Venom-Induced Blood Disturbances by Palearctic Viperid Snakes, and Their Relative Neutralization by Antivenoms and Enzyme-Inhibitors.

Palearctic vipers are medically significant snakes in the genera Daboia, Macrovipera, Montivipera, and Vipera which occur throughout Europe, Central Asia, Near and Middle East. While the ancestral condition is that of a small-bodied, lowland species, extensive diversification has occurred in body size, and niche specialization. Using 27 venom samples and a panel of in vitro coagulation assays, we evaluated the relative coagulotoxic potency of Palearctic viper venoms and compared their neutralization by three antivenoms (Insoserp Europe, VIPERFAV and ViperaTAb) and two metalloprotease inhibitors (prinomastat and DMPS). We show that variation in morphology parallels variation in the Factor X activating procoagulant toxicity, with the three convergent evolutions of larger body sizes (Daboia genus, Macrovipera genus, and Vipera ammodytes uniquely within the Vipera genus) were each accompanied by a significant increase in procoagulant potency. In contrast, the two convergent evolutions of high altitude specialization (the Montivipera genus and Vipera latastei uniquely within the Vipera genus) were each accompanied by a shift away from procoagulant action, with the Montivipera species being particularly potently anticoagulant. Inoserp Europe and VIPERFAV antivenoms were both effective against a broad range of Vipera species, with Inoserp able to neutralize additional species relative to VIPERFAV, reflective of its more complex antivenom immunization mixture. In contrast, ViperaTAb was extremely potent in neutralizing V. berus but, reflective of this being a monovalent antivenom, it was not effective against other Vipera species. The enzyme inhibitor prinomastat efficiently neutralized the metalloprotease-driven Factor X activation of the procoagulant venoms. In contrast, DMPS (2,3-dimercapto-1-propanesulfonic acid), which as been suggested as another potential treatment option in the absence of antivenom, DMPS failed against all venoms tested. Overall, our results highlight the evolutionary variations within Palearctic vipers and help to inform clinical management of viper envenomation.

Examination of the Efficacy and Cross-Reactivity of a Novel Polyclonal Antibody Targeting the Disintegrin Domain in SVMPs to Neutralize Snake Venom.

Snake envenomation can result in hemorrhage, local necrosis, swelling, and if not treated properly can lead to adverse systemic effects such as coagulopathy, nephrotoxicity, neurotoxicity, and cardiotoxicity, which can result in death. As such, snake venom metalloproteinases (SVMPs) and disintegrins are two toxic components that contribute to hemorrhage and interfere with the hemostatic system. Administration of a commercial antivenom is the common antidote to treat snake envenomation, but the high-cost, lack of efficacy, side effects, and limited availability, necessitates the development of new strategies and approaches for therapeutic treatments. Herein, we describe the neutralization ability of anti-disintegrin polyclonal antibody on the activities of isolated disintegrins, P-II/P-III SVMPs, and crude venoms. Our results show disintegrin activity on platelet aggregation in whole blood and the migration of the SK-Mel-28 cells that can be neutralized with anti-disintegrin polyclonal antibody. We characterized a SVMP and found that anti-disintegrin was also able to inhibit its activity in an in vitro proteolytic assay. Moreover, we found that anti-disintegrin could neutralize the proteolytic and hemorrhagic activities from crude Crotalus atrox venom. Our results suggest that anti-disintegrin polyclonal antibodies have the potential for a targeted approach to neutralize SVMPs in the treatment of snakebite envenomations.

Circulating Secretory Phospholipase A2 Activity following Snakebites and Its Relationship with Envenomation Status and Progression of Local Swelling.

