Anti-HIV Activity of Snake Venom Phospholipase A2s: Updates for New Enzymes and Different Virus Strains.

Since the beginning of the HIV epidemic, lasting more than 30 years, the main goal of scientists was to develop effective methods for the prevention and treatment of HIV infection. Modern medicines have reduced the death rate from AIDS by 80%. However, they still have side effects and are very expensive, dictating the need to search for new drugs. Earlier, it was shown that phospholipases A2 (PLA2s) from bee and snake venoms block HIV replication, the effect being independent on catalytic PLA2 activity. However, the antiviral activity of human PLA2s against Lentiviruses depended on catalytic function and was mediated through the destruction of the viral membrane. To clarify the role of phospholipolytic activity in antiviral effects, we analyzed the anti-HIV activity of several snake PLA2s and found that the mechanisms of their antiviral activity were similar to that of mammalian PLA2. Our results indicate that snake PLA2s are capable of inhibiting syncytium formation between chronically HIV-infected cells and healthy CD4-positive cells and block HIV binding to cells. However, only dimeric PLA2s had pronounced virucidal and anti-HIV activity, which depended on their catalytic activity. The ability of snake PLA2s to inactivate the virus may provide an additional barrier to HIV infection. Thus, snake PLA2s might be considered as candidates for lead molecules in anti-HIV drug development.

Antiangiogenic effects of phospholipase A2 Lys49 BnSP-7 from Bothrops pauloensis snake venom on endothelial cells: An in vitro and ex vivo approach.

Antiangiogenic strategies are promising tools for cancer treatment and several other disorders. In this sense, phospholipases A2 (PLA2s) from snake venom have been described to possess antiangiogenic properties. In this study, we evaluated both in vitro and ex vivo antiangiogenic effects induced by BnSP-7, a Lys49 PLA2 isolated from Bothrops pauloensis snake venom. BnSP-7 was able to inhibit endothelial cell (HUVEC) proliferation, which was indeed confirmed by a modulation of cell cycle progression. Interestingly, BnSP-7 also inhibited the adhesion and migration of HUVECs and blocked in vitro angiogenesis in a VEGF-dependent manner, an important proangiogenic factor. Finally, BnSP-7 was capable of inhibiting sprouting angiogenic process through an ex vivo aortic ring assay. Taken together, these results indicate that BnSP-7 has potent in vitro and ex vivo antiangiogenic effect.

Antimicrobial Activity of Snake ß-Defensins and Derived Peptides.

ß-defensins are antimicrobial peptides presenting in vertebrate animals. They participate in innate immunity, but little is known about them in reptiles, including snakes. Although several ß-defensin genes were described in Brazilian snakes, their function is still unknown. The peptide sequence from these genes was deduced, and synthetic peptides (with approximately 40 amino acids and derived peptides) were tested against pathogenic bacteria and fungi using microbroth dilution assays. The linear peptides, derived from ß-defensins, were designed applying the bioisosterism strategy. The linear ß-defensins were more active against Escherichia coli, Micrococcus luteus, Citrobacter freundii, and Staphylococcus aureus. The derived peptides (7-14 mer) showed antibacterial activity against those bacteria and on Klebsiella pneumoniae. Nonetheless, they did not present activity against Candida albicans, Cryptococcus neoformans, Trychophyton rubrum, and Aspergillus fumigatus showing that the cysteine substitution to serine is deleterious to antifungal properties. Tryptophan residue showed to be necessary to improve antibacterial activity. Even though the studied snake ß-defensins do not have high antimicrobial activity, they proved to be attractive as template molecules for the development of antibiotics.

Isolation and Characterization of CD39-like Phosphodiesterase (Cc-PDE) from Cerastes cerastes Venom: Molecular Inhibitory Mechanism of Antiaggregation and Anticoagulation.

