Antihyaluronidase and Alkaline Phosphatase (ALP) Activities of Medicinal Plants to Combat Echis carinatus Venom-Induced Toxicities.

Snakebite is one of the most neglected diseases of developing countries. Deaths due to snakebite envenoming are quite high in Pakistan, and many deaths are caused by Echis carinatus envenomation. Traditional use of medicinal plants against snakebites is a common practice in Pakistan due to countless benefits. The current study was performed with the objective to evaluate eighteen Pakistani medicinal plants inhibitory potential against hyaluronidase and alkaline phosphatase enzymes of Pakistani Echis carinatus venom. Hyaluronidase activity (0.2-1.6 mg/0.1 mL) and alkaline phosphatase activity (0.1-0.8 mg/0.1 mL) were measured in dose-dependent manner. Crude methanolic extracts of medicinal plants were used for in vitro investigation of their inhibitory activity against toxic enzymes. All active plants were fractioned using different solvents and were again analyzed for inhibitory activity of same enzymes. Results indicated all plants were able to neutralize hyaluronidase that Swertia chirayita (Roxb. ex Flem.) Karst., Terminalia arjuna Wight and Arn, Rubia cordifolia Thumb., and Matthiola incana (L.) R.Br. inhibited maximum hyaluronidase activity equivalent to standard reference (p > 0.5). Pakistani medicinal plants are dense with natural neutralizing metabolites and other active phytochemicals which could inhibit hyaluronidase activity of Pakistani Echis carinatus venom. Further advanced studies at molecular level could lead us to an alternative for envenoming of Pakistani Echis carinatus venom.

The synthetic varespladib molecule is a multi-functional inhibitor for PLA2 and PLA2-like ophidic toxins.

BACKGROUND: The treatment for snakebites is early administration of antivenom, which can be highly effective in inhibiting the systemic effects of snake venoms, but is less effective in the treatment of extra-circulatory and local effects. To complement standard-of-care treatments such as antibody-based antivenoms, natural and synthetic small molecules have been proposed for the inhibition of key venom components such as phospholipase A2 (PLA2) and PLA2-like toxins. Varespladib (compound LY315920) is a synthetic molecule developed and clinically tested aiming to block inflammatory cascades of several diseases associated with high PLA2s. Recent studies have demonstrated this molecule is able to potently inhibit snake venom catalytic PLA2 and PLA2-like toxins. METHODS: In vivo and in vitro techniques were used to evaluate the inhibitory effect of varespladib against MjTX-I. X-ray crystallography was used to reveal details of the interaction between these molecules. A new methodology that combines crystallography, mass spectroscopy and phylogenetic data was used to review its primary sequence. RESULTS: Varespladib was able to inhibit the myotoxic and cytotoxic effects of MjTX-I. Structural analysis revealed a particular inhibitory mechanism of MjTX-I when compared to other PLA2-like myotoxin, presenting an oligomeric-independent function. CONCLUSION: Results suggest the effectiveness of varespladib for the inhibition of MjTX-I, in similarity with other PLA2 and PLA2-like toxins. GENERAL SIGNIFICANCE: Varespladib appears to be a promissory molecule in the treatment of local effects led by PLA2 and PLA2-like toxins (oligomeric dependent and independent), indicating that this is a multifunctional or broadly specific inhibitor for different toxins within this superfamily.

Sulfur Compounds as Inhibitors of Enzymatic Activity of a Snake Venom Phospholipase A2: Benzyl 4-nitrobenzenecarbodithioate as a Case of Study.