We studied whether circulating secretory phospholipase A2 (sPLA2) activity reliably distinguished patients with snakebite envenomation from those with nonvenomous/dry snakebites, and whether patients with progressive local swelling had persistence of circulating sPLA2 activity despite antivenom treatment. We prospectively enrolled adults presenting to the emergency with a history of snakebite in the past 24 hours. We estimated circulating sPLA2 activity at baseline before antivenom administration and after 48 hours in those with envenomation. We enrolled 52 patients with snakebites (mean age 39.3 ± 12.6 years; 35 [67%] men), and 16 patients with infective cellulitis as controls. Thirty patients had local ± systemic envenomation; 15 were classified as dry/nonvenomous bites; and envenomation status was unclear in seven patients. Baseline sPLA2 activity was significantly higher in snakebite patients than that in those with infective cellulitis (4.64 [3.38-5.91] versus 3.38 [1.69-4.01] nmol/minute/mL; P = 0.005). Among patients with snakebites, sPLA2 activity in the highest quartile was significantly associated with envenomation (12 of 27 versus two of 22; P = 0.010). However, median sPLA2 activity did not differ significantly between patients with envenomation and the rest. Baseline sPLA2 activity was significantly associated with the maximum extent of limb swelling (P = 0.031 for trend). In envenomed patients, circulating sPLA2 activity significantly decreased after 48 hours compared with the baseline (5.49 [3.38-8.86] versus 3.38 [2.53-4.64]; P = 0.003) including those with progressive swelling. Although circulating sPLA2 activity was elevated following snakebites, its sensitivity to diagnose envenomation appears to be limited. Administration of more antivenom after systemic manifestations had reversed might not benefit patients with progressive local swelling.

Physiological Stress Integrates Resistance to Rattlesnake Venom and the Onset of Risky Foraging in California Ground Squirrels.

Using venom for predation often leads to the evolution of resistance in prey. Understanding individual variation in venom resistance is key to unlocking basic mechanisms by which antagonistic coevolution can sustain variation in traits under selection. For prey, the opposing challenges of predator avoidance and resource acquisition often lead to correlated levels of risk and reward, which in turn can favor suites of integrated morphological, physiological and behavioral traits. We investigate the relationship between risk-sensitive behaviors, physiological resistance to rattlesnake venom, and stress in a population of California ground squirrels. For the same individuals, we quantified foraging decisions in the presence of snake predators, fecal corticosterone metabolites (a measure of "stress"), and blood serum inhibition of venom enzymatic activity (a measure of venom resistance). Individual responses to snakes were repeatable for three measures of risk-sensitive behavior, indicating that some individuals were consistently risk-averse whereas others were risk tolerant. Venom resistance was lower in squirrels with higher glucocorticoid levels and poorer body condition. Whereas resistance failed to predict proximity to and interactions with snake predators, individuals with higher glucocorticoid levels and in lower body condition waited the longest to feed when near a snake. We compared alternative structural equation models to evaluate alternative hypotheses for the relationships among stress, venom resistance, and behavior. We found support for stress as a shared physiological correlate that independently lowers venom resistance and leads to squirrels that wait longer to feed in the presence of a snake, whereas we did not find evidence that resistance directly facilitates latency to forage. Our findings suggest that stress may help less-resistant squirrels avoid a deadly snakebite, but also reduces feeding opportunities. The combined lethal and non-lethal effects of stressors in predator-prey interactions simultaneously impact multiple key traits in this system, making environmental stress a potential contributor to geographic variation in trait expression of toxic predators and resistant prey.

PLA2 Inhibitor Varespladib as an Alternative to the Antivenom Treatment for Bites from Nikolsky's Viper Vipera berus nikolskii.

Although envenoming by a small East European species of viper is rarely severe, and only exceptionally fatal, lack of specific antivenom stocks in a few areas within this region and possible severe side effects of antivenom application leave most bites to be treated only with antihistamines and supportive therapy. Varespladib is an effective inhibitor of snake phospholipase, and, as such, it could be considered as first-line therapy. The Nikolsky's viper venom contains an extremely high concentration of phospholipase A2 (PLA2), responsible for the toxic effects of the venom, as well as minor amounts of other toxins. If Varespladib can successfully inhibit PLA2 activity, the Nikolsky's viper could be one of the first venomous snakes having an antitoxin-specific treatment regimen. To assess that, Varespladib was administered alone subcutaneously to adult male CD-1 mice (8 mg/kg) and compared to mice exposed to Vipera berus nikolskii crude venom (8 mg/kg = 10 LD50) or a combination of Varespladib and the same amount of the venom. Experimental animals were monitored for the presence of envenoming symptoms and mortality for 48 h after injection. Eighty percent of mice receiving both Varespladib and venom survived, while 100% of the control group receiving venom alone died within 4 h. Experimental results are consistent with Varespladib acting as an effective antitoxin in the mouse model against Nikolsky's viper venom. Further studies are needed under experimental conditions that more closely resemble natural envenoming (i.e., delayed administration).

Understanding the Snake Venom Metalloproteinases: An Interview with Jay Fox and José María Gutiérrez.

