Inhibitory Effects of Varespladib, CP471474, and Their Potential Synergistic Activity on Bothrops asper and Crotalus durissus cumanensis Venoms.

Snakebite is a neglected tropical disease that causes extensive mortality and morbidity in rural communities. Antivenim sera are the currently approved therapy for snake bites; however, they have some therapeutic limitations that have been extensively documented. Recently, small molecule toxin inhibitors have received significant attention as potential alternatives or co-adjuvant to immunoglobulin-based snakebite therapies. Thus, in this study, we evaluated the inhibitory effects of the phospholipase A2 inhibitor varespladib and the metalloproteinase inhibitor CP471474 and their synergistic effects on the lethal, edema-forming, hemorrhagic, and myotoxic activities of Bothrops asper and Crotalus durissus cumanensis venoms from Colombia. Except for the preincubation assay of the lethal activity with B. asper venom, the mixture showed the best inhibitory activity. Nevertheless, the mix did not display statistically significant differences to varespladib and CP471474 used separately in all assays. In preincubation assays, varespladib showed the best inhibitory activity against the lethal effect induced by B. asper venom. However, in independent injection assays, the mix of the compounds partially inhibited the lethal activity of both venoms (50%). In addition, in the assays to test the inhibition of edema-forming activity, the mixture exhibited the best inhibitory activity, followed by Varespladib, but without statistically significant differences (p > 0.05). The combination also decreased the myotoxic activity of evaluated venoms. In these assays, the mix showed statistical differences regarding CP471474 (p < 0.05). The mixture also abolished the hemorrhagic activity of B. asper venom in preincubation assays, with no statistical differences to CP471474. Finally, the mixture showed inhibition in studies with independent administration in a time-dependent manner. To propose a mode of action of varespladib and CP471474, molecular docking was performed. PLA2s and SVMPs from tested venoms were used as targets. In all cases, our molecular modeling results suggested that inhibitors may occupy the substrate-binding cleft of the enzymes, which was supported by specific interaction with amino acids from the active site, such as His48 for PLA2s and Glu143 for the metalloproteinase. In addition, varespladib and CP471474 also showed interaction with residues from the hydrophobic channel in PLA2s and substrate binding subsites in the SVMP. Our results suggest a synergistic action of the mixed inhibitors and show the potential of varespladib, CP471474, and their mixture to generate new treatments for snakebite envenoming with application in the field or as antivenom co-adjuvants.

Phenotypic and functional variation in venom and venom resistance of two sympatric rattlesnakes and their prey.

Predator-prey interactions often lead to the coevolution of adaptations associated with avoiding predation and, for predators, overcoming those defences. Antagonistic coevolutionary relationships are often not simple interactions between a single predator and prey but rather a complex web of interactions between multiple coexisting species. Coevolution between venomous rattlesnakes and small mammals has led to physiological venom resistance in several mammalian taxa. In general, viperid venoms contain large quantities of snake venom metalloproteinase toxins (SVMPs), which are inactivated by SVMP inhibitors expressed in resistant mammals. We explored variation in venom chemistry, SVMP expression, and SVMP resistance across four co-distributed species (California Ground Squirrels, Bryant's Woodrats, Southern Pacific Rattlesnakes, and Red Diamond Rattlesnakes) collected from four different populations in Southern California. Our aim was to understand phenotypic and functional variation in venom and venom resistance in order to compare coevolutionary dynamics of a system involving two sympatric predator-prey pairs to past studies that have focused on single pairs. Proteomic analysis of venoms indicated that these rattlesnakes express different phenotypes when in sympatry, with Red Diamonds expressing more typical viperid venom (with a diversity of SVMPs) and Southern Pacifics expressing a more atypical venom with a broader range of non-enzymatic toxins. We also found that although blood sera from both mammals were generally able to inhibit SVMPs from both rattlesnake species, inhibition depended strongly on the snake population, with snakes from one geographic site expressing SVMPs to which few mammals were resistant. Additionally, we found that Red Diamond venom, rather than woodrat resistance, was locally adapted. Our findings highlight the complexity of coevolutionary relationships between multiple predators and prey that exhibit similar offensive and defensive strategies in sympatry.

CatroxMP-II: a heme-modulated fibrinogenolytic metalloproteinase isolated from Crotalus atrox venom.