BACKGROUND: Cerastes cerastes venom contains several bioactive proteins with inhibitory potential of platelet aggregation and blood coagulation. OBJECTIVE: The current study deals with purification, characterization and determination of structural properties of Cc-PDE, the first phosphodiesterase from Cerastes cerastes venom. MATERIAL AND METHODS: The purification process consists of three successive chromatographies including G75-Sephadex size exclusion, DEAE exchange chromatography and affinity using Sildenafil as a main PDEs' specific inhibitor. The amino acid sequence of purified Cc-PDE was determined by liquid chromatography coupled off line to MALDI-TOF/TOF. Modeling and structural features were obtained using several bioinformatics tools. In vivo and in vitro antiplatelet aggregation and anticoagulant assays were performed. RESULTS: Cc-PDE (73 506.42 Da) is a 654-residue single polypeptide with 1-22 signal peptide and it is characterized by the presence of predominant basic amino acids suitable to alkaline pI (8.17). Cc-PDE structure is composed of ß-strands (17%) and α-helices (24%) and it shares a high identity with homologous snake venom PDEs. Cc-PDE hydrolyzes both Bis-p-nitrophenyl phosphate (Km = 2.60 ± 0.95 mM, Vmax = 0.017 ± 0.002569 µmol.min-1) and p-nitrophenyl phosphate (Km = 7.13 mM ± 0.04490 mM, Vmax = 0.053 ±0.012 µmol.min-1). Cc-PDE prevents ADP- and ATP-induced platelet aggregation by hydrolyzing ADP and ATP, reducing surface P-selectin expression and attenuating platelet function. In addition, Cc-PDE inhibits coagulation factors involved in the intrinsic pathway demonstrated by a significant prolongation of activated partial thromboplastin time and in vivo long-lasting anticoagulation. CONCLUSION: The obtained results revealed that Cc-PDE may have a therapeutic potential and could be a remedy for thromboembolic diseases as an alternative of anticoagulant and antiplatelet aggregation chemical origins.

Study of the venom proteome of Vipera ammodytes ammodytes (Linnaeus, 1758): A qualitative overview, biochemical and biological profiling.

Vipera ammodytes (Va), is the European venomous snake of the greatest medical importance. We analyzed whole venom proteome of the subspecies V. ammodytes ammodytes (Vaa) from Serbia for the first time using the shotgun proteomics approach and identified 99 proteins belonging to four enzymatic families: serine protease (SVSPs), L-amino acid oxidase (LAAOs), metalloproteinases (SVMPs), group II phospholipase (PLA2s), and five nonenzymatic families: cysteine-rich secretory proteins (CRISPs), C-type lectins (snaclecs), growth factors -nerve (NGFs) and vascular endothelium (VEGFs), and Kunitz-type protease inhibitors (SPIs). Considerable enzymatic activity of LAAO, SVSPs, and SVMPs and a high acidic PLA2 activity was measured implying potential of Vaa to produce haemotoxic, myotoxic, neuro and cardiotoxic effects. Moreover, significant antimicrobial activity of Vaa venom against Gram-negative (Klebsiella pneumoniae, Pseudomonas aeruginosa) and Gram-positive bacteria (Staphylococcus aureus) was found. The crude venom shows considerable potential cytotoxic activity on the C6 and HL60 and a moderate level of potency on B16 cell lines. HeLa cells showed the same sensitivity, while DU 145 and PC-3 are less sensitive than as normal cell line. Our data demonstrated a high complexity of Vaa and considerable enzymatic, antibacterial and cytotoxic activity, implying a great medical potential of Vaa venom as a promising source for new antibacterial and cytostatic agents.

Antibiofilm Activity of Acidic Phospholipase Isoform Isolated from Bothrops erythromelas Snake Venom.

