Assessing Target Specificity of the Small Molecule Inhibitor MARIMASTAT to Snake Venom Toxins: A Novel Application of Thermal Proteome Profiling.

New treatments that circumvent the pitfalls of traditional antivenom therapies are critical to address the problem of snakebite globally. Numerous snake venom toxin inhibitors have shown promising cross-species neutralization of medically significant venom toxins in vivo and in vitro. The development of high-throughput approaches for the screening of such inhibitors could accelerate their identification, testing, and implementation and thus holds exciting potential for improving the treatments and outcomes of snakebite envenomation worldwide. Energetics-based proteomic approaches, including thermal proteome profiling and proteome integral solubility alteration (PISA) assays, represent "deep proteomics" methods for high throughput, proteome-wide identification of drug targets and ligands. In the following study, we apply thermal proteome profiling and PISA methods to characterize the interactions between venom toxin proteoforms in Crotalus atrox (Western Diamondback Rattlesnake) and the snake venom metalloprotease (SVMP) inhibitor marimastat. We investigate its venom proteome-wide effects and characterize its interactions with specific SVMP proteoforms, as well as its potential targeting of non-SVMP venom toxin families. We also compare the performance of PISA thermal window and soluble supernatant with insoluble precipitate using two inhibitor concentrations, providing the first demonstration of the utility of a sensitive high-throughput PISA-based approach to assess the direct targets of small molecule inhibitors for snake venom.

The anti-myotoxic effects and mechanisms of Sinonatrix annularis serum and a novel plasma metalloproteinase inhibitor towards Deinagkistrodon acutus envenomation.

Non-venomous snakes commonly evolve natural resistance to venom to escape predators. Sinonatrix annularis serum has been shown to inhibit Deinagkistrodon acutus venom-induced hemorrhage and upregulation of serum CK, CK-MB, LDH, AST and ALT levels. Using TMT-labeled proteomics analysis, 168 proteins were found to be altered significantly in the envenomed gastrocnemius muscle and categorized into pathways such as complement and coagulation cascades, leukocyte transendothelial migration, and JAK/STAT signaling. These alterations were mitigated by S. annularis serum. Subsequently, a novel metalloproteinase inhibitor, SaMPI, was isolated from S. annularis serum by two-step chromatography. It showed strong antidotal effects against D. acutus envenomation, including inhibition of subcutaneous bleeding caused by crude venom and DaMP (a metalloproteinase derived from D. acutus) activity in a 1:1 ratio. Histology and immunoblotting analyses demonstrated that SaMPI mitigated myonecrosis, reduced neutrophil infiltration and local inflammatory factor release, and retarded JAK/STAT and MAPK signaling activation. Analysis of the SaMPI gene cloned by 5'-RACE revealed a shared sequence identity of 58-79% with other SVMP inhibitors. These findings demonstrate the protective effects of SaMPI and indicate its potential value as a candidate for viper bite adjuvant therapy.

Gamma irradiated protease from Echis pyramidum venom: A promising immunogen to improve viper bites treatment.

Echis pyramidum (Epy) is a venomous snake belongs to Viperidae family; it causes fetal coagulopathy systemic effects and death. Searching for more effective and safe antivenom is mandatory for viper bites treatment. Proteases are the most lethal components in viper venom inducing hemorrhage, edema and coagulation problems. Thus, the study aims to evaluate the potency of the prepared antisera and their neutralizing properties against the biological activities induced by whole Epy venom individually. Echis pyramidum metalloprotease enzyme (60 kDa) was purified using size-exclusion followed by DEAE-ion exchange chromatography. The purified Epy metalloprotease enzyme (SVMP) was detoxified with 1.5 kGy gamma rays from cobalt60 gamma cell and used for immunization. 1.5 kGy irradiated Epy metalloprotease (SVMPi) showed less lethal activity (LD50) compared to the corresponding native immunogen. The prepared antisera boosted against whole Epy venom (WV), 1.5 kGy irradiated whole Epy venom (WVi), SVMP and SVMPi were tested for neutralization of lethality and biological activities induced by Epy venom. The antibodies elicited against WVi and SVMPi were 30,000 and 20,000 EU, respectively. The anti-SVMPi serum showed the highest neutralization of lethality (ED50) compared to the other prepared antisera. In addition, it prolonged the clotting time from 49.0 ± 2.5 to 176.2 ± 1.4 s. Furthermore, it demonstrated a highly neutralizing activity against edema induction and hemorrhage of Epy venom by 66.8% and 94.3%, respectively compared with the other prepared antisera. These findings would encourage further studies for using gamma irradiated purified fraction(s) from different snake venoms as safe antigen(s) to produce more effective antivenoms.