It has been recently demonstrated that the hemotoxic venom activity of several species of snakes can be inhibited by carbon monoxide (CO) or a metheme forming agent. These and other data suggest that the biometal, heme, may be attached to venom enzymes and may be modulated by CO. A novel fibrinogenolytic metalloproteinase, named CatroxMP-II, was isolated and purified from the venom of a Crotalus atrox viper, and subjected to proteolysis and mass spectroscopy. An ion similar to the predicted singly charged m/z of heme at 617.18 was identified. Lastly, CORM-2 (tricarbonyldichlororuthenium (II) dimer, a CO releasing molecule) inhibited the fibrinogenolytic effects of CatroxMP-II on coagulation kinetics in human plasma. In conclusion, we present the first example of a snake venom metalloproteinase that is heme-bound and CO-inhibited.

Inhibition of a Snake Venom Metalloproteinase by the Flavonoid Myricetin.

Most of the snakebite envenomations in Central and South America are caused by species belonging to Bothrops genus. Their venom is composed mainly by zinc-dependent metalloproteinases, responsible of the hemorrhage characteristic of these envenomations. The aim of this study was to determine the inhibitory ability of ten flavonoids on the in-vitro proteolytic activity of Bothrops atrox venom and on the hemorrhagic, edema-forming and myonecrotic activities of Batx-I, the most abundant metalloproteinase isolated from this venom. Myricetin was the most active compound, exhibiting an IC 50 value of 150 µ M and 1021 µ M for the inhibition of proteolytic and hemorrhagic activity, respectively. Independent injection experiments, with a concentration of 1600 µ M of myricetin administered locally, immediately after toxin injection, demonstrated a reduction of 28 ± 6 % in the hemorrhagic lesion. Additionally, myricetin at concentrations 800, 1200 and 1600 µ M promoted a reduction in plasma creatine kinase activity induced by Batx-I of 21 ± 2 % , 60 ± 5 % and 63 ± 2 % , respectively. Molecular dynamics simulations coupled with the adaptive biasing method suggest that myricetin can bind to the metalloproteinase active site via formation of hydrogen bonds between the hydroxyl groups 3', 4' and 5' of the benzyl moiety and amino acid Glu143 of the metalloproteinase. The hydroxyl substitution pattern of myricetin appears to be essential for its inhibitory activity. Based on this evidence, myricetin constitutes a candidate for the development of inhibitors to reduce local tissue damage in snakebite envenomations.

Effects of purified human fibrinogen modified with carbon monoxide and iron on coagulation in rabbits injected with Crotalus atrox venom.

While snake venom derived enzymes, such as the thrombin-like activity possessing ancrod, have been used to treat thrombotic disease by defibrinogenating patients, the therapeutic potential of fibrinogenolytic snake venom enzymes, such as those derived from Crotalus atrox, have not been fully explored. However, one of the potential risks of administering fibrinogenolytic enzymes to effect defibrinogenation is hemorrhage secondary to hypofibrinogenemia. The present investigation sought to determine if human fibrinogen modified with carbon monoxide (CO) and iron (Fe) could resist degradation by C. atrox venom as has been seen in vitro in a recently developed rabbit model of envenomation. Compared with unmodified human fibrinogen, CO/Fe modified fibrinogen administered prior to envenomation had significantly shorter onset of coagulation and greater strength; however, when administered after envenomation, there was no differences between the two types of fibrinogen. Of interest, when administered after envenomation, both types of fibrinogen delayed the onset of coagulation while increasing plasma clot strength, a mixed effect likely secondary to formation of fibrinogen degradation products. Further preclinical investigations are needed to further define the benefits and risks of the use of fibrinogenolytic enzymes as defibrinogenating agents, as well as the risks of the "biochemical brakes" used to modulate the activity or substrate of the fibrinogenolytic enzyme.

Structures and Functions of Snake Venom Metalloproteinases (SVMP) from Protobothrops venom Collected in Japan.