INTRODUCTION: Bacterial resistance is a worldwide public health problem, requiring new therapeutic options. An alternative approach to this problem is the use of animal toxins isolated from snake venom, such as phospholipases A2 (PLA2), which have important antimicrobial activities. Bothropserythromelas is one of the snake species in the northeast of Brazil that attracts great medical-scientific interest. Here, we aimed to purify and characterize a PLA2 from B. erythromelas, searching for heterologous activities against bacterial biofilms. METHODS: Venom extraction and quantification were followed by reverse-phase high-performance liquid chromatography (RP-HPLC) in C18 column, matrix-assisted ionization time-of-flight (MALDI-ToF) mass spectrometry, and sequencing by Edman degradation. All experiments were monitored by specific activity using a 4-nitro-3-(octanoyloxy) benzoic acid (4N3OBA) substrate. In addition, hemolytic tests and antibacterial tests including action against Escherichiacoli, Staphylococcusaureus, and Acinetobacterbaumannii were carried out. Moreover, tests of antibiofilm action against A. baumannii were also performed. RESULTS: PLA2, after one purification step, presented 31 N-terminal amino acid residues and a molecular weight of 13.6564 Da, with enzymatic activity confirmed in 0.06 µM concentration. Antibacterial activity against S. aureus (IC50 = 30.2 µM) and antibiofilm activity against A. baumannii (IC50 = 1.1 µM) were observed. CONCLUSIONS: This is the first time that PLA2 purified from B. erythromelas venom has appeared as an alternative candidate in studies of new antibacterial medicines.

Functional Mining of the Crotalus Spp. Venom Protease Repertoire Reveals Potential for Chronic Wound Therapeutics.

Chronic wounds are a major health problem that cause millions of dollars in expenses every year. Among all the treatments used, active wound treatments such as enzymatic treatments represent a cheaper and specific option with a fast growth category in the market. In particular, bacterial and plant proteases have been employed due to their homology to human proteases, which drive the normal wound healing process. However, the use of these proteases has demonstrated results with low reproducibility. Therefore, alternative sources of proteases such as snake venom have been proposed. Here, we performed a functional mining of proteases from rattlesnakes (Crotalus ornatus, C. molossus nigrescens, C. scutulatus, and C. atrox) due to their high protease predominance and similarity to native proteases. To characterize Crotalus spp. Proteases, we performed different protease assays to measure and confirm the presence of metalloproteases and serine proteases, such as the universal protease assay and zymography, using several substrates such as gelatin, casein, hemoglobin, L-TAME, fibrinogen, and fibrin. We found that all our venom extracts degraded casein, gelatin, L-TAME, fibrinogen, and fibrin, but not hemoglobin. Crotalus ornatus and C. m. nigrescens extracts were the most proteolytic venoms among the samples. Particularly, C. ornatus predominantly possessed low molecular weight proteases (P-I metalloproteases). Our results demonstrated the presence of metalloproteases capable of degrading gelatin (a collagen derivative) and fibrin clots, whereas serine proteases were capable of degrading fibrinogen-generating fibrin clots, mimicking thrombin activity. Moreover, we demonstrated that Crotalus spp. are a valuable source of proteases that can aid chronic wound-healing treatments.

Insights into the antiviral activity of phospholipases A2 (PLA2s) from snake venoms.

Viruses are associated with several human diseases that infect a large number of individuals, hence directly affecting global health and economy. Owing to the lack of efficient vaccines, antiviral therapy and emerging resistance strains, many viruses are considered as a potential threat to public health. Therefore, researches have been developed to identify new drug candidates for future treatments. Among them, antiviral research based on natural molecules is a promising approach. Phospholipases A2 (PLA2s) isolated from snake venom have shown significant antiviral activity against some viruses such as Dengue virus, Human Immunodeficiency virus, Hepatitis C virus and Yellow fever virus, and have emerged as an attractive alternative strategy for the development of novel antiviral therapy. Thus, this review provides an overview of remarkable findings involving PLA2s from snake venom that possess antiviral activity, and discusses the mechanisms of action mediated by PLA2s against different stages of virus replication cycle. Additionally, molecular docking simulations were performed by interacting between phospholipids from Dengue virus envelope and PLA2s from Bothrops asper snake venom. Studies on snake venom PLA2s highlight the potential use of these proteins for the development of broad-spectrum antiviral drugs.

Crotalus neutralising factor and its role in human leukocyte modulation.

