also observed at the fingertip of loop II amino acid residues Lys25, Trp27, Trp28, Ser29, His31, Gly33, Thr34 and Arg38 Mordvintsev et al.

The structure of neurotoxin b NTb , a long neurotoxin from Ophiophagus hannah , has been elucidated Peng et al. Conserved residues in loop II also play an important role in the toxicity of the long neurotoxins by making ionic interactions between toxin and receptor.

Positively charged residues Trp27, Lys24 and Asp28 are highly conserved residues in the long neurotoxins. Furthermore, a modification of the Trp27 in the long neurotoxin analog of NTb from king cobra venom led to a significant loss in neurotoxicity. The additional disulphide bridge in loop II of long neurotoxins does not affect the toxin activity.

Nevertheless, cleavage of the additional disulphide bridge in loop II can disrupt the positively charged cluster at the tip of loop II. Changes in loop II conformation will affect the binding of the long neurotoxin to the target receptor resulting the loss of neurotoxicity Peng et al.

Long and short neurotoxins show sequence homology and similar structure. Previous studies show that many residues located at the tip of loop II are conserved in both short and long neurotoxins. It is consisting of the long central β-sheets forming three loops and globular core.

From the studies of α-bungarotoxin and α-cobratoxin, the least conserved regions of the long neurotoxin are the C-terminal and the first loop Walkinshaw et al.

However, significant differences between long-chain neurotoxin and short chain neurotoxin are indicated by the immunological reactivity.

Many of the residues involved in the antibody-long neurotoxins binding are located in loop II, loop III, and in the C-terminal, while in short neurotoxins the antibody's epitope makes interactions with the loop I and loop II Engmark et al.

Animal-derived antivenoms are considered the only specific therapy available for treating snakebite envenoming Maduwage and Isbister, ; Slagboom et al. These consist of polyclonal immunoglobulins, such as intact IgGs or F ab' 2 , or Fab fragments Ouyang et al. Antivenoms can be classified as monovalent or polyvalent depending on the immunogen used during production.

Monovalent antivenoms are produced by immunizing animals with venom from a single snake species, whereas polyvalent antivenoms contain antibodies produced from a cocktail of venoms of several medically relevant snakes from a particular geographical region.

Polyvalent antivenoms are therefore designed to address the limited paraspecific cross-reactivity of monovalent antivenoms by stimulating the production of antibodies against diverse venom toxins found in different snake species, and to avoid issues relating to the wrong antivenom being given due to a lack of existing snakebite diagnostic tools O'leary and Isbister, ; Abubakar et al.

However, polyvalent therapies come with disadvantages—larger therapeutic dose are required to effect cure, potentially resulting in an increased risk of adverse reactions, and in turn increasing cost to impoverished snakebite victims Hoogenboom, ; O'leary and Isbister, ; Deshpande et al.

Variation in venom constituents therefore causes a great challenge for the development of broadly effective snakebite therapeutics. The diversity of toxins found in the venom of any one species represents considerable complexity, which is further enhanced when trying to neutralize the venom of multiple species, particularly given variations in the immunogenicity of the multi-functional toxins described in this review.

Antivenom efficacy is therefore, typically limited to those species whose venoms were used as immunogens and, in a number of cases, closely-related snake species that share sufficient toxin overlap for the generated antibodies to recognize and neutralize the key toxic components Casewell et al.

Because variation in venom composition is ubiquitous at every level of snake taxonomy e. Such studies have revealed surprising cross-reactivity of antivenoms against distinct, non-targeted, snake species, such as: i the potential utility of Asian antivenoms developed against terrestrial elapid snakes at neutralizing the venom toxicity of potent sea snake venoms Tan et al.

The later of these studies demonstrated cross-neutralization between distinct snake lineages e. Thus, detailed knowledge of venom composition can greatly inform studies assessing the geographical utility of antivenoms. Such studies have stimulated much research into the development of novel therapeutic approaches to tackle snakebite.

These include the use of monoclonal antibody technologies to target key pathogenic toxins found in certain snake species Laustsen et al. It is anticipated that in the future these new therapeutics may offer superior specificities, neutralizing capabilities, affordability and safety over conventional antivenoms.

However, the translation of their early research promise into the mainstay of future snakebite treatments will ultimately rely on further research on the toxins that they are designed to neutralize. Specifically, the selection, testing and optimization of new tools to combat snake envenoming is reliant upon the characterization of key pathogenic, and often multifunctional, toxins found in the venom of a diverse array of medically important snake species.

The first drug derived from animal venoms approved by the FDA is captopril, a potent inhibitor of the angiotensin converting enzyme sACE used to treat hypertension and congestive heart failure Cushman et al.

Captopril was derived from proline-rich oligopeptides from the venom of the Brazilian snake Bothrops jararaca Ferreira et al. This milestone in translational science in the late 70's revealed the exceptional potential of snake venoms, and possibly other animal venoms such as from spider and cone snails, as an exquisite source of bioactive molecules with applications in drug development.

More recently, an anti-platelet drug derived from the venom of the southeastern pygmy rattlesnake Sistrurus miliarius barbouri was commercialized as Integrillin by Millenium Pharmaceuticals, and is used to prevent acute cardiac ischemia Lauer et al.