Snakebite is a neglected disease with a high impact in tropical and subtropical countries. Therapy based on antivenom has limited efficacy in local tissue damage caused by venoms. Phospholipases A2 (PLA2) are enzymes that abundantly occur in snake venoms and induce several systemic and local effects. Furthermore, sulfur compounds such as thioesters have an inhibitory capacity against a snake venom PLA2. Hence, the objective of this work was to obtain a carbodithioate from a thioester with known activity against PLA2 and test its ability to inhibit the same enzyme. Benzyl 4-nitrobenzenecarbodithioate (I) was synthesized, purified, and characterized using as precursor 4-nitrothiobenzoic acid S-benzyl ester (II). Compound I showed inhibition of the enzymatic activity a PLA2 isolated from the venom of the Colombian rattlesnake Crotalus durissus cumanensis with an IC50 of 55.58 µM. This result is comparable with the reported inhibition obtained for II. Computational calculations were performed to support the study, and molecular docking results suggested that compounds I and II interact with the active site residues of the enzyme, impeding the normal catalysis cycle and attachment of the substrate to the active site of the PLA2.

Metabolites from the citrus extracts inhibit the activity of selected proteins in Indian Cobra (Naja naja) venom.

ETHNOPHARMACOLOGICAL RELEVANCE: Snakebite is a severe problem in many parts of the world, specifically in tropical and subtropical regions. A range of medicinal plant extracts are administered for treating snake bite. Of the many common plants, extracts of Citrus species have been documented to be used for treating snake bite and have been shown to decrease the snake venom toxicity. AIM: The aim of the current work is to evaluate the utility of citrus peel extracts (Citrus aurantium L. and Citrus reticulate Blanco) in the management of Indian cobra envenomation. MATERIALS AND METHODS: Peels of citrus species were evaluated for their phospholipase A2, protease and haemolytic inhibition properties. The phytochemicals present in the extract were inferred using GC-MS. In-vivo studies, using mice model, were done to confirm the inhibitory effect of the extracts. Molecular docking was used to understand the possible binding modes of selected phytochemicals to snake venom phospholipase. RESULTS: Citrus peel extracts are rich in polyphenols, flavonoids and tannins. The methanolic extract of Citrus aurantium L. and Citrus reticulate Blanco inhibits phospholipase (75%), protease (71%) and hemolysis (80%) activity of the venom. GC-MS analyses indicate the presence of ß-sitosterol, n-hexadecanoic acid, eicosanoic acid, and flavone in both the extracts. In addition, C. reticulate extract contains α-tocopherol and squalene. Molecular docking revealed that α-tocopherol, spiro [androst-5-ene-17,1'-cyclobutan]-2'-one,3-hydroxy-(3ß,17ß)- and ß-sitosterol acetate bind with moderate affinity to the catalytic site of phospholipase A2. CONCLUSION: The present study provides new molecular insight and scientific evidence on the utility of the methanolic extracts of citrus peels to neutralize the venom toxins of Naja naja.

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.

First look into the venom of Roatan Island's critically endangered coral snake Micrurus ruatanus: Proteomic characterization, toxicity, immunorecognition and neutralization by an antivenom.

A proteomic and toxicological study of the venom from one specimen of Micrurus ruatanus, a critically endangered coral snake species endemic to Roatan Island, Honduras, was carried out. Immunorecognition and neutralization of venom lethality by an anticoral antivenom was also evaluated. Forty peaks were collected from RP-HPLC fractionation of the venom. After SDS-PAGE analysis, fifty-eight bands were examined by MALDI-TOF/TOF mass spectrometry. Micrurus ruatanus venom displayed a three-finger toxin (3FTx)-rich venom phenotype, as well as a significant amount of phospholipases A2 (PLA2s). Various other proteins were identified, including Kunitz-type inhibitor proteins, L-amino acid oxidases, C-type lectin/lectin-like, metalloproteinases, serine proteinases, vespryn/ohanin, 5'-nucleotidases, glutathione peroxidases, and phosphodiesterases. Micrurus ruatanus venom displayed significant PLA2 activity in vitro and myotoxicity in vivo. The venom showed high lethal potency in mice, being one of the most lethal in Central America. The anticoral antivenom (SAC-ICP) produced by Instituto Clodomiro Picado neutralized the lethal activity of the venom. Major fractions with relevant lethal activity were also identified by a screening analysis. SIGNIFICANCE: The proteomic characterization, toxicity, immunorecognition and neutralization of Micrurus ruatanus venom have been determined for the first time. This coral snake is endemic to Roatan Island and contains a three-finger toxin-rich venom that displayed a potent lethal activity in mice. The anticoral antivenom produced by Instituto Clodomiro Picado neutralized the lethal activity of this venom in vivo, and therefore should be effective in the treatment of envenomings by this snake.