Crotalus neutralising factor (CNF) is an endogenous γ-type phospholipase A2 (PLA2) inhibitor that inhibits the toxic action of crotoxin, a neurotoxin present in Crotalus durissus terrificus venom. However, its effects on the activation and modulation of immune cells, which play a major role in the development of inflammation, is not known. The objective of the present study was to assess the effects of CNF on human leukocyte modulation in vitro by analysing the following parameters: cell viability, phagocytic capacity, lipid droplet formation, reactive oxygen species production, nitric oxide production, p38 MAPK activation, and cytosolic PLA2 (cPLA2) gene expression. Neutrophils and peripheral blood mononuclear cells from healthy donors were isolated via the density gradient method, resuspended in RPMI medium, and incubated with RPMI (negative control), LPS, or PMA (positive control) or CNF (sample test) at a concentration of 50 µg/mL. Results showed that CNF was not toxic to human neutrophils after 48 and 72 h of incubation. CNF treatment induced an increase in PBMCs and neutrophil phagocytic capacity, as well as the formation of lipid droplets within these cells after 1 h of incubation. However, CNF did not induce the formation of reactive oxygen and nitric oxide species. Moreover, CNF induced p38 MAPK protein phosphorylation and cPLA2 gene expression in neutrophils. The data obtained herein showed that CNF action modulates human leukocytes, CNF activates important signalling pathways for human leukocytes, and it is pro-inflammatory. These findings also complement previous studies on CNF action on human peripheral blood leukocyte function.

Disintegrins extracted from totonacan rattlesnake (Crotalus totonacus) venom and their anti-adhesive and anti-migration effects on MDA-MB-231 and HMEC-1 cells.

Disintegrins are low molecular weight cysteine-rich proteins (4-14 kDa) that are isolated mainly from viperid snake venom. Due to their potential as lead compounds for binding and blocking integrin receptors, snake venom disintegrins have become one of the most studied venom protein families. The aim of this study was to obtain disintegrins from C. totonacus venom and evaluate their capability to bind and block integrin receptors. The C. totonacus disintegrin fraction (totonacin) represents two disintegrin isoforms obtained from C. totonacus venom. These disintegrins showed extracellular-matrix (ECM) protein adhesion and migration inhibitory effects on MDA-MB-231 and HMEC-1 cells. Totonacin (3 µM) inhibited MDA-MB-231 cell adhesion to the ECM proteins, fibronectin, vitronectin, and laminin by 31.2, 44.0, and 32.1, respectively. Adhesion inhibition to fibronectin, vitronectin, and laminin observed on HMEC-1 cells was 42.8, 60.8, and 51%, respectively. In addition, totonacin (3 µM) significantly inhibited MDA-MB-231 and HMEC-1 cell migration (41.4 and 48.3%, respectively). Totonacin showed more potent cell adhesion inhibitory activity toward vitronectin in both cell lines. These results suggest a major affinity of totonacin toward αVß3, α8ß1, αVß5, αVß1, and αIIbß3 integrins. In addition, the inhibitory effect observed on MDA-MB-231 and HMEC-1 cell migration reinforces the evidence of an interaction between these disintegrins and αVß3 integrin, which plays a key role in migration and angiogenesis.

Bothrops moojeni venom and BmooLAAO-I downmodulate CXCL8/IL-8 and CCL2/MCP-1 production and oxidative burst response, and upregulate CD11b expression in human neutrophils.

Bothrops snake venoms contain biologically active components, including L-amino acid oxidases (LAAO) that induce significant leukocyte accumulation at inflammatory sites characterized by early neutrophil infiltration. As it remains unclear how snake venoms modulate neutrophil activation and chemokine production, here we examined whether Bothrops moojeni crude venom (BmV) and its LAAO (BmooLAAO-I) affect expression of the surface activation markers CD11b and CD66b, production of the chemokines CCL2/MCP-1, CCL5/RANTES, CXCL8/IL-8, CXCL9/MIG, and CXCL-10/IP-10, and activation of oxidative burst in human neutrophils. Cell viability, expression of activation markers, and chemokine production were assessed by flow cytometry, while the oxidative burst response was measured by chemiluminescence. BmV at 50 and 75 µg/mL reduced CXCL8/IL-8 (p < 0.001 and p < 0.01, respectively) and CCL2/MCP-1 production (p < 0.05), while BmooLAAO-I at the same concentrations reduced only CCL2/MCP-1 production (p < 0.01). These effects were accompanied by CD11b upregulation (p < 0.05 for 50 and 75 µg/mL BmV; p < 0.01 for 50 and 75 µg/mL BmooLAAO-I) and CD66b downregulation (p < 0.05 for 50 and 75 µg/mL BmV). Both BmV and BmooLAAO-I at concentrations ranging from 0.625 to 5 µg/mL suppressed the oxidative burst of neutrophils stimulated with phorbol 12-myristate 13-acetate, while BmooLAAO-I at 2.5 and 5 µg/mL also suppressed the neutrophil response stimulated with opsonized zymosan. Considering that neutrophils participate in the pathogenesis of autoimmune and inflammatory diseases, the findings reported herein indicate that BmV and BmooLAAO-I are potential immunomodulating agents.