Snake venom metalloproteinases (SVMP) are widely distributed among the venoms of Crotalinae and Viperidae, and are organized into three classes (P-I, P-II and P-III) according to their size and domain structure. P-I SVMP are the smallest SVMP, as they only have a metalloproteinase (M) domain. P-II SVMP contain a disintegrin-like (D) domain, which is connected by a short spacer region to the carboxyl terminus of the M domain. P-III SVMP contain a cysteine-rich (C) domain, which is attached to the carboxyl terminus of the D domain. Some SVMP exhibit hemorrhagic activity, whereas others do not. In addition, SVMP display fibrinolytic/fibrinogenolytic (FL) activity, and the physiological functions of SVMP are controlled by their structures. Furthermore, these proteinases also demonstrate fibrinogenolytic and proteolytic activity against synthetic substrates for matrix metalloproteinases and ADAM (a disintegrin and metalloproteinase). This article describes the structures and FL, hemorrhagic, and platelet aggregation-inhibiting activity of SVMP derived from Protobothrops snake venom that was collected in Japan.

Thrombolytic protein from cobra venom with anti-adhesive properties.

A metalloproteinase anticoagulant toxin of molecular weight 66 kDa has been purified from the venom of Indian monocled cobra (Naja kaouthia). This toxin named as NKV 66 cleaved fibrinogen in a dose and time dependent manner. The digestion process was specific to Aα chain and cleaved fibrinogen to peptide fragments. NKV 66 completely liquefied the fibrin clots developed in vitro in 18 h. Plasma recalcification time and thrombin time were significantly prolonged following treatment of plasma with NKV 66. NKV 66 significantly inhibited ADP and collagen induced platelet aggregation in a dose dependent manner. It showed disintegrin like activity on A549 cells cultured in vitro. About 40% inhibition of adherence of A549 cells to matrix was observed following NKV 66 treatment also NKV 66 treated A549 cells were drastically inhibited from passing through the matrix in cell invasion assays in vitro, suggesting anti-adhesive properties of NKV 66.

Recombinant expression of the precursor of the hemorrhagic metalloproteinase HF3 and its non-catalytic domains using a cell-free synthesis system.

Snake venom metalloproteinases (SVMPs) participate in snakebite pathology such as hemorrhage, inflammation, and necrosis. They are synthesized as latent multi-domain precursors whose processing generates either catalytically active enzymes or free non-enzymatic domains. Recombinant expression of the precursor of P-III class SVMPs has failed due to the instability of the multi-domain polypeptide structure. Conversely, functional recombinant non-catalytic domains were obtained by prokaryotic expression systems. Here, we show for the first time the recombinant expression of the precursor of HF3, a highly hemorrhagic SVMP from Bothrops jararaca, and its non-catalytic domains, using an E. coli-based cell-free synthesis system. The precursor of HF3, composed of pro-, metalloproteinase-, disintegrin-like-, and cysteine-rich domains, and containing 38 Cys residues, was successfully expressed and purified. A protein composed of the disintegrin-like and cysteine-rich domains (DC protein) and the cysteine-rich domain alone (C protein) were expressed in vitro individually and purified. Both proteins were shown to be functional in assays monitoring the interaction with matrix proteins and in modulating the cleavage of fibrinogen by HF3. These data indicate that recombinant expression using prokaryotic-based cell-free synthesis emerges as an attractive alternative for the study of the structure and function of multi-domain proteins with a high content of Cys residues.

Decreased snake venom metalloproteinase effects via inhibition of enzyme and modification of fibrinogen.

Since the introduction of antivenom administration 120 years ago to treat venomous snake bit, it has been the gold standard for saving life and limb. However, this therapeutic approach is not always effective and not without potential life-threatening side effects. We tested a new paradigm to abrogate the plasmatic anticoagulant effects of fibrinogenolytic snake venom metalloproteinases by modification of fibrinogen with iron and carbon monoxide and by inhibiting these Zn(2+) dependent metalloproteinases directly with carbon monoxide exposure. Assessment of the fibrinogenolytic effects of venoms collected from Puff adder, Gaboon viper and Indian cobra snakes on plasmatic coagulation kinetics was performed with thrombelastography. Pretreatment of plasma with iron and carbon monoxide exposure markedly attenuated the effects of all three venoms, and direct pretreatment of each venom with carbon monoxide also significantly decreased the ability to compromise coagulation. These results demonstrated that the introduction of a transition metal (e.g., modulation of the α-chain of fibrinogen with iron), modulation of transition metal in heme (e.g., carbon monoxide modulation of fibrinogen-bound heme iron), and direct inhibition of transition metal containing venom enzymes (e.g., CO binding to Zn(2+) or displacing Zn(2+) from the catalytic site) significantly decreased fibrinogenolytic activity. This biometal modulation strategy to attenuate the anticoagulant effects of snake venom metalloproteinases could potentially diminish hemostatic injury in envenomed patients until antivenom can be administered.