Deep Profiling of the Cleavage Specificity and Human Substrates of Snake Venom Metalloprotease HF3 by Proteomic Identification of Cleavage Site Specificity (PICS) Using Proteome Derived Peptide Libraries and Terminal Amine Isotopic Labeling of Substrates (TAILS) N-Terminomics.

Snakebite is a major medical concern in many parts of the world with metalloproteases playing important roles in the pathological effects of Viperidae venoms, including local tissue damage, hemorrhage, and coagulopathy. Hemorrhagic Factor 3 (HF3), a metalloprotease from Bothrops jararaca venom, induces local hemorrhage and targets extracellular matrix (ECM) components, including collagens and proteoglycans, and plasma proteins. However, the full substrate repertoire of this metalloprotease is unknown. We report positional proteomic studies identifying >2000 N-termini, including neo-N-termini of HF3 cleavage sites in mouse embryonic fibroblast secretome proteins. Terminal amine isotopic labeling of substrates (TAILS) analysis identified a preference for Leu at the P1' position among candidate HF3 substrates including proteins of the ECM and focal adhesions and the cysteine protease inhibitor cystatin-C. Interestingly, 190 unique peptides matched to annotated cleavage sites in the TopFIND N-termini database, suggesting that these cleavages occurred at a site prone to cleavage or might have been generated by other proteases activated upon incubation with HF3, including caspases-3 and -7, cathepsins D and E, granzyme B, and MMPs 2 and 9. Using Proteomic identification of cleavage site specificity (PICS), a tryptic library derived from THP-1 monocytic cells was used as HF3 substrates for identifying protease cleavage sites and sequence preferences in peptides. A total of 799 unique cleavage sites were detected and, in accordance with TAILS analysis using native secreted protein substrates of MEF cells, revealed a clear preference for Leu at P1'. Taken together, these results greatly expand the known substrate degradome of HF3 and reveal potential new targets, which may serve as a basis to better elucidate the complex pathophysiology of snake envenomation.

Deep profiling of the cleavage specificity and human substrates of snake venom metalloprotease HF3 by proteomic identification of cleavage site specificity (PICS) using proteome derived peptide libraries and terminal amine isotopic labeling of substrates (TAILS) n-terminomics

Snakebite is a major medical concern in many parts of the world with metalloproteases playing important roles in the pathological effects of Viperidae venoms, including local tissue damage, hemorrhage, and coagulopathy. Hemorrhagic Factor 3 (HF3), a metalloprotease from Bothrops jararaca venom, induces local hemorrhage and targets extracellular matrix (ECM) components, including collagens and proteoglycans, and plasma proteins. However, the full substrate repertoire of this metalloprotease is unknown. We report positional proteomic studies identifying >2000 N-termini, including neo-N-termini of HF3 cleavage sites in mouse embryonic fibroblast secretome proteins. Terminal amine isotopic labeling of substrates (TAILS) analysis identified a preference for Leu at the P1′ position among candidate HF3 substrates including proteins of the ECM and focal adhesions and the cysteine protease inhibitor cystatin-C. Interestingly, 190 unique peptides matched to annotated cleavage sites in the TopFIND N-termini database, suggesting that these cleavages occurred at a site prone to cleavage or might have been generated by other proteases activated upon incubation with HF3, including caspases-3 and -7, cathepsins D and E, granzyme B, and MMPs 2 and 9. Using Proteomic identification of cleavage site specificity (PICS), a tryptic library derived from THP-1 monocytic cells was used as HF3 substrates for identifying protease cleavage sites and sequence preferences in peptides. A total of 799 unique cleavage sites were detected and, in accordance with TAILS analysis using native secreted protein substrates of MEF cells, revealed a clear preference for Leu at P1′. Taken together, these results greatly expand the known substrate degradome of HF3 and reveal potential new targets, which may serve as a basis to better elucidate the complex pathophysiology of snake envenomation.

Differential procoagulant effects of saw-scaled viper (Serpentes: Viperidae: Echis) snake venoms on human plasma and the narrow taxonomic ranges of antivenom efficacies.

