Crotalus Adamanteus Research Articles

Molecular genetic evidence for alternative reproductive strategies in North American pitvipers (Serpentes: Viperidae): long-term sperm storage and facultative parthenogenesis

The first documentation of facultative parthenogenesis (FP) in non-avian reptiles (snakes) occurred in 1997, following the application of molecular genetic methods for parentage analysis, and since has been described in only four additional snake species. Here, in two species of live-bearing (viviparous) North American pitviper snakes, using microsatellite DNA fingerprinting, we describe the first record of a virgin birth by FP in the copperhead (Agkistrodon contortrix), and the first genetically confirmed case of long-term sperm storage (LTSS) of exceptional duration (5 years) in the eastern diamond-backed rattlesnake (Crotalus adamanteus). The capacity of female squamate reptiles (snakes, lizards, and amphisbaenians) to store viable sperm for extended periods of time is a competing hypothesis to FP, and it is often inferred when interpreting certain births where adult females have been isolated from adult male mates for prolonged periods. On reviewing the literature of certain cases of LTSS in snakes, we provide compelling support for the prevalence of FP, casting doubt over the widespread acceptance of LTSS. Accordingly, with the discovery of FP in squamates and the results of the present study, we advocate that in order to differentiate between LTSS and FP, especially under natural conditions, rigorous molecular testing will be required. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 104, 934–942.

Therapeutic Potential of Peptides with Neutralizing Ability Towards the Venom and Toxin (CaTx-I) of Crotalus adamanteus

The CaTx-I (PLA2) toxin of Crotalus adamanteus venom is responsible for most of the symptoms observed during envenomation. Synthetic peptides were designed and screened for venom (0.8 μg/ml) and CaTx-I (0.1 μM) inhibition at varying doses of the peptide (10000- 0.0001 μM) using a Cayman chemical human secretory phospholipase A2 (sPLA2, Type II) assay kit. Further, in vitro neutralization studies were evaluated by a fixed dose of peptide (1 μM) against venom (0.8 μg/ml) and toxin (0.1 μM). Among the linear peptides (PIP-18, cyclic C and PIP59-67) that showed potent neutralizing effects against the venom/toxin of C. adamanteus. PIP-18 [IC50, 1.23 μM] and cyclic C [IC50, 1.27 μM] peptides possessed the strongest inhibitory effect against CaTx-I. A fixed dose of CaTx-I (75 μg/kg) was administered intraperitoneally (i.p.) into mice followed by an i.p. injection of peptides PIP-18 and cyclic C at (6 μg/mouse), venom (150 μg/kg) and toxin CaTx-I alone served as references. Mice treated with PIP-18 and cyclic C showed a very strong neutralizing effect and markedly reduced mortality compared to the control after 24 h. The CA venom and CaTx-I injected mice showed severe toxicity after 24 h. Peptides PIP-18 and cyclic C were non-hemolytic at 100 μM. They produced a significant decrease in lipid peroxidase (LPx) and enhancement of superoxide dismutase (SOD), catalase (CAT) and Glutathione-s-transferase (GST) levels indicating their antioxidant property against venom-induced changes in mice. This study confirmed the potent snake venom neutralizing properties of peptides.

Crotalidae Polyvalent Immune Fab

Crotalidae polyvalent immune Fab is an antivenom comprising purified, sheep-derived, Fab IgG fragments and is indicated for use in patients with North American crotaline envenomation. Crotalidae polyvalent immune Fab is produced using four North American snake venoms: Crotalus atrox, Crotalus adamanteus, Crotalus scutulatus, and Agkistrodon piscivorus. Intravenous crotalidae polyvalent immune Fab was effective in patients aged ≥10 years who had minimal or moderate envenomation by a North American crotaline, who presented within 6 hours of the snakebite, and who had progression of the envenomation syndrome, according to the results of two prospective trials. One trial was a noncomparative, multicenter pilot study and the other trial was a randomized, open-label, multicenter trial in which patients received scheduled or 'as needed' administration of crotalidae polyvalent immune Fab after initial control had been achieved. A prospective, postmarketing trial demonstrated the efficacy of crotalidae polyvalent immune Fab in confirmed Crotalus viridis helleri envenomation (indicating cross-protection against a venom not used in its production). Results of these prospective trials are supported by the findings of additional (mainly retrospective) studies demonstrating the efficacy of crotalidae polyvalent immune Fab in patients with crotaline envenomation, including patients with severe envenomation, pediatric patients, and patients with symptoms of neurotoxicity. Despite treatment with crotalidae polyvalent immune Fab, patients may experience delayed-onset or recurrent venom effects (e.g. coagulopathy). Intravenous crotalidae polyvalent immune Fab was generally well tolerated; acute hypersensitivity reactions (e.g. urticaria, rash, pruritus) were the most commonly occurring adverse event.

Reproduction in Male Crotalus adamanteus Beauvois (Eastern Diamond-Backed Rattlesnake): Relationship of Plasma Testosterone to Testis and Kidney Dimensions and the Mating Season

The reproductive ecology of male Crotalus adamanteus Beauvois (Eastern Diamond-backed Rattlesnake) from southwestern Georgia (Joseph W. Jones Ecological Research Center, Baker County) was studied from 14 August 2003 to 14 August 2006. Few studies provide information on reproduction of free-living Eastern Diamondbacked Rattlesnakes, and no information is available on the seasonal relationship of plasma sex steroids of males to changes in the reproductive organs (e.g., mass of the testis and kidney) and the mating season. Here, the main goals were to determine whether: (i) males show variation in concentrations of plasma testosterone (T) during the active season (late March through November), (ii) males have elevated (peak) concentrations of plasma T during the single mating season, which has been documented to occur from late summer through early fall in nearby populations, and (iii) there are seasonal changes in length, width, and mass of the testis and kidney during the active season, particularly during the breeding period. There was a significant difference in mean concentrations of plasma T among seasons, with levels in summer significantly greater than those of spring, and levels in spring and fall were not significantly different. Testis mass and width, but not length, varied significantly across seasons. Testis mass paralleled elevated levels of plasma T, with peak mass occurring in the summer. Similarly, testis width was significantly greater in summer than in fall, but there was no significant difference between summer and spring, nor between spring and fall. We found no significant seasonal changes in any of the kidney measurements. Bisexual pairings were coincident or followed the occurrence of elevated levels of plasma T and changes in testis size; however, despite frequent observations, copulations were not observed. Nonetheless, our results support a mating season of late summer/early fall for the present population of Eastern Diamond-backed Rattlesnakes.

A high-throughput venom-gland transcriptome for the Eastern Diamondback Rattlesnake (Crotalus adamanteus) and evidence for pervasive positive selection across toxin classes

Despite causing considerable human mortality and morbidity, animal toxins represent a valuable source of pharmacologically active macromolecules, a unique system for studying molecular adaptation, and a powerful framework for examining structure-function relationships in proteins. Snake venoms are particularly useful in the latter regard as they consist primarily of a moderate number of proteins and peptides that have been found to belong to just a handful of protein families. As these proteins and peptides are produced in dedicated glands, transcriptome sequencing has proven to be an effective approach to identifying the expressed toxin genes. We generated a venom-gland transcriptome for the Eastern Diamondback Rattlesnake (Crotalus adamanteus) using Roche 454 sequencing technology. In the current work, we focus on transcripts encoding toxins. We identified 40 unique toxin transcripts, 30 of which have full-length coding sequences, and 10 have only partial coding sequences. These toxins account for 24% of the total sequencing reads. We found toxins from 11 previously described families of snake-venom toxins and have discovered two putative, previously undescribed toxin classes. The most diverse and highly expressed toxin classes in the C. adamanteus venom-gland transcriptome are the serine proteinases, metalloproteinases, and C-type lectins. The serine proteinases are the most abundant class, accounting for 35% of the toxin sequencing reads. Metalloproteinases are the most diverse; 11 different forms have been identified. Using our sequences and those available in public databases, we detected positive selection in seven of the eight toxin families for which sufficient sequences were available for the analysis. We find that the vast majority of the genes that contribute directly to this vertebrate trait show evidence for a role for positive selection in their evolutionary history.

Squamous Cell Carcinoma with Vascular Invasion in a Diamondback Rattlesnake (Crotalus adamanteus)

Squamous cell carcinoma (SCC) is a common neoplasm diagnosed in domestic and wild animals, including several species of reptiles. However, reports of SCC invading vasculature or metastasizing in snakes are lacking. This report documents a case of SCC in an adult male eastern diamondback rattlesnake (Crotalus adamanteus) with a unique presentation and invasion into several small- to medium-sized vessels, suggestive of a metastatic process. What was initially suspected to be an abscessed tail was ultimately determined to be SCC originating at the base of the rattle.

A Localizing Circumferential Compression Device Delayed Death After Artificial Eastern Diamondback Rattlesnake Envenomation to the Torso of an Animal Model in a Pilot Study

Nearly all prior studies to delay onset of systemic toxicity and death after snake bite use a model of distal extremity envenomation. In the first of a series of planned studies using snake venoms with different toxicity profiles, the application of a novel device in a new model of torso envenomation in the setting of Eastern Coral Snake (Micrurus fulvius) venom (a potent neurotoxin) envenomation showed promise by delaying systemic intoxication. In this pilot study, we investigated this novel localizing circumferential compression (LoCC) device's ability to delay onset of life threatening systemic toxicity after Eastern Diamondback Rattlesnake (Crotalus adamanteus) envenomation, a potent hemotoxic and myotoxic venom. With university approval, four juvenile female pigs (22-25 kg) were anesthetized, sedated, and intubated but not paralyzed to allow for spontaneous respirations. Each animal was injected subcutaneously with 50 mg of C. adamanteus venom in identical preselected areas of the trunk. After 1 min, two treatment animals had the LoCC device applied; two control animals had no intervention. Vital signs were recorded every 10 min for the first 2 h and every 30 min thereafter. Endpoints included cardiovascular collapse (fatal arrhythmia, loss of mean arterial pressure, or pulse) or respiratory arrest (<3 breaths/min, saturation < 80%) or survival to 7 h. The pigs in the treatment group reached an endpoint at an average time of 355 (+/-65) min compared with control 32 (+/-3.5) min (p < 0.04). In this pilot study, the LoCC device significantly delayed onset of systemic symptoms and death after torso envenomation with Eastern Diamondback Rattlesnake venom in this model.

Multiscale Influences of Landscape Composition and Configuration on the Spatial Ecology of Eastern Diamond-backed Rattlesnakes (Crotalus adamanteus)

Landscape composition and configuration affect ecological processes at the population and community levels, but few studies have demonstrated the effects of landscape pattern on individuals. Because heterogeneity influences abundance and distribution of critical resources, it is hypothesized that it indirectly affects home range size of individuals. To examine the spatial ecology of the declining Eastern Diamond-backed Rattlesnake (Crotalus adamanteus; EDB), we conducted a two-year study in southwestern Georgia. We obtained home range estimates via radio-telemetry, employed Euclidean distance analysis to examine habitat associations at two spatial scales, and used the software program FRAGSTATS to analyze landscape heterogeneity and examine its effect on home range size. Although no significant habitat associations were detected, there were trends for a positive association with pine habitat at the landscape scale and a negative association with agriculture within the home range. Home range size was negatively correlated with several landscape metrics representing heterogeneity in patch configuration, such that individuals in heterogeneous landscapes had small home ranges. This relationship was strongest at three spatial scales: the first was similar to mean home range size of EDBs; and the others were three and four times as large as the largest home range recorded. Together, these results suggest that management regimes to enhance population densities of EDBs emphasize the preservation of pine uplands, while maintaining a mosaic of other habitat types, and limit the conversion of forest to agriculture. Also, our results underscore the importance of using robust analytical tools and multiscale approaches in studies of spatial ecology.

The Herpetofauna of the Camp Shelby Joint Forces Training Center in the Gulf Coastal Plain of Mississippi

Abstract The Camp Shelby Joint Forces Training Center (CSJFTC) is a 54,315-ha National Guard training installation located in the Piney Woods subprovince of the Gulf Coastal Plain physiographic region of south-central Mississippi. A list of 108 native amphibian and reptile species possibly occurring on the CSJFTC was generated using known geographic distribution ranges and museum records. General herpetofaunal field-collecting techniques were used to survey the CSJFTC's aquatic and terrestrial habitats from April 2004-April 2007. Eighty-three native and one introduced species were identified, comprising 59% of the state's known (excluding marine species) and 77% of the CSJFTC's suspected herpetofauna. Ten species listed as needing special protection by state or federal agencies occur at the site, including Crotalus adamanteus, Gopherus polyphemus, Lampropeltis calligaster, Masticophis flagellum, Micrurus fulvius, Ophisaurus attenuatus, Pituophis melanoleucus, Pseudacris ornata, Pseudotriton ruber, and Reg...

The effect of different snake venoms and anti-venoms on thrombin clotting time in human plasma

Background Crotaline snake species, or pit vipers, are distributed throughout Asia and America. While much is known about the clinical effect of these snake venoms, there is a lack of evidence related to the various anti-venoms available and their effectiveness in reversing the effect of different venoms. Aim This study aimed to determine the interaction of the venoms of the following species: Crotalus unicolor, Crotalus adamanteus, Crotalus vegrandis, Trimeresurus spp, Calloselasma rhodostoma, Bothriechis schlegelii and Agkistrodon and the following anti-venoms: Anticrotalico, Antivipmyn, Antibotropico, Antifidico and SAIMR by evaluating their effect on the thrombin clotting time in human plasma. Method The interactions of venoms and anti-venoms were evaluated using thrombin clotting time in human plasma. Results The results demonstrate that Anticrotalico anti-venom was most effective for the Crotalid species (Crotalus unicolor, Crotalus adamanteus, Crotalus vegrandis). Anticrotalico extended the time to clot formation 2.7 fold for Crotalus Unicolor, 3 fold for Crotalus Adamanteus and 4.6 fold for Crotalus Vegrandis. The anti-venoms most efficient in reversing the effect of the Trimeresurus spp venom, were Anticrotalico, Antivipmyn, Antibotropico and Antifidico anti-venoms, which all completely reversed the effect of clot formation as evident by no clot formation within the 999 seconds measurement limit. Bothriechis schlegelii venom was neutralized by all anti-venoms tested. Calloselasma rhodostoma venom was neutralized by Antifidico as well as Anticrotalico. The most efficient anti-venoms against the Agkistrodon venom were Anticrotalico and Antibotropico. In general, monovalent anti-venoms had improved efficiency for their corresponding snake species, depending highly on the composition of the snake venom. This study confirms the importance of considering the choice of anti-venom in a clinical setting, to reverse the effect of specific snake venoms. In addition, this study suggests that some anti-venoms can be considered for use against a variety of snake-venoms.

Catalytic Domain Architecture of Metzincin Metalloproteases

Metalloproteases cleave proteins and peptides, and deregulation of their function leads to pathology. An understanding of their structure and mechanisms of action is necessary to the development of strategies for their regulation. Among metallopeptidases are the metzincins, which are mostly multidomain proteins with approximately 130-260-residue globular catalytic domains showing a common core architecture characterized by a long zinc-binding consensus motif, HEXXHXXGXX(H/D), and a methionine-containing Met-turn. Metzincins participate in unspecific protein degradation such as digestion of intake proteins and tissue development, maintenance, and remodeling, but they are also involved in highly specific cleavage events to activate or inactivate themselves or other (pro)enzymes and bioactive peptides. Metzincins are subdivided into families, and seven such families have been analyzed at the structural level: the astacins, ADAMs/adamalysins/reprolysins, serralysins, matrix metalloproteinases, snapalysins, leishmanolysins, and pappalysins. These families are reviewed from a structural point of view.

Polyphosphatase Activity of CthTTM, a Bacterial Triphosphate Tunnel Metalloenzyme

Triphosphate tunnel metalloenzymes (TTMs) are a superfamily of phosphotransferases with a distinctive active site located within an eight-stranded beta barrel. The best understood family members are the eukaryal RNA triphosphatases, which catalyze the initial step in mRNA capping. The RNA triphosphatases characteristically hydrolyze nucleoside 5'-triphosphates in the presence of manganese and are inept at cleaving inorganic tripolyphosphate. We recently identified a TTM protein from the bacterium Clostridium thermocellum (CthTTM) with the opposite substrate preference. Here we report that CthTTM catalyzes hydrolysis of guanosine 5'-tetraphosphate to yield GTP and P(i) (K(m) = 70 microm, k(cat) = 170 s(-1)) much more effectively than it converts GTP to GDP and P(i) (K(m) = 70 microm, k(cat) = 0.3 s(-1)), implying that a nucleoside interferes when positioned too close to the tunnel entrance. CthTTM is capable of quantitatively cleaving diadenosine hexaphosphate but has feeble activity with shorter derivatives diadenosine tetraphosphate and diadenosine pentaphosphate. We propose that the tunnel opens to accommodate the dumbbell-shaped diadenosine hexaphosphate and then closes around it to perform catalysis. We find that CthTTM can exhaustively hydrolyze a long-chain inorganic polyphosphate, a molecule that plays important roles in bacterial physiology. CthTTM differs from other known polyphosphatases in that it yields a approximately 2:1 mixture of P(i) and PP(i) end products. Bacterial/archaeal TTMs have a C-terminal helix located near the tunnel entrance. Deletion of this helix from CthTTM exerts pleiotropic effects. (i) It suppresses hydrolysis of guanosine 5'-tetraphosphate and inorganic PPP(i); (ii) it stimulates NTP hydrolysis; and (iii) it biases the outcome of the long-chain polyphosphatase reaction more strongly in favor of P(i) production. We discuss models for substrate binding in the triphosphate tunnel.

Fatality in a case of envenomation byCrotalus adamanteus initially successfully treated with polyvalent ovine antivenom followed by recurrence of defibrinogenation syndrome

Recurrences of clinical or laboratory manifestations of North American pit viper envenomation may happen despite control of the envenomation syndrome by prompt and adequate antivenom therapy. Recurrences of coagulopathy in victims of Eastern diamondback rattlesnake envenomation are generally regarded as benign. The vast majority suffer no actual bleeding despite florid coagulation laboratory abnormalities due to selective defibrinogenation. We report what we believe to be the first fatality following successful control of the envenomation syndrome following ovine antivenom treatment resulting from envenomation by a bite from the Eastern diamondback rattlesnake. This case raises the question of whether such recurrences are in fact benign, causal, or coincidental. This patient sustained significant brain hemorrhage and death ensued due to generalized cerebral edema. Defibrinogenation occurred 4 days after treatment with ovine antivenom. Coagulation abnormalities following Eastern diamondback rattlesnake envenomation are due to selective defibrinogenation. This is separate from disseminated intravascular coagulation (DIC). Thrombin generation, thus hemostasis, are generally considered normal. This case may cause reexamination of this belief.

Vegetation structure and the habitat specificity of a declining North American reptile: A remnant of former landscapes

Although all species provide some spatial information about past environments, remnant populations of habitat specialists can serve as biological legacies and natural archives of historical landscapes. The endangered longleaf pine ecosystem is home to an array of imperiled fauna that specialize on the habitat. Often referred to as pine savanna, the ecosystem was characterized by longleaf pine ( Pinus palustris), but included an array of open-canopy habitats within a grassland matrix dominated by a variety of tree species. In this study, we used a coarse scale of description to quantify habitat associations of a declining reptile, the eastern diamondback rattlesnake ( Crotalus adamanteus), historically associated with pine savannas of the southeastern United States. We made cross-scale habitat comparisons and controlled for land use and geographic variability. Habitat models of within home range and microhabitat selection indicated that the species was associated with an open-canopy savanna community structure. We identified the eastern diamondback rattlesnake as a remnant of the historical southeastern savanna, which is important for species conservation and broader management of the southeastern savanna community. Given their longevity and habitat specificity, remnant eastern diamondback rattlesnake populations are biological legacies of the southeastern savanna community and act as a surrogate for the prioritization of land conservation. Thus, the species’ presence provides spatial information that can be used by conservationists to identify habitats that have high restoration potential, and also increases the probability that other species associated with pine savanna occur locally.

Correspondence

Dear Editor Following Dr Schaer's letter we have checked our reference list and found that indeed we have inadvertently referenced the wrong study with regard to the prevalence of cardiac arrhythmias in dogs with Eastern diamondback rattlesnake bites. We obviously had every intention of referencing the original study and of giving the authors the full credit they deserved. Apparently there was an error while transferring this reference (“Willey JR, Schaer M. Eastern diamondback rattlesnake (Crotalus adamanteus) envenomation of dogs: 31 cases (1982–2002). J Am Anim Hosp Assoc 2005;41:22–33”) from the Endnote program to the list of references. Obviously, reference (10) that was cited in relation to snakebites had nothing to with that subject and was erroneously cited there. We would like to apologize and are also grateful for the opportunity to correct this unfortunate mistake. Yours sincerely

Correspondence

Dear Editor I am writing this letter in reference to the paper titled “Cardiac arrhythmias and serum cardiac troponins in Vipera palaestinae envenomations in dogs” by Segev G., et al in J Vet Intern Med 2008;22:106–113. I appreciate the work that made this study possible, and I enjoyed reading it. If it were not for my great interest in venomous snake bites in dogs, I probably would not have detected the following accidental oversight. On the bottom of p. 106 and top of p. 107 the authors make reference to a 42–47% incidence of cardiac arrhythmias in dogs envenomated by the Eastern diamondback rattlesnake, Crotalus adamanteus, but their reference (10) is in error because I and my colleagues authored the papers that contain that particular information.1, 2 The paper that is used as reference (10) describes serum troponin elevations in dogs with gastric dilatation-volvulus and chest trauma. We all make mistakes, but at the same time all authors should take every effort to quote the original source of scientific information. The original sources are provided should there be any interest by the JVIM readers.

Mathematical modelling of amino acid resolution catalyzed by l-amino acid oxidases from Crotalus adamanteus and Crotalus atrox

Amino acid resolution was studied in this paper. l-Methionine and dl-methionine were used as substrates for the two snake venom l-amino acid oxidases (from Crotalus adamanteus and Crotalus atrox). Even though these enzymes have similar background, they exhibit differences as well as similarities. It was found that both enzymes are active towards l-methionine as substrate ( V m = 0.78 mmol min −1 g −1) and have similar affinities ( K m L - met ( C. adamanteus) = 0.19 mM, K m L - met ( C. atrox) = 0.25 mM). Both enzymes were inhibited in the presence of 2- oxo-4-methylthiobutyric acid ( K i 2 - oxo ( C. adamanteus) = 0.83 mM, K i 2 - oxo ( C. atrox) = 2.04 mM), a reaction product of l-methionine oxidative deamination. l-Amino acid oxidase from C. atrox was found to be inhibited by the substrate as well. l-Amino acid oxidase from C. adamanteus was inhibited by d-enantiomer of l-methionine ( K i D - met = 1.41 mM ). Both enzymes were successful in l-methionine oxidation in the batch reactor (100% conversion). This was not the case in the continuously operated enzyme membrane reactor where enzyme deactivation occurred.

Mechanism and Regulation of the Two-component FMN-dependent Monooxygenase ActVA-ActVB from Streptomyces coelicolor

The ActVA-ActVB system from Streptomyces coelicolor is a two-component flavin-dependent monooxygenase involved in the antibiotic actinorhodin biosynthesis. ActVB is a NADH:flavin oxidoreductase that provides a reduced FMN to ActVA, the monooxygenase that catalyzes the hydroxylation of dihydrokalafungin, the precursor of actinorhodin. In this work, using stopped-flow spectrophotometry, we investigated the mechanism of hydroxylation of dihydrokalafungin catalyzed by ActVA and that of the reduced FMN transfer from ActVB to ActVA. Our results show that the hydroxylation mechanism proceeds with the participation of two different reaction intermediates in ActVA active site. First, a C(4a)-FMN-hydroperoxide species is formed after binding of reduced FMN to the monooxygenase and reaction with O(2). This intermediate hydroxylates the substrate and is transformed to a second reaction intermediate, a C(4a)-FMN-hydroxy species. In addition, we demonstrate that reduced FMN can be transferred efficiently from the reductase to the monooxygenase without involving any protein.protein complexes. The rate of transfer of reduced FMN from ActVB to ActVA was found to be controlled by the release of NAD(+) from ActVB and was strongly affected by NAD(+) concentration, with an IC(50) of 40 microm. This control of reduced FMN transfer by NAD(+) was associated with the formation of a strong charge.transfer complex between NAD(+) and reduced FMN in the active site of ActVB. These results suggest that, in Streptomyces coelicolor, the reductase component ActVB can act as a regulatory component of the monooxygenase activity by controlling the transfer of reduced FMN to the monooxygenase.

Molecular modeling and inhibition of phospholipase A 2 by polyhydroxy phenolic compounds

Phospholipases A 2 are enzymes responsible for the hydrolysis of membrane phospholipids that release arachidonic acid, which serves as substrate for pro-inflammatory mediators, such as prostaglandins and leucotriens. The design of specific inhibitors for PLA 2 might help in the development of new anti-inflammatory drugs. Polyhydroxy phenolic compounds, such as flavonoids, vitamin E, rosmarinic acid and aristolochic acid, are able to inhibit PLA 2 from different sources. Herein, we have studied the kinetic behavior and the capacity of inhibiting edema formation induced by PLA 2 of five different polyhydroxy phenolic compounds (two phenolic derivatives and three acetophenone hydroxylated derivatives) extracted from the venom of Crotalus adamanteus. The results showed that compounds 1,3-dihydroxy benzene, 1,3,5-trihydroxy benzene and 2,4,6-trihydroxy acetophenone were the most efficient in the inhibition of the enzymatic activity and edema induction by PLA 2. It was also verified that the number of hydroxyls in each molecule is not a limiting factor for the inhibition capacity of these compounds. Molecular modeling studies indicated that the most active compounds are linked to the amino acid Asp 49 and that they destabilize the coordination of the calcium atom, which is essential to the catalytic activity. The study of potential surfaces showed that there are conditions in which the potential values must be adequate for enzyme complex formation with polyhydroxy phenolic compounds. When the potential over the hydroxyl surfaces is very high, formation of stable complexes does not occur and the enzyme does not act intensely. These results might be helpful in the design of a drug that specifically inhibits PLA 2.

Molecular modeling of the inhibition of enzyme PLA2 from snake venom by dipyrone and 1‐phenyl‐3‐methyl‐5‐pyrazolone

AbstractPhospholipases A2 (PLA2) are enzymes that trigger the degradation cascade of the arachidonic acid, leading to the formation of pro‐inflammatory eicosanoids. The selective inhibition of PLA2s is crucial in the search for a more efficient anti‐inflammatory drug with fewer side effects than the drugs currently used. Hence, we studied the influences caused by two pyrazolonic inhibitors: dipyrone (DIP) and 1‐phenyl‐3‐methyl‐5‐pyrazolone (PMP) on the kinetic behavior of PLA2 from Crotalus adamanteus venom. Molecular modeling results, by DFT and MM approaches, showed that DIP is strongly associated to the active site of PLA2 through three hydrogen bonds, whereas PMP is associated to