Effect of Isolated Proteins from Crotalus Durissus Terrificus Venom on Leishmania (Leishmania) Amazonensis-Infected Macrophages.

BACKGROUND: Cutaneous and mucocutaneous leishmaniasis are parasitic diseases characterized by skin manifestations. In Brazil, Leishmania (Leishmania) amazonensis is one of the etiological agents of cutaneous leishmaniasis. The therapeutic arsenal routinely employed to treat infected patients is unsatisfactory, especially for pentavalent antimonials, as they are often highly toxic, poorly tolerated and of variable effectiveness. This study aimed to evaluate in vitro the leishmanicidal activity of toxins isolated from Crotalus durissus terrificus venom as a new approach for the treatment of leishmaniasis. METHODS: The comparative effects of crotamine, crotoxin, gyrotoxin, convulxin and PLA2 on bone marrow-derived macrophages infected with L. (L.) amazonensis as well as the release of TGF-ß from the treated macrophages were studied. RESULTS AND DISCUSSION: Crotamine had the strongest inhibitory effect on parasite growth rate (IC50: 25.65±0.52 µg/mL), while convulxin showed the weakest inhibitory effect (IC50: 52.7±2.21 µg/mL). In addition, TGF-ß was significantly reduced after the treatment with all toxins evaluated. CONCLUSION: The Crotalus durissus terrificus toxins used in this study displayed significant activity against L. (L.) amazonensis, indicating that all of them could be a potential alternative for the treatment of cutaneous leishmaniasis.

Dimeric Disintegrins from the Steppe Viper V. ursinii Venom.

Four dimeric disintegrins were isolated from the venom of the steppe viper V. ursinii using liquid chromatography. Disintegrins prevented adhesion of MCF7 cells to fibronectin, which indicates their interaction with integrin receptors of the αVß1 type. According to mass spectrometry data, the molar masses of disintegrins are about 14 kDa. The method of peptide mapping established the structure of a new heterodimeric disintegrin weighing 13 995.5 Da and shows that it belongs to the class of RGD/KGD-containing disintegrins.

Antiviral activity of cathelicidin 5, a peptide from Alligator sinensis, against WSSV in caridean shrimp Exopalaemon modestus.

White spot disease caused by white spot syndrome virus (WSSV) is responsible for harming shrimp aquaculture industry and results in a pandemic throughout the world. Cathelicidin 5 treatment enhanced immune parameters including antioxidant enzyme activity and immune-related genes expression in shrimp Exopalaemon modestus. Shrimp treated with cathelicidin 5 and inoculated with white spot syndrome virus (WSSV) exhibited a significantly lower mortality rate and lower viral VP28 amplification and expression than control. This study addresses the role of cathelicidin 5 in immune stimulatory and antiviral activities that could protect E. modestus from WSSV infection.

Variation in the Protein Composition and Biological Activity of King Cobra (Ophiophagus hannah) Venoms.