Saw-scaled vipers (genus Echis) are one of the leading causes of snakebite morbidity and mortality in parts of Sub-Saharan Africa, the Middle East, and vast regions of Asia, constituting a public health burden exceeding that of almost any other snake genus globally. Venom-induced consumption coagulopathy, owing to the action of potent procoagulant toxins, is one of the most relevant clinical manifestations of envenomings by Echis spp. Clinical experience and prior studies examining a limited range of venoms and restricted antivenoms have demonstrated for some antivenoms an extreme lack of antivenom cross-reactivity between different species of this genus, sometimes resulting in catastrophic treatment failure. This study undertook the most comprehensive testing of Echis venom effects upon the coagulation of human plasma, and also the broadest examination of antivenom potency and cross-reactivity, to-date. 10 Echis species/populations and four antivenoms (two African, two Asian) were studied. The results indicate that the venoms are, in general, potently procoagulant but that the relative dependence on calcium or phospholipid cofactors is highly variable. Additionally, three out of the four antivenoms tested demonstrated only a very narrow taxonomic range of effectiveness in preventing coagulopathy, with only the SAIMR antivenom displaying significant levels of cross-reactivity. These results were in conflict with previous studies using prolonged preincubation of antivenom with venom to suggest effective cross-reactivity levels for the ICP Echi-Tab antivenom. These findings both inform upon potential clinical effects of envenomation in humans and highlight the extreme limitations of available treatment. It is hoped that this will spur efforts into the development of antivenoms with more comprehensive coverage for bites not only from wild snakes but also from specimens widely kept in zoological collections.

Disseminated intravascular coagulation caused by moojenactivase, a procoagulant snake venom metalloprotease.

Snake venom toxins that activate coagulation factors are key players in the process of venom-induced coagulopathy, and account for severe clinical manifestations. The present study applies a variety of biochemical, hematological, and histopathological approaches to broadly investigate the intravascular and systemic effects of moojenactivase (MooA), the first described PIIId subclass metalloprotease isolated from Bothrops sp. venom that activates coagulation factors. MooA induced consumption coagulopathy with high toxic potency, characterized by prolongation of prothrombin and activated partial thromboplastin time, consumption of fibrinogen and the plasma coagulation factors X and II, and thrombocytopenia. MooA promoted leukocytosis and expression of the proinflammatory cytokines interleukin-6 and tumor necrosis factor-α, accompanied by tissue factor-dependent procoagulant activity in peripheral blood mononuclear cells. This metalloprotease also caused intravascular hemolysis, elevated plasma levels of creatine kinase-MB, aspartate transaminase, and urea/creatinine, and induced morphopathological alterations in erythrocytes, heart, kidney, and lungs associated with thrombosis and hemorrhage. Diagnosis of MooA-induced disseminated intravascular coagulation represents an important approach to better understand the pathophysiology of Bothrops envenomation and develop novel therapeutic strategies targeting hemostatic disturbances.

Isolation and characterization of a novel metalloprotease inhibitor from Bothrops alternatus snake serum.

Resistance of snakes and some other animals to snake envenomation has been attributed to soluble factors present in their tissues. Here we report the isolation of a novel metalloprotease inhibitor from Bothrops alternatus snake serum (named BaltMPI) with high purity, using a four-step chromatographic method. BaltMPI has molecular weights of 60.5 and 42.4kDa, as determined by SDS-PAGE and mass spectrometry, respectively, and pI=5.27. The first 60 amino acids from the N-terminal region of BaltMPI, determined by Edman's degradation, showed high homology (97%) with the snake venom metalloprotease inhibitor (SVMPI) BJ46a and other SVMPIs (78-82%). The chromatographic fractions and purified BaltMPI exhibited anti-hemorrhagic activity against Batroxase and BjussuMP-I. BaltMPI was stable over wide ranges of pH (1, 5, 8, and 9) and temperature (-80, -20, 4, 60, and 100°C), and suppressed the fibrinogenolytic, fibrinolytic, and azocaseinolytic activities of Batroxase. BaltMPI specifically inhibited the activity of metalloproteases, without affecting the activity of serine proteases. Together, our results suggest that BaltMPI and other SVMPIs are promising molecules for the treatment of snake envenomation, in particular that caused by Bothrops sp.

rACLF, a recombinant snake venom metalloprotease, activates endothelial cells in vitro

Snake venom metalloproteases (SVMPs) comprise a family of snake venom toxins responsible for most of local and systemic effects observed during envenomation by snakes from the Viperidae family. The vascular system and more specifically the endothelium seem to be the preferential targets of these proteins. This work describes the effects of rACLF, a recombinant SVMP from Agkistrodon contortrix laticinctus on human umbilical vein endothelial cells (HUVECs) in vitro. Our results showed that rACLF activates HUVECs by the release of mediators involved in inflammation and hemostasis such as prostacyclin and interleukin-8. We also demonstrated that rACLF increased the expression of ICAM-I and decay accelerating factor (DAF). Moreover, rACLF protects the HUVECs against apoptosis induced by serum deprivation. These results suggest that the endothelial cell activation induced by SVMPs may have a significant role in the development of the local inflammatory lesion observed in Viperidae envenomation.