P-I class metalloproteinase from Bothrops moojeni venom is a post-proline cleaving peptidase with kininogenase activity: insights into substrate selectivity and kinetic behavior.

Snake venom metalloproteinases (SVMPs) belonging to P-I class are able to hydrolyze extracellular matrix proteins and coagulation factors triggering local and systemic reactions by multiple molecular mechanisms that are not fully understood. BmooMPα-I, a P-I class SMVP from Bothrops moojeni venom, was active upon neuro- and vaso-active peptides including angiotensin I, bradykinin, neurotensin, oxytocin and substance P. Interestingly, BmooMPα-I showed a strong bias towards hydrolysis after proline residues, which is unusual for most of characterized peptidases. Moreover, the enzyme showed kininogenase activity similar to that observed in plasma and cells by kallikrein. FRET peptide assays indicated a relative promiscuity at its S2-S'2 subsites, with proline determining the scissile bond. This unusual post-proline cleaving activity was confirmed by the efficient hydrolysis of the synthetic combinatorial library MCA-GXXPXXQ-EDDnp, described as resistant for canonical peptidases, only after Pro residues. Structural analysis of the tripeptide LPL complexed with BmooMPα-I, generated by molecular dynamics simulations, assisted in defining the subsites and provided the structural basis for subsite preferences such as the restriction of basic residues at the S2 subsite due to repulsive electrostatic effects and the steric impediment for large aliphatic or aromatic side chains at the S1 subsite. These new functional and structural findings provided a further understanding of the molecular mechanisms governing the physiological effects of this important class of enzymes in envenomation process.

Structural analysis and characterization of new small serum proteins from the serum of a venomous snake (Gloydius blomhoffii).

Some snakes have several anti-toxic proteins in their sera that neutralize their own venom. Five new small serum proteins (SSPs) were isolated from Japanese mamushi (Gloydius blomhoffii) serum by gel-filtration and RP-HPLC, and their N-Terminal sequences were determined. The amino acid sequences of the precursor proteins were deduced from the nucleotide sequences of cDNAs encoding them. Due to the sequence similarity to those of SSPs in habu snake (Protobothrops flavoviridis) serum (>75% identity), these proteins were designated mSSP-1 to mSSP-5 as the homologs of habu proteins. mSSP-1 was stable at 100 °C and in the pH range of 1-10, and inhibited the proteolytic activity of a certain snake venom metalloproteinase. The inhibitory activity was extinguished by modifying the amino groups of mSSP-1. mSSP-1 is the first prostate secretory protein of the 94 amino acid-family protein with a carbohydrate chain in the Asn37 residue.

Action of BjussuMP-II, a snake venom metalloproteinase isolated from Bothrops jararacussu venom, on human neutrophils.

Snake venom metalloproteinases (SVMPs) are enzymatic proteins present in large amounts in snake venoms presenting proteolytic, hemorrhagic, and coagulant activities. BjussuMP-II, a class P-I SVMP, isolated from the Bothrops jararacussu snake venom does not have relevant hemorrhagic activity but presents fibrinolytic, fibrinogenolytic, antiplatelet, gelatinolytic, and collagenolytic action. This study aimed to verify the action of BjussuMP-II on human neutrophil functionality focusing on the lipid bodies formation and hydrogen peroxide production, the release of dsDNA through colorimetric and microscopic assays, and cytokines by immunoenzymatic assays. Results showed that BjussuMP-II at concentrations of 1.5 up to 50 µg/mL for 24 h is not toxic to human neutrophils using an MTT assay. Under non-cytotoxic concentrations, BjussuMP-II can induce an increase in the formation of lipid bodies, production of hydrogen peroxide and cytokines [tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and interleukin-8 (IL-8)] liberation and, the release of dsDNA to form NETs. Taken together, the data obtained show for the first time that BjussuMP-II has a pro-inflammatory action and activates human neutrophils that can contribute to local damage observed in snakebite victims.

Type I-like metalloproteinase in the venom of the West African saw-scaled carpet viper (Echis ocellatus) has anti-trypanosomal activity against African trypanosomes.