Neutralizing properties of LY315920 toward snake venom group I and II myotoxic phospholipases A2.

A need exists to develop specific and clinically useful inhibitors of toxic enzymes present in snake venoms, responsible for severe tissue damage and life-threatening effects occurring in thousands of people suffering envenomations globally. LY315920 (Varespladib, S-5920, A-001), a low molecular weight drug developed to inhibit several human secreted phospholipases A2 (PLA2s), was recently shown to also inhibit PLA2s in whole snake venoms with high potency, yet no studies have examined its direct effect on purified snake venom PLA2s. This work evaluated the ability of LY315920 to neutralize the enzymatic and toxic activities of three isolated PLA2 toxins of structural groups I (pseudexin) and II (crotoxin B and myotoxin I), and their corresponding whole venoms. In vitro, LY315920 inhibited the catalytic activity of these three enzymes upon a synthetic substrate. The drug also blocked their cytotoxic effect on cultured murine myotubes. In mice, preincubation of the toxins or venoms with LY315920, followed by their intramuscular injection, resulted in significant inhibition of muscle damage. Finally, immediate, independent injection of LY315920 at the site of toxin or venom inoculation also resulted in a large reduction of myonecrosis in the case of pseudexin and myotoxin-I, and of Pseudechis colletti and Bothrops asper whole venoms, suggesting a possible method of drug delivery in emergency situations. Present findings add evidence to suggest the possibility of using LY315920 as a field antidote in snakebites, aiming to limit the myonecrosis induced by many venom PLA2s in the clinical setting.

Of molecules, memories and migration: M1 acetylcholine receptors facilitate spatial memory formation and recall during migratory navigation.

Many animals use complex cognitive processes, including the formation and recall of memories, for successful navigation. However, the developmental and neurological processes underlying these cognitive aspects of navigation are poorly understood. To address the importance of the formation and recollection of memories during navigation, we pharmacologically manipulated turtles (Chrysemys picta) that navigate long distances using precise, complex paths learned during a juvenile critical period. We treated freely navigating turtles both within and outside of their critical learning period with a specific M1 acetylcholine receptor antagonist, a drug known to disrupt spatial cognition. Experienced adult turtles lost all navigational ability under the influence of the drug, while naive juveniles navigated successfully. We retested these same juveniles the following year (after they had passed their critical period). The juveniles that initially navigated successfully under the influence of the antagonist (but were unable to form spatial memories) were unable to do so subsequently. However, the control animals (who had the opportunity to form memories previously) exhibited typical navigational precision. These results suggest that the formation of spatial memories for navigation occur during a critical period, and successful navigation after the critical period is dependent upon the recall of such memories.

Biochemical characterization of venom from Pseudoboa neuwiedii (Neuwied's false boa; Xenodontinae; Pseudoboini).