Jay W. Fox and José María Gutiérrez recently finished editing a Special Issue on the topic "Snake Venom Metalloproteinases" in Toxins. The Special Issue covers a wide range of topics, including the molecular evolution and structure of snake venom metalloproteinases (SVMPs), the mechanisms involved in the generation of diversity of SVMPs, the mechanism of action of SVMPs, and their role in the pathophysiology of envenomings, with implications for improving the therapy of envenomings. In this interview, we discussed with Jay W. Fox and José María Gutiérrez their research on the SVMPs and their perspectives on the future trends and challenges for studying snake venoms.

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.

Bothrops fonsecai snake venom activities and cross-reactivity with commercial bothropic venom.

In this work, we examined some biochemical and biological activities of Bothrops fonsecai venom, a pitviper endemic to southeastern Brazil, and assessed their neutralization by commercial bothropic antivenom (CAv). Cross-reactivity of venom with CAv was also assessed by immunoblotting and size-exclusion high performance chromatography (SE-HPLC). Bothrops fonsecai venom had PLA2, proteolytic and esterase activities that were neutralized to varying extents by venom:antivenom ratios of 5:1 and 5:2 (PLA2 and esterase activities) or not significantly by either venom:antivenom ratio (proteolytic activity). The minimum hemorrhagic dose (69.2µg) was totally neutralized by both ratios. Clotting time in rat citrated plasma was 33±10.5s (mean±SD; n=5) and was completely neutralized by a 5:2 ratio. Edema formation was dose-dependent (1-30µg/site) and significantly inhibited by both ratios. Venom (10-300µg/mL) caused neuromuscular blockade in extensor digitorum longus preparations; this blockade was inhibited best by a 5:2 ratio. Venom caused myonecrosis and creatine kinase release in vivo (gastrocnemius muscle) and in vitro (extensor digitorum longus) that was effectively neutralized by both venom:antivenom ratios. Immunoblotting showed that venom components of ~25-100kDa interacted with CAv. SE-HPLC profiles for venom incubated with CAv or specific anti-B. fonsecai antivenom raised in rabbits (SAv) indicated that CAv had a higher binding capacity than SAv, whereas SAv had higher affinity than CAv. These findings indicate that B. fonsecai venom contains various activities that are neutralized to different extents by CAv and suggest that CAv could be used to treat envenoming by B. fonsecai.

Viper venom hyaluronidase and its potential inhibitor analysis: a multipronged computational investigation.

Viper venom hyaluronidase (VV-HYA) inhibitors have long been used as therapeutic agents for arresting the local and systemic effects caused during its envenomation. Henceforth, to understand its structural features and also to identify the best potential inhibitor against it the present computational study was undertaken. Structure-based homology modeling of VV-HYA followed by its docking and free energy-based ranking analysis of ligand, the MD simulations of the lead complex was also performed. The sequence analysis and homology modeling of VV-HYA revealed a distorted (ß/α)8 folding as in the case of hydrolases family of proteins. Molecular docking of the resultant 3D structure of VV-HYA with known inhibitors (compounds 1-25) revealed the importance of molecular recognition of hotspot residues (Tyr 75, Arg 288, and Trp 321) other than that of the active site residues. It also revealed that Trp 321 of VV-HYA is highly important for mediating π-π interactions with ligands. In addition, the molecular docking and comparative free energy binding analysis was investigated for the VV-HYA inhibitors (compounds 1-25). Both molecular docking and relative free energy binding analysis clearly confirmed the identification of sodium chromoglycate (compound 1) as the best potential inhibitor against VV-HYA. Molecular dynamics simulations additionally confirmed the stability of their binding interactions. Further, the information obtained from this work is believed to serve as an impetus for future rational designing of new novel VV-HYA inhibitors with improved activity and selectivity.

Antitoxin activity of aqueous extract of Cyclea peltata root against Naja naja venom.