African trypanosomiasis is an infectious disease caused by hemoparasites of the genus Trypanosoma and remains a major health problem in Africa - killing around 4000 people and animals worth an estimated $5 billion, annually. The absence of a vaccine and satisfactory drug against African trypanosomiasis (AT) necessitates the continued search for new chemotherapy options. Owing to the rich biochemical diversity in snake venom, it has recently become a source of therapeutic peptides that are being explored for the development of novel drug candidates for diverse ailments such as cancers and infectious diseases. To explore this, Echis ocellatus venom (EOV) was investigated for the presence of an anti-Trypanosoma factor, with the subsequent aim to isolate and identify it. Crude EOV was collected and tested in vitro on the bloodstream form (BSF) i.e. long and slender morphological form of Trypanosoma brucei and T. congolense. This initial testing was followed by a sequential anti-trypanosomal assay guided purification of EOV using ethanol precipitation, distillation, and ion exchange (IEX) chromatography to obtain the active trypanocidal component. The purified anti-Trypanosoma factor, estimated to be a 52-kDa protein on SDS-PAGE, was subjected to in-gel trypsin digestion and 2D RP HPLC-MS/MS to identify the protein. The anti-Trypanosoma factor was revealed to be a zinc-dependent metalloproteinase that contains the HEXXHXXGXXH adamalysin motif. This protein may provide a conceptual framework for the possible design of a safe and effective anti-trypanosomal peptide for the treatment of AT.

Rhomb-I, a P-I metalloproteinase from Lachesis muta rhombeata venom degrades vessel extra cellular matrix components and impairs platelet aggregation.

Rhomb-I, a 23-kDa metalloproteinase was isolated from L. m. rhombeata venom. Its dimethylcasein proteolysis was abolished by metal chelators, and slightly enhanced by Ca2+ and Mg2+ ions, but inhibited by Co2+, Zn2+ and α2-macroglobulin. In aqueous solution, rhomb-I autoproteolyzed to a 20- and 11-kDa fragments at 37 °C. The amino acid sequence showed high homology with other snake venom metalloproteinases. Rhomb-I causes hemorrhage that may be ascribed to hydrolysis of essential basement membrane, extracellular matrix and plasma proteins. It preferentially cleaves the α-chains of fibrin (ogen). Rhomb-I inhibited convulxin- and von Willebrand factor (vWF)-induced aggregation on human platelets without significant effect on collagen-stimulated aggregation or other effectors. It digests vWF into a low-molecular-mass multimers of vWF and a rvWF-A1 domain to a 27-kDa fragment as revealed by western blotting with mouse anti-rvWF A1-domain IgG. Incubation of platelets with rhomb-I resulted in adhesion to and cleavage of platelet receptors glycoprotein (GP)Ibα and GPVI to release a 55-kDa soluble form. Both membrane glycoproteins GPIbα that binds vWF, together with GPVI which binds collagen, play a key role in mediating platelet adhesion/activation and can initiate (patho)physiological thrombus formation. Conclusions: rhomb-I is implicated in the pathophysiology of Lachesis envenoming by disrupting vasculature, hemostasis and platelet aggregation through impairing vWF-GPIb axis and blocking GPVI-collagen binding.

Protein-protein interaction of LDH and CRP-1 with hematotoxin snake venom proteins of all species of snake: An in silico approach.

Objective: Snake bite-induced elevation of serum LDH and CRP-1 is considered as useful biomarkers of hemotoxic. The snake venom contains proteins and may result in various envenomation such as bleeding, inflammation, and pain, cytotoxic, cardiotoxic, or neurotoxic effects. This in silico study was aimed to screen the snake venom proteins and to find out the most interactive hemotoxic venom protein against LDH and CRP-1 proteins as biomarkers. Materials and Methods: To validate the hypothesis of the prospective interaction of snake venom proteins, molecular docking analysis was used in the current work by deploying a cutting-edge docking program. Snake venom peptides were screened from literature and both peptide as well as target protein were obtained from PDB. HDOCK online server was used for the molecular docking analysis of target proteins with hemotoxic snake venom peptides. Further, the toxicity properties of each docked complex of target proteins were subjected for ADME/T analysis. Results: The selected snake venom peptides were subjected to molecular docking study and the results generated from computational-based approach reveals that all the hematotoxin snake venom proteins are interactive with LDH and CRP-1 peptide. Further, this study indicates that snake venom metalloproteinase (SVMPS) peptide may be considered as the best interactive protein with both LDH and CRP-1 proteins; also, ADME/T screening revealed that all docked complex are safe and follow toxicity properties. Conclusion: This in silico study clearly shows that the greatest interaction of SVMPS peptide with LDH and CRP-1 may be due to strong binding in the active site of the target proteins LDH and CRP-1 with SVMPS. Results, further, confirmed LDH and CRP-1 as potential biomarkers against hemotoxic snake venoms. This study should be validated by in vitro and in vivo analysis as well as specific species snake venom should be assessed. For further studies, SVMPS can be consider as therapeutic point of view.