The biochemical properties and biological activities of the venom from three individual Ophiophagus hannah (King cobra) specimens was compared. The toxicity against mice, the cytotoxicity against five cell lines, and the antioxidant activity were measured. The KV2 venom showed a higher cytotoxicity than the KV6 and the non-cytotoxic KV9 venoms. Comparative analysis of the O. hannah venom proteins was performed after 2-dimensional (2-D) denaturing gel electrophoresis and reverse phase high performance liquid chromatography (RP-HPLC). 2-D analysis by isoelectric focusing (IEF) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) resolution of the venoms revealed significant differences between all three venoms, with most spots being unique to that venom. Only 2 out of the 13-16 distinct spots were common to all three venoms, and four spots were common to KV6 and KV9. KV2 had the highest proportion of low molecular mass spots, and KV6 and KV9 appeared more related to each other than to KV9. From peptide mass mapping by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and MASCOT-based amino acid sequence database searching, the two venom proteins that were common to all three specimens are likely to be ophanin and acidic phospholipase A2 (PLA2), whilst the proteins unique to the cytotoxic KV2 venom, included three other PLA2 proteins. The RP-HPLC pattern of KV2 was different from the other two venoms with a higher protein concentration eluting in the 31-41% (v/v) acetonitrile (ACN) fraction than for the other two venoms.

Mutacytin-1, a New C-Type Lectin-Like Protein from the Venezuelan Cuaima (Lachesis muta muta Linnaeus, 1766) (Serpentes: Viperidae) Snake Venom Inducing Cardiotoxicity in Developing Zebrafish (Danio rerio) Embryos.

Envenomation by the Venezuelan bushmaster snake (Lachesis muta muta) (Serpentes: Viperidae) is characterized by local and cardiac alterations. This study investigates the in vivo cardiac dysfunction, tissue destruction, and cellular processes triggered by Lachesis muta muta snake crude venom and a C-type lectin (CTL)-like toxin named Mutacytin-1 (MC-1). The 28 kDa MC-1 was obtained by molecular exclusion, ion exchange, and C-18 (checking pureness) reverse-phase chromatographies. N-terminal sequencing of the first eight amino acids (NNCPQ LLM) revealed 100% identity with Mutina (CTL-like) isolated from Lachesis stenophrys, which is a Ca2+-dependent-type galactoside-binding lectin from Bothrops jararaca and CTL BpLec from Bothrops pauloensis. The cardiotoxicity in zebrafish of MC-1 was evaluated by means of specific phenotypic expressions and larvae behavior at 5, 15, 30, 40 and 60 min post-treatment. The L. muta muta venom and MC-1 also produced heart rate/rhythm alterations, circulation modifications, and the presence of thrombus and apoptotic phenomenon with pericardial damages. Acridine orange (100 µg/mL) was used to visualize apoptosis cellular process in control and treated whole embryos. The cardiotoxic alterations happened in more than 90% of all larvae under the action of L. muta muta venom and MC-1. The findings have demonstrated the potential cardiotoxicity by L. muta muta venom, suggesting the possibility of cardiovascular damages to patients after bushmaster envenoming.

Intervention on immunodeficiency mice and structural identification of enzymatic peptides from Mauremys mutica and Cuora trifasciata.

ETHNOPHARMACOLOGY RELEVANCY: Mauremys mutica (Asian yellow pond turtle, YPT) and Cuora trifasciata (Chinese three-striped box turtle, TBT) are traditional Chinese medicine. They possess many biological characteristics, such as immune-enhancement, anti-inflammatory, anti-cancer effects. They have been used as folk anti-cancer drugs in central and southern China for a long time. However, there was no reports of comparing the immune-enhancement effect of YPT and TBT, nor of identifying the structures of YPT peptides and TBT peptides. AIMS OF THE STUDY: The aim of this study was to evaluate the protective efficacy of YPT and TBT on immunodeficient mice and to compare the primary structures of YPT peptides and TBT peptides. MATERIALS AND METHODS: The protein extracts were extracted using 100 °C water, and peptides were obtained by hydrolyzing protein extracts using alkaline protease. Cyclophosphamide (CTX) was used to induce immunodeficiency in mice. The immune enhancement effect was evaluated by measuring body weight gain curve, thymus index, spleen index, serum SOD activity and GSH-Px activity. Primary structure of peptides was identified by HPLC-ESI-MS/MS. RESULTS: The protein extracts and peptides of the YPT and TBT had certain recovery effects on immunodeficient mice. YPT peptide has the best effect on the recovery of damaged immune organs and the improvement of SOD and GSH-Px activities in mice. In the identification of the primary structure of the polypeptide, we find that YPT and TBT contain some similar peptides as well as different peptides, and the concentration of the peptide segments in HPLC data is very different. The difference of biological activity may be determined by both the difference of specific peptide structure and concentration. CONCLUSIONS: Two kinds of healthy turtle protein extracts and peptides could have immune-enhancement function, and peptides obtained by enzymatic hydrolysis of YPT protein extracts have the best immune-enhancement effect. The identification of the primary structure of the peptide segment preliminarily showed that its biological activity was affected by the amino acid sequence and the concentration of part of the peptide segment. It laid a foundation for the follow-up search of immune-enhancement peptides and the development of high-value YPT products.