In this work, we examined the proteolytic and phospholipase A2 (PLA2) activities of venom from the opisthoglyphous colubrid Pseudoboa neuwiedii. Proteolytic activity (3 and 10 µg of venom) was comparable to that of Bothrops neuwiedii venom but less than Bothrops atrox. This activity was inhibited by EDTA and 1,10-phenanthroline but only slightly affected (≤30% inhibition) by PMSF and AEBSF, indicating it was mediated by snake venom metalloproteinases (SVMPs). The pH and temperature optima for proteolytic activity were 8.0 and 37 °C, respectively. The venom had no esterase activity, whereas PLA2 activity was similar to B. atrox, greater than B. neuwiedii but less than B. jararacussu. SDS-PAGE revealed venom proteins >100 kDa, 45-70 kDa, 21-24 kDa and ~15 kDa, and mass spectrometry of protein bands revealed SVMPs, cysteine-rich secretory proteins (CRISPs) and PLA2, but no serine proteinases. In gelatin zymography, the most active bands occurred at 65-68 kDa (seen with 0.05-0.25 µg of venom). Caseinolytic activity occurred at 50-66 kDa and was generally weaker than gelatinolytic activity. RP-HPLC of venom yielded 15 peaks, five of which showed gelatinolytic activity; peak 7 was the most active and apparently contained a P-III class SVMP. The venom showed α-fibrinogenase activity, without affecting the ß and γ chains; this activity was inhibited by EDTA and 1,10-phenanthroline. The venom did not clot rat citrated plasma but reduced the rate and extent of coagulation after plasma recalcification. In conclusion, P. neuwiedii venom is highly proteolytic and could potentially affect coagulation in vivo by degrading fibrinogen via SVMPs.

Venom proteomics and antivenom neutralization for the Chinese eastern Russell's viper, Daboia siamensis from Guangxi and Taiwan.

The eastern Russell's viper (Daboia siamensis) causes primarily hemotoxic envenomation. Applying shotgun proteomic approach, the present study unveiled the protein complexity and geographical variation of eastern D. siamensis venoms originated from Guangxi and Taiwan. The snake venoms from the two geographical locales shared comparable expression of major proteins notwithstanding variability in their toxin proteoforms. More than 90% of total venom proteins belong to the toxin families of Kunitz-type serine protease inhibitor, phospholipase A2, C-type lectin/lectin-like protein, serine protease and metalloproteinase. Daboia siamensis Monovalent Antivenom produced in Taiwan (DsMAV-Taiwan) was immunoreactive toward the Guangxi D. siamensis venom, and effectively neutralized the venom lethality at a potency of 1.41 mg venom per ml antivenom. This was corroborated by the antivenom effective neutralization against the venom procoagulant (ED = 0.044 ± 0.002 µl, 2.03 ± 0.12 mg/ml) and hemorrhagic (ED50 = 0.871 ± 0.159 µl, 7.85 ± 3.70 mg/ml) effects. The hetero-specific Chinese pit viper antivenoms i.e. Deinagkistrodon acutus Monovalent Antivenom and Gloydius brevicaudus Monovalent Antivenom showed negligible immunoreactivity and poor neutralization against the Guangxi D. siamensis venom. The findings suggest the need for improving treatment of D. siamensis envenomation in the region through the production and the use of appropriate antivenom.

Rattling the border wall: Pathophysiological implications of functional and proteomic venom variation between Mexican and US subspecies of the desert rattlesnake Crotalus scutulatus.

While some US populations of the Mohave rattlesnake (Crotalus scutulatus scutulatus) are infamous for being potently neurotoxic, the Mexican subspecies C. s. salvini (Huamantlan rattlesnake) has been largely unstudied beyond crude lethality testing upon mice. In this study we show that at least some populations of this snake are as potently neurotoxic as its northern cousin. Testing of the Mexican antivenom Antivipmyn showed a complete lack of neutralisation for the neurotoxic effects of C. s. salvini venom, while the neurotoxic effects of the US subspecies C. s. scutulatus were time-delayed but ultimately not eliminated. These results document unrecognised potent neurological effects of a Mexican snake and highlight the medical importance of this subspecies, a finding augmented by the ineffectiveness of the Antivipmyn antivenom. These results also influence our understanding of the venom evolution of Crotalus scutulatus, suggesting that neurotoxicity is the ancestral feature of this species, with the US populations which lack neurotoxicity being derived states.

Evaluation of Rhamnetin as an Inhibitor of the Pharmacological Effect of Secretory Phospholipase A2.