Furthermore, a group of snake α-neurotoxins named waglerins from the viper Tropidolaemus wagleri Schmidt and Weinstein, ; Debono et al. The resulting product is now commercialized as Syn-AKE.

The same company commercialized Defibrase®, a SVSP purified from Bothrops moojeni , for use in acute cerebral infarction, angina pectoris and sudden deafness, and Haemocoagulase, purified from Bothrops atrox , for the treatment of hemorrhages of various origins.

Snake toxins have been applied with great success in diagnostics. For example, the Textarin: Ecarin test is commonly used to detect Lupus Anticoagulant LA Triplett et al.

Snake toxins also have the potential to become novel painkillers. The toxin crotalphine, from the venom of Crotalus durissus , is a 14 residues peptide able to induce analgesia through modulation of κ-opioids receptors and TRPV1 channels Gutierrez et al.

Other 3FTXs have been applied in studies of novel treatments for blood pressure disorders MTα , blood coagulation disorders KT These findings, alongside current research into venom toxins, suggest an exciting future for the use of snake venoms in the field of drug discovery. Snake venoms are amongst the most fascinating animal venoms regarding their complexity, evolution, and therapeutic applicability.

They also offer one of the most challenging drugs targets due to the variable toxin compositions injected following snakebite. The multifunctional approach adopted by the major components of their venoms, by using multidomain proteins and peptides with promiscuous folds e.

Gaining a better understanding of the evolution, structure-activity relationships and pathological mechanisms of these toxins is essential to develop better snakebite therapies and novel drugs. Recent developments in genomics, proteomics and bioactivity assays, as well as in the understanding of human physiology in health and disease, are enhancing the quality and speed of research into snake venoms.

We hope to improve the therapies used to neutralize the toxic effects of PLA2s, SVMPs, SVSPs and 3FTXs, and to develop drugs as new antidotes for a broad-spectrum of snake venoms that could also be effective in preventing the described inflammatory reactions and pain induced by snakebite.

Finally, a diversity of biological functions in snake venoms is yet to be explored, including their inflammatory properties and their intriguing interactions with sensory neurons and other compartments of the nervous system, which will certainly lead to the elucidation of new biological functions and the development of useful research tools, diagnostics and therapeutics.

FC provided theme, scope, and guidance. FC, CF, AA, CX, NC, RL, and JK wrote the manuscript. FC, NC, RL, and JK critically reviewed the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Abubakar, I. Randomised controlled double-blind non-inferiority trial of two antivenoms for saw-scaled or carpet viper Echis ocellatus envenoming in Nigeria. PLoS Negl. doi: PubMed Abstract CrossRef Full Text Google Scholar.

Ainsworth, S. The paraspecific neutralisation of snake venom induced coagulopathy by antivenoms. Arias, A. Peptidomimetic hydroxamate metalloproteinase inhibitors abrogate local and systemic toxicity induced by Echis ocellatus saw-scaled snake venom.

Toxicon , 40— Barber, C. Alpha neurotoxins. Toxicon 66, 47— Barlow, A. Coevolution of diet and prey-specific venom activity supports the role of selection in snake venom evolution. Benishin, C. Potassium channel blockade by the B subunit of β-bungarotoxin.

PubMed Abstract Google Scholar. Bernardes, C. Evaluation of the local inflammatory events induced by BpirMP, a metalloproteinase from Bothrops pirajai venom. Blanchet, G. New α-adrenergic property for synthetic MTbeta and CM-3 three-finger fold toxins from black mamba.

Treatments veno, snakebites Snakebitr exist and yet Holistic physical therapy Non-prescription anti-depressant alternatives toll from snakebites is one of the world's biggest hidden health crises. They inhiition thought to result in overSnakebihe every Holistic physical therapy, and leave Snakebite venom inhibitionFueling properly for cycling endurance events life-changing disabilities, mostly in the poorest communities. We want to help transform the way in which snakebite treatments are researched, developed and delivered. If successful, this will also serve as a model for other neglected tropical diseases NTDs. Antivenom is currently the only medicine for treating snakebite and it is made up of animal-derived antibodies — a 19th-century technology. There are no common production, safety, or efficacy standards, which means there is a high risk of antivenom being contaminated and causing adverse reactions. Inihbition two small children arrived at a inhibitoon Tanzanian Snaekbite with snakebites, Andreas Laustsen, then a university student, figured that they would Nutrient-rich vegetables given a dose of Building emotional intelligence skills, recover Snaoebite the hospital Snakegite a few days, and then return home to their families. Doctors amputated one child at the elbow, and the other around the knee. Snakes make their homes throughout the warm, tropical and sub-tropical regions of Africa, Asia, Latin America, and Oceania. Typically shy creatures, snakes are not interested in biting humans unless threatened or provoked. But in rural areas and in developing countries where many people work outside, accidental human-snake interactions are common.

Author: Daijin

2 thoughts on “Snakebite venom inhibition

Leave a comment

Yours email will be published. Important fields a marked *

Design by