Systemic toxicity of snake venom metalloproteinases: Multi-omics analyses of kidney and blood plasma disturbances in a mouse model.

Snakebite envenomation is classified as a Neglected Tropical Disease. Bothrops jararaca venom induces kidney injury and coagulopathy. HF3, a hemorrhagic metalloproteinase of B. jararaca venom, participates in the envenomation pathogenesis. We evaluated the effects of HF3 in mouse kidney and blood plasma after injection in the thigh muscle, mimicking a snakebite. Transcriptomic analysis showed differential expression of 31 and 137 genes related to kidney pathology after 2 h and 6 h, respectively. However, only subtle changes were observed in kidney proteome, with differential abundance of 15 proteins after 6 h, including kidney injury markers. N-terminomic analysis of kidney proteins showed 420 proteinase-generated peptides compatible with meprin specificity, indicating activation of host proteinases. Plasma analysis revealed differential abundance of 90 and 219 proteins, respectively, after 2 h and 6 h, including coagulation-cascade and complement-system components, and creatine-kinase, whereas a semi-specific search of N-terminal peptides indicated activation of endogenous proteinases. HF3 promoted host reactions, altering the gene expression and the proteolytic profile of kidney tissue, and inducing plasma proteome imbalance driven by changes in abundance and proteolysis. The overall response of the mouse underscores the systemic action of a hemorrhagic toxin that transcends local tissue damage and is related to known venom-induced systemic effects.

Montane Rattlesnakes in México: Venoms of Crotalus tancitarensis and Related Species within the Crotalus intermedius Group.

The Crotalus intermedius group is a clade of rattlesnakes consisting of several species adapted to a high elevation habitat, primarily in México. Crotalus tancitarensis was previously classified as C. intermedius, until individuals occurring on Cerro Tancítaro in Michoacán, México, were reevaluated and classified as a new species (C. tancitarensis) based on scale pattern and geographic location. This study aimed to characterize the venom of C. tancitarensis and compare the venom profile to those of other species within the Crotalus intermedius group using gel electrophoresis, biochemical assays, reverse-phase high performance liquid chromatography, mass spectrometry, and lethal toxicity (LD50) assays. Results show that the venom profiles of species within the Crotalus intermedius group are similar, but with distinct differences in phospholipase A2 (PLA2), metalloproteinase PI (SVMP PI), and kallikrein-like serine proteinase (SVSP) activity and relative abundance. Proteomic analysis indicated that the highland forms produce venoms with 50-60 protein isoforms and a composition typical of type I rattlesnake venoms (abundant SVMPs, lack of presynaptic PLA2-based neurotoxins), as well as a diversity of typical Crotalus venom components such as serine proteinases, PLA2s, C-type lectins, and less abundant toxins (LAAOs, CRiSPs, etc.). The overall venom profile of C. tancitarensis appears most similar to C. transversus, which is consistent with a previous mitochondrial DNA analysis of the Crotalus intermedius group. These rattlesnakes of the Mexican highlands represent a radiation of high elevation specialists, and in spite of divergence of species in these Sky Island habitats, venom composition of species analyzed here has remained relatively conserved. The majority of protein family isoforms are conserved in all members of the clade, and as seen in other more broadly distributed rattlesnake species, differences in their venoms are largely due to relative concentrations of specific components.

The secretory phenotypes of envenomed cells: Insights into venom cytotoxicity.