Rhamnetin (Rhm), 3-O-methylquercetin (3MQ), and Rhamnazin (Rhz) are methylated derivatives of quercetin commonly found in fruits and vegetables that possess antioxidant and anti-inflammatory properties. Phospholipase A2 (PLA2) displays several important roles during acute inflammation; therefore, this study aimed at investigating new compounds able to inhibit this enzyme, besides evaluating creatine kinase (CK) levels and citotoxicity. Methylated quercetins were compared with quercetin (Q) and were incubated with secretory PLA2 (sPLA2) from Bothrops jararacussu to determine their inhibitory activity. Cytotoxic studies were performed by using the J774 cell lineage incubated with quercertins. In vivo tests were performed with Swiss female mice to evaluate decreasing paw edema potential and compounds' CK levels. Structural modifications on sPLA2 were made with circular dichroism (CD). Despite Q and Rhz showing greater enzymatic inhibitory potential, high CK was observed. Rhm exhibited sPLA2 inhibitory potential, no toxicity and, remarkably, it decreased CK levels. The presence of 3OH on the C-ring of Rhm may contribute to both its anti-inflammatory and enzymatic inhibition of sPLA2, and the methylation of ring A may provide the increase in cell viability and low CK level induced by sPLA2. These results showed that Rhm can be a candidate as a natural compound for the development of new anti-inflammatory drugs.

Na+/K+-ATPase activity in the anoxic turtle (Trachemys scripta) brain at different acclimation temperature.

Survival of prolonged anoxia requires a balance between cellular ATP demand and anaerobic ATP supply from glycolysis, especially in critical tissues such as the brain. To add insight into the ATP demand of the brain of the anoxia-tolerant red-eared slider turtle (Trachemys scripta) during prolonged periods of anoxic submergence, we quantified and compared the number of Na+-K+-ATPase units and their molecular activity in brain tissue from turtles acclimated to either 21°C or 5°C and exposed to either normoxia or anoxia (6h 21°C; 14days at 5°C). Na+-K+-ATPase activity and density per g tissue were similar at 21°C and 5°C in normoxic turtles. Likewise, anoxia exposure at 21°C did not induce any change in Na+-K+-ATPase activity or density. In contrast, prolonged anoxia at 5°C significantly reduced Na+-K+-ATPase activity by 55%, which was largely driven by a 50% reduction of the number of Na+-K+-ATPase units without a change in the activity of existing Na+-K+-ATPase pumps or α-subunit composition. These findings are consistent with the "channel arrest" hypothesis to reduce turtle brain Na+-K+-ATPase activity during prolonged, but not short-term anoxia, a change that likely helps them overwinter under low temperature, anoxic conditions.

Direct Inhibitory Effects of Carbon Monoxide on Six Venoms Containing Fibrinogenolytic Metalloproteinases.

Since the introduction of antivenom administration over a century ago to treat venomous snake bite, it has been the most effective therapy for saving life and limb. However, this treatment 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 (SVMP) by inhibiting these Zn+2 -dependent enzymes directly with carbon monoxide (CO) exposure. Assessment of the fibrinogenolytic effects of venoms collected from the Arizona black rattlesnake, Northern Pacific rattlesnake, Western cottonmouth, Eastern cottonmouth, Broad-banded copperhead and Southern copperhead on human plasmatic coagulation kinetics was performed with thrombelastography in vitro. Isolated exposure of all but one venom (Southern copperhead) to CO significantly decreased the ability of the venoms to compromise coagulation. These results demonstrated that direct inhibition of transition metal-containing venom enzymes by yet to be elucidated mechanisms (e.g. CO, binding to Zn+2 or displacing Zn+2 from the catalytic site, CO binding to histidine residues) can in many instances significantly decrease fibrinogenolytic activity. This new paradigm of CO-based inhibition of the anticoagulant effects of SVMP could potentially diminish haemostatic compromise in envenomed patients until antivenom can be administered.