OBJECTIVES: Snakebites are a significant and severe global health problem. Till date, anti-snake venom serum is the only beneficial remedy existing on treating the snakebite victims. As antivenom was reported to induce early or late adverse reactions to human beings, snake venom neutralizing potential for Cyclea peltata root extract was tested for the present research by ex vivo and in vivo approaches on Naja naja toxin. MATERIALS AND METHODS: Ex vivo evaluation of venom toxicity and neutralization assays was carried out. The root extracts from C. peltata were used to evaluate the Ex vivo neutralization tests such as acetylcholinesterase, protease, direct hemolysis assay, phospholipase activity, and procoagulant activity. Gas chromatography-mass spectrometry (GC-MS) analysis from root extracts of C. peltata was done to investigate the bioactive compounds. RESULTS: The in vivo calculation of venom toxicity (LD50) of N. naja venom remained to be 0.301 µg. C. peltata root extracts were efficiently deactivated the venom lethality, and effective dose (ED50) remained to be 7.24 mg/3LD50 of N. naja venom. C. peltata root extract was found effective in counteracting all the lethal effects of venom. GC-MS analysis of the plant extract revealed the presence of antivenom compounds such as tetradecanoic and octadecadienoic acid which have neutralizing properties on N. naja venom. CONCLUSION: The result from the ex vivo and in vivo analysis indicates that C. peltata plant root extract possesses significant compounds such as tetradecanoic acid hexadecanoic acid, heptadecanoic acid, and octadecadienoic acid which can counteract the toxins present in N. naja.

Clinical and Pharmacological Investigation of Myotoxicity in Sri Lankan Russell's Viper (Daboia russelii) Envenoming.

BACKGROUND: Sri Lankan Russell's viper (Daboia russelii) envenoming is reported to cause myotoxicity and neurotoxicity, which are different to the effects of envenoming by most other populations of Russell's vipers. This study aimed to investigate evidence of myotoxicity in Russell's viper envenoming, response to antivenom and the toxins responsible for myotoxicity. METHODOLOGY AND FINDINGS: Clinical features of myotoxicity were assessed in authenticated Russell's viper bite patients admitted to a Sri Lankan teaching hospital. Toxins were isolated using high-performance liquid chromatography. In-vitro myotoxicity of the venom and toxins was investigated in chick biventer nerve-muscle preparations. Of 245 enrolled patients, 177 (72.2%) had local myalgia and 173 (70.6%) had local muscle tenderness. Generalized myalgia and muscle tenderness were present in 35 (14.2%) and 29 (11.8%) patients, respectively. Thirty-seven patients had high (>300 U/l) serum creatine kinase (CK) concentrations in samples 24h post-bite (median: 666 U/l; maximum: 1066 U/l). Peak venom and 24h CK concentrations were not associated (Spearman's correlation; p = 0.48). The 24h CK concentrations differed in patients without myotoxicity (median 58 U/l), compared to those with local (137 U/l) and generalised signs/symptoms of myotoxicity (107 U/l; p = 0.049). Venom caused concentration-dependent inhibition of direct twitches in the chick biventer cervicis nerve-muscle preparation, without completely abolishing direct twitches after 3 h even at 80 µg/ml. Indian polyvalent antivenom did not prevent in-vitro myotoxicity at recommended concentrations. Two phospholipase A2 toxins with molecular weights of 13kDa, U1-viperitoxin-Dr1a (19.2% of venom) and U1-viperitoxin-Dr1b (22.7% of venom), concentration dependently inhibited direct twitches in the chick biventer cervicis nerve-muscle preparation. At 3 µM, U1-viperitoxin-Dr1a abolished twitches, while U1-viperitoxin-Dr1b caused 70% inhibition of twitch force after 3h. Removal of both toxins from whole venom resulted in no in-vitro myotoxicity. CONCLUSION: The study shows that myotoxicity in Sri Lankan Russell's viper envenoming is mild and non-life threatening, and due to two PLA2 toxins with weak myotoxic properties.

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.

Quercetin-3-O-rhamnoside from Euphorbia hirta protects against snake Venom induced toxicity.

BACKGROUND: The plant Euphorbia hirta is widely used against snake envenomations in rural areas and it was proved to be effective in animal models. Therefore, the scientific validation of its phytoconstituents for their antiophidian activity is aimed in the present study. METHODS: E. hirta extract was subjected to bioactivity guided fractionation and the fractions that inhibited different enzyme activities of Naja naja venom in vitro was structurally characterized using UV, FT-IR, LC-MS and NMR spectroscopy. Edema, hemorrhage and lethality inhibition activity of the compound were studied in mice model. In addition, molecular docking and molecular dynamic simulations were also performed in silico. RESULTS: The bioactive fraction was identified as Quercetin-3-O-α-rhamnoside (QR, 448.38 Da). In vitro experiments indicated that protease, phospholipase-A(2), hemolytic activity and hemorrhage inducing activity of the venom were inhibited completely at a ratio of 1:20 (venom: QR) w/w. At the same concentration, the edema ratio was drastically reduced from 187% to 107%. Significant inhibition (93%) of hyaluronidase activity was also observed at a slightly higher concentration of QR (1:50). Further, in in vivo analysis, QR significantly prolonged the survival time of mice injected with snake venom. CONCLUSION: For the first time Quercetin-3-O-α-rhamnoside, isolated from E. hirta, has been shown to exhibit anti-snake venom activity against Naja naja venom induced toxicity. GENERAL SIGNIFICANCE: Exploring such multifunctional lead molecules with anti-venom activity would help in developing complementary medicine for snakebite treatments especially in rural areas where anti-snake venom is not readily available.