The kallikrein-like activity of Heloderma venom is inhibited by carbon monoxide.

Lizards in the genus Heloderma are the most ancient venomous reptiles, with a traceable lineage nearly 100 million years old. The proteome of the venom of three of the remaining species (Heloderma suspectum, H. exasperatum, H. horridum) are very conserved, with kallikrein-like activity present to cause critical hypotension to immobilize and outright kill prey. Kallikrein-like activity would be expected to activate the contact protein pathway of coagulation, which would be detectable with thrombelastography in human plasma. Thus, it was proposed to determine if kallikrein-like activity could be detected with thrombelastography, and if this activity could be inhibited by carbon monoxide (CO) via a putative heme-based mechanism. Procoagulant activity of each venom was assessed via thrombelastography with normal plasma, and kallikrein-like activity confirmed with FX-depleted plasma. Venom was then exposed to carbon monoxide releasing molecule-2 (CORM-2) or its inactive releasing molecule to assess CO inhibition. All three venoms demonstrated kallikrein-like activity with the same potency and inhibition of activity by CO. In conclusion, the present work documented that procoagulant, kallikrein-like activity containing venoms of the oldest species of venomous reptiles was inhibited by CO, potentially via heme modulation. This is also the first identification and characterization of a kallikrein-like enzyme utilizing coagulation factor-depleted plasma to assess venom that inflicts hypotension. Future investigations will continue to define the vulnerability of venom enzymatic activities to CO.

The isolation and characterization of a new snake venom cysteine-rich secretory protein (svCRiSP) from the venom of the Southern Pacific rattlesnake and its effect on vascular permeability.

A novel snake venom cysteine-rich secretory protein (svCRiSP), Hellerin, was purified from C. o. helleri venom using sequential reverse phase and cation-exchange chromatography. Gel electrophoresis, N-terminal sequencing, and LC-MS/MS sequencing identified a single protein with a molecular mass of approximately 24.8 kDa and confirmed its identity as a svCRiSP. Hellerin had cytotoxic effects on human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner but not in human dermal lymphatic endothelial cells (HDLECs) and human dermal blood endothelial cells (HDBECs). Hellerin produced a dramatic increase in both blood vascular permeability in vivo, and in the trans-epithelial permeability of cultured HDLEC and HDBEC cells. This is the first study that describes the effect of a svCRiSP on vascular, blood and lymphatic permeability.

Malaysian Cobra Venom: A Potential Source of Anti-Cancer Therapeutic Agents.

Cancer is a deadly disease and there is an urgent need for the development of effective and safe therapeutic agents to treat it. Snake venom is a complex mixture of bioactive proteins that represents an attractive source of novel and naturally-derived anticancer agents. Malaysia is one of the world's most biodiverse countries and is home to various venomous snake species, including cobras. Naja kaouthia, Naja sumatrana, and Ophiophagus hannah are three of the most common cobra species in Malaysia and are of medical importance. Over the past decades, snake venom has been identified as a potential source of therapeutic agents, including anti-cancer agents. This present review highlights the potential anticancer activity of the venom and purified venom protein of N. kaouthia, N. sumatrana, and O. hannah. In conclusion, this review highlights the important role of the venom from Malaysian cobras as an important resource that researchers can exploit to further investigate its potential in cancer treatment.