Varespladib (LY315920) Appears to Be a Potent, Broad-Spectrum, Inhibitor of Snake Venom Phospholipase A2 and a Possible Pre-Referral Treatment for Envenomation.

Snakebite remains a neglected medical problem of the developing world with up to 125,000 deaths each year despite more than a century of calls to improve snakebite prevention and care. An estimated 75% of fatalities from snakebite occur outside the hospital setting. Because phospholipase A2 (PLA2) activity is an important component of venom toxicity, we sought candidate PLA2 inhibitors by directly testing drugs. Surprisingly, varespladib and its orally bioavailable prodrug, methyl-varespladib showed high-level secretory PLA2 (sPLA2) inhibition at nanomolar and picomolar concentrations against 28 medically important snake venoms from six continents. In vivo proof-of-concept studies with varespladib had striking survival benefit against lethal doses of Micrurus fulvius and Vipera berus venom, and suppressed venom-induced sPLA2 activity in rats challenged with 100% lethal doses of M. fulvius venom. Rapid development and deployment of a broad-spectrum PLA2 inhibitor alone or in combination with other small molecule inhibitors of snake toxins (e.g., metalloproteases) could fill the critical therapeutic gap spanning pre-referral and hospital setting. Lower barriers for clinical testing of safety tested, repurposed small molecule therapeutics are a potentially economical and effective path forward to fill the pre-referral gap in the setting of snakebite.

Natural Inhibitors of Snake Venom Metalloendopeptidases: History and Current Challenges.

The research on natural snake venom metalloendopeptidase inhibitors (SVMPIs) began in the 18th century with the pioneering work of Fontana on the resistance that vipers exhibited to their own venom. During the past 40 years, SVMPIs have been isolated mainly from the sera of resistant animals, and characterized to different extents. They are acidic oligomeric glycoproteins that remain biologically active over a wide range of pH and temperature values. Based on primary structure determination, mammalian plasmatic SVMPIs are classified as members of the immunoglobulin (Ig) supergene protein family, while the one isolated from muscle belongs to the ficolin/opsonin P35 family. On the other hand, SVMPIs from snake plasma have been placed in the cystatin superfamily. These natural antitoxins constitute the first line of defense against snake venoms, inhibiting the catalytic activities of snake venom metalloendopeptidases through the establishment of high-affinity, non-covalent interactions. This review presents a historical account of the field of natural resistance, summarizing its main discoveries and current challenges, which are mostly related to the limitations that preclude three-dimensional structural determinations of these inhibitors using "gold-standard" methods; perspectives on how to circumvent such limitations are presented. Potential applications of these SVMPIs in medicine are also highlighted.

No safety in the trees: Local and species-level adaptation of an arboreal squirrel to the venom of sympatric rattlesnakes.

Within some species, squirrels respond to variable selection from venomous snake predators by showing population-level variation in resistance, while between species, some rattlesnakes possess venom that is more effective at overcoming venom resistance in different species of squirrels. A functional evaluation of resistance variation to venom within and between species of squirrels and snakes can link resistance variation to its evolutionary causes across these different evolutionary scales. To do this, we compared the effectiveness of squirrel sera in inhibiting rattlesnake (Crotalus spp.) venom metalloproteinase activity between populations and between species to test for a response to local variation in selection from a single rattlesnake predator and for specialization of two resistant squirrel species to each of their distinct sympatric snake predators. We found that Timber Rattlesnake (Crotalus horridus) venom inhibition by Eastern gray squirrels (Sciurus carolinensis) is higher at a site where the rattlesnakes are present, which suggests selection may maintain venom resistance in populations separated by short distances. Next, we performed a reciprocal cross of venoms and sera from two rattlesnake and two squirrel species. This showed that squirrel resistance is lower when tested against venom from allopatric compared to sympatric rattlesnake species, demonstrating that squirrel inhibitors are specialized to sympatric venom and suggesting a tradeoff in terms of specialization to the venom of a specific species of rattlesnake predator. This pattern can be explained if inhibitors must recognize venom proteins and resistance evolution tracks venom evolution.

Functional characterization of six aspartate (D) recombinant mojastin mutants (r-Moj): A second aspartate amino acid carboxyl to the RGD in r-Moj-D_ peptides is not sufficient to induce apoptosis of SK-Mel-28 cells.

Disintegrins are small peptides produced in viper venom that act as integrin antagonists. When bound to integrins, disintegrins induce altered cellular behaviors, such as apoptotic induction. Disintegrins with RGDDL or RGDDM motifs induce apoptosis of normal and cancer cells. We hypothesized that a second aspartate (D) carboxyl to the RGD is sufficient to induce apoptosis. Five recombinant mojastin D mutants were produced by site-directed mutagenesis (r-Moj-DA, r-Moj-DG, r-Moj-DL, r-Moj-DN, and r-Moj-DV). Stable αv integrin knockdown and shRNA scrambled control SK-Mel-28 cell lines were produced to test a second hypothesis: r-Moj-D_ peptides bind to αv integrin. Only r-Moj-DL, r-Moj-DM, and r-Moj-DN induced apoptosis of SK-Mel-28 cells (at 29.4%, 25.6%, and 36.2%, respectively). Apoptotic induction was significantly reduced in SK-Mel-28 cells with a stable αv integrin knockdown (to 2%, 17%, and 2%, respectively), but not in SK-Mel-28 cells with a stable scrambled shRNA. All six r-Moj-D_ peptides inhibited cell proliferation; ranging from 49.56% (r-Moj-DN) to 75.6% (r-Moj-DA). Cell proliferation inhibition by r-Moj-D_ peptides was significantly reduced in SK-Mel-28 cells with a stable αv integrin knockdown. All six r-Moj-D_ peptides inhibited SK-Mel-28 cell migration at high levels (69%-100%). As a consequence, rac-1 mRNA expression levels were significantly reduced as early as 1 h after treatment, suggesting that rac-1 is involved in the cell migration activity of SK-Mel-28.

Mass spectrometry guided venom profiling and bioactivity screening of the Anatolian Meadow Viper, Vipera anatolica.

This contribution reports on the first characterization of the venom proteome and the bioactivity screening of Vipera anatolica, the Anatolian Meadow Viper. The crude venom as well as an isolated dimeric disintegrin showed remarkable cytotoxic activity against glioblastoma cells. Due to the rare occurrence and the small size of this species only little amount of venom was available, which was profiled by means of a combination of bottom-up and top-down mass spectrometry. From this analysis we identified snake venom metalloproteases, cysteine-rich secretory protein isoforms, a metalloprotease inhibitor, several type A2 phospholipases, disintegrins, a snake venom serine protease, a C-type lectin and a Kunitz-type protease inhibitor. Furthermore, we detected several isoforms of above mentioned proteins as well as previously unknown proteins, indicating an extensive complexity of the venom which would have remained undetected with conventional venomic approaches.

Structural Basis for the Inhibition of a Phospholipase A2-Like Toxin by Caffeic and Aristolochic Acids.

One of the main challenges in toxicology today is to develop therapeutic alternatives for the treatment of snake venom injuries that are not efficiently neutralized by conventional serum therapy. Venom phospholipases A2 (PLA2s) and PLA2-like proteins play a fundamental role in skeletal muscle necrosis, which can result in permanent sequelae and disability. This leads to economic and social problems, especially in developing countries. In this work, we performed structural and functional studies with Piratoxin-I, a Lys49-PLA2 from Bothropspirajai venom, complexed with two compounds present in several plants used in folk medicine against snakebites. These ligands partially neutralized the myotoxic activity of PrTX-I towards binding on the two independent sites of interaction between Lys49-PLA2 and muscle membrane. Our results corroborate the previously proposed mechanism of action of PLA2s-like and provide insights for the design of structure-based inhibitors that could prevent the permanent injuries caused by these proteins in snakebite victims.