Snake envenomation is listed as Category A Neglected Tropical Diseases (NTD) by World Health Organization, indicates a severe public health problem. The global figures for envenomation cases are estimated to be more than 1.8 million annually. Even if the affected victims survive the envenomation, they might suffer from permanent morbidity due to local envenomation. One of the most prominent local envenomation is dermonecrosis. Dermonecrosis is a pathophysiological outcome of envenomation that often causes disability in the victims due to surgical amputations, deformities, contracture, and chronic ulceration. The key venom toxins associated with this local symptom are mainly attributed to substantial levels of enzymatic and non-enzymatic toxins as well as their possible synergistic actions. Despite so, the severity of the local tissue damage is based on macroscopic observation of the bite areas. Furthermore, limited knowledge is known about the key biomarkers involved in the pathogenesis of dermonecrosis. The current immunotherapy with antivenom is also ineffective against dermonecrosis. These local effects eventually end up as sequelae. There is also a global shortage of toxins-targeted therapeutics attributed to inadequate knowledge of the actual molecular mechanisms of cytotoxicity. This chapter discusses the characterization of secretory phenotypes of dermonecrosis as an advanced tool to indicate its severity and pathogenesis in envenomation. Altogether, the secretory phenotypes of envenomed cells and tissues represent the precise characteristics of dermonecrosis caused by venom toxins.

A Metalloproteinase Cocktail from the Venom of Protobothrops flavoviridis Cleaves Amyloid Beta Peptides at the α-Cleavage Site.

A disintegrin and metalloproteinase (ADAM) family proteins are a major class of membrane-anchored multidomain proteinases that are responsible for the shedding of cell surface protein ectodomains, including amyloid precursor protein (APP). Human ADAM 9, 10, and 17 proteolyze APPs and produce non-amyloid-genic p3 peptides, instead of neurotoxic amyloid-ß peptides (Aßs; Aß40 and Aß42), which form fibrils and accumulate in the brain of patients with Alzheimer's disease (AD). The ADAM family is closely related to snake venom metalloproteinases (SVMPs), which are derived from ancestral ADAMs but act as soluble proteinases. To test the therapeutic potential of SVMPs, we purified SVMPs from Protobothrops flavoviridis venom using metal ion affinity and pooled into a cocktail. Thus, 9 out of 11 SVMPs in the P. flavoviridis genome were identified in the cocktail. SVMPs inhibited Aß secretion when added to human cell culture medium without affecting APP proteolysis. SVMPs degraded synthetic Aß40 and Aß42 peptides at the same cleavage site (α-site of APP) as ADAM9, 10, and 17. SVMPs did not degrade Aß fibrils but interfered with their formation, assessed using thioflavin-T. Thus, SVMPs have therapeutic potential for AD as an Aß-degrading protease, and the finding adds to the discovery of bioactive peptides from venoms as novel therapeutics.

In vitro and in vivo preclinical venom inhibition assays identify metalloproteinase inhibiting drugs as potential future treatments for snakebite envenoming by Dispholidus typus.

Snakebite envenoming affects more than 250,000 people annually in sub-Saharan Africa. Envenoming by Dispholidus typus (boomslang) results in venom-induced consumption coagulopathy (VICC), whereby highly abundant prothrombin-activating snake venom metalloproteinases (SVMPs) consume clotting factors and deplete fibrinogen. The only available treatment for D. typus envenoming is the monovalent SAIMR Boomslang antivenom. Treatment options are urgently required because this antivenom is often difficult to source and, at US$6000/vial, typically unaffordable for most snakebite patients. We therefore investigated the in vitro and in vivo preclinical efficacy of four SVMP inhibitors to neutralise the effects of D. typus venom; the matrix metalloproteinase inhibitors marimastat and prinomastat, and the metal chelators dimercaprol and DMPS. The venom of D. typus exhibited an SVMP-driven procoagulant phenotype in vitro. Marimastat and prinomastat demonstrated equipotent inhibition of the SVMP-mediated procoagulant activity of the venom in vitro, whereas dimercaprol and DMPS showed considerably lower potency. However, when tested in preclinical murine models of envenoming using mixed sex CD1 mice, DMPS and marimastat demonstrated partial protection against venom lethality, demonstrated by prolonged survival times of experimental animals, whereas dimercaprol and prinomastat failed to confer any protection at the doses tested. The preclinical results presented here demonstrate that DMPS and marimastat show potential as novel small molecule-based therapeutics for D. typus snakebite envenoming. These two drugs have been previously shown to be effective against Echis ocellatus VICC in preclinical models, and thus we conclude that marimastat and DMPS should be further explored as potentially valuable early intervention therapeutics to broadly treat VICC following snakebite envenoming in sub-Saharan Africa.