Comparative Studies of Structural and Functional Properties of Snake Venom Metalloproteinases.

Snake venom metalloproteinases (SVMPs) induces local and systemic effects on patients suffering from snakebite, degrading extracellular matrix (ECM) proteins such as collagen, gelatin, elastin, laminin, fibronectin, nidogen (entactin), and thrombospondin that cause local hemorrhage and tissue damage. They cleave or activate coagulation factors such as fibrinogen, fibrin, prothrombin, factor V, factor IX, factor X and protein C that bring about systemic coagulopathy. SVMPs and their truncated forms cleave or interfere with platelet adhesive proteins such as vWF, fibrinogen and collagen, and cleave or interfere with platelet receptors such as GPVI, alpha2beta1, GPIb, GPIX, and GPIIbIIIa that result in platelet aggregation defect. SVMPs induce cancer cell line to form morphological changes and apoptosis in vitro concordant with skin necrosis after snakebite in some cases. These local effects caused by SVMPs have no certain treatments, even with commercial anti-venom. SVMPs researches are focusing on their inhibitors, measurement and replacement of blood coagulation factor defects, or anti-cancer drug.

Harpalycin 2 inhibits the enzymatic and platelet aggregation activities of PrTX-III, a D49 phospholipase A2 from Bothrops pirajai venom.

BACKGROUND: Harpalycin 2 (HP-2) is an isoflavone isolated from the leaves of Harpalyce brasiliana Benth., a snakeroot found in northeast region of Brazil and used in folk medicine to treat snakebite. Its leaves are said to be anti-inflammatory. Secretory phospholipases A2 are important toxins found in snake venom and are structurally related to those found in inflammatory conditions in mammals, as in arthritis and atherosclerosis, and for this reason can be valuable tools for searching new anti-phospholipase A2 drugs. METHODS: HP-2 and piratoxin-III (PrTX-III) were purified through chromatographic techniques. The effect of HP-2 in the enzymatic activity of PrTX-III was carried out using 4-nitro-3-octanoyloxy-benzoic acid as the substrate. PrTX-III induced platelet aggregation was inhibited by HP-2 when compared to aristolochic acid and p-bromophenacyl bromide (p-BPB). In an attempt to elucidate how HP-2 interacts with PrTX-III, mass spectrometry, circular dichroism and intrinsic fluorescence analysis were performed. Docking scores of the ligands (HP-2, aristolochic acid and p-BPB) using PrTX-III as target were also calculated. RESULTS: HP-2 inhibited the enzymatic activity of PrTX-III (IC50 11.34 ± 0.28 µg/mL) although it did not form a stable chemical complex in the active site, since mass spectrometry measurements showed no difference between native (13,837.34 Da) and HP-2 treated PrTX-III (13,856.12 Da). A structural analysis of PrTX-III after treatment with HP-2 showed a decrease in dimerization and a slight protein unfolding. In the platelet aggregation assay, HP-2 previously incubated with PrTX-III inhibited the aggregation when compared with untreated protein. PrTX-III chemical treated with aristolochic acid and p-BPB, two standard PLA2 inhibitors, showed low inhibitory effects when compared with the HP-2 treatment. Docking scores corroborated these results, showing higher affinity of HP-2 for the PrTX-III target (PDB code: 1GMZ) than aristolochic acid and p-BPB. HP-2 previous incubated with the platelets inhibits the aggregation induced by untreated PrTX-III as well as arachidonic acid. CONCLUSION: HP-2 changes the structure of PrTX-III, inhibiting the enzymatic activity of this enzyme. In addition, PrTX-III platelet aggregant activity was inhibited by treatment with HP-2, p-BPB and aristolochic acid, and these results were corroborated by docking scores.

Lipoic acid effects on renal function, aminopeptidase activities and oxidative stress in Crotalus durissus terrificus envenomation in mice.

Crotalus durissus terrificus envenomation has been associated with direct nephrotoxicity, rhabdomyolysis, hyperuricemia, urinary hypoosmolality, alterations in aminopeptidase activities (AP) and oxidative stress. This study evaluated the effects of lipoic acid (LA) on renal function, lethality, AP and GSSG/GSH in mice injected with C. d. terrificus venom (vCdt). The doses and routes of administration of LA and vCdt promoted no systemic myotoxicity. LA did not alter significantly the lethality of vCdt. In nonenvenomed, LA induced hypercreatinemia, urinary hyperosmolality, decrease of urinary urea and creatinine, increase of protein in plasma and in soluble fraction (SF) and decrease in membrane-bound fraction (MF) of cortex and medulla. Decreased levels of all AP (except neutral-AP in MF-medulla) were also induced by LA in nonenvenomed. LA associated with vCdt decreased plasma osmolality, hematocrit, urinary uric acid, but increased urinary and SF-medullar protein. LA mitigated the increase of protein in SF-cortex and corrected hyperuricemia, GSSG/GSH and protein in MF-cortex and MF-medulla, as well as decreased plasma neutral AP and acid AP in MF-medulla of envenomed mice. Data suggest that LA contributes to the solubilization/remotion of proteins in MF with impairment of most AP, but it could be beneficial for the treatment of the direct nephrotoxicity of vCdt.

Absence of phospholipase A(2) in most Crotalus horridus venom due to translation blockage: comparison with Crotalus horridus atricaudatus venom.

To investigate the peculiar absence of phospholipases A(2) (PLA(2)s) in most Crotalus horridus (CH) venom, we cloned and sequenced the venom PLA(2)s of three CH specimens from different regions. The results revealed that all the venom glands contained mRNAs that encoded an acidic PLA(2) (designated as either CH-E6 or CH-E6'). The predicted CH-E6 from the Iowan CH and CH-E6' from the South Carolinian CH differed by only one amino acid residue, while the PLA(2) cDNA cloned from the Kentuckian CH contained an early stop codon instead of a Tyr(22) codon. Only the individual South Carolinian CH venom was found to contain the CH-E6' protein whose mass was confirmed by MALDI-TOF analysis. Our results suggest that low PLA(2) expression levels in most CH venom can be attributed to translation blockage. We also purified two acidic PLA(2)s and canebrake toxin from the pooled venom of Crotalus horridus atricaudatus (neurotoxic CH subspecies). One of the acidic PLA(2)s was identical to CH-E6 and showed high lipolytic activity and weak anti-platelet activities. The possibility that C. h. atricaudatus could be a hybrid between CH and Crotalus scutulatus is discussed.

Protease inhibitors: a panacea?

With the increasing evidence of protease involvement in several diseases, novel strategies for drug development involve the use of protease inhibitors (PIs). The local balance between protease inhibitors and proteases is an important determinant of the occurrence and progression of a particular disease. Hence, enzymes and their cognate inhibitors are finding their applications as diagnostic and prognostic markers. PIs are widely implicated for their use in host defense against infection, tissue repair and matrix production, blood coagulation, cancer, and they are, therefore, the current focus as therapeutic alternatives for major diseases such as AIDS and Alzheimer's diseases. This review is a brief summary of the varied role of protein protease inhibitors in controlling the activity of aberrant enzymes in several diseases afflicting mankind today.

The pharmacological role of nucleotidases in snake venoms.

Several hydrolytic enzymes of snake venom have evolved to interfere in various physiological processes, which are well defined. However, hydrolytic enzymes such as nucleotidases (5'nucleotidase, ATPase, and ADPase) are less studied and their pharmacological role in venoms is not clearly defined. Very few studies have shown the pharmacological importance of these endogenous purine release related enzymes in venoms. The near-ubiquitous distribution of these enzymes in venoms, suggests a significant role for these enzymes in envenomation. It is suggested that their major function is in the generation of purines (mainly adenosine)-a multitoxin. Therefore, it appears that these enzymes play a central role in liberating adenosine and through the action of adenosine help in prey immobilization. However, apart from this, these enzymes could also possess other pharmacological activities. Further research is needed to biologically characterize these enzymes in snake venoms, such that their role in venom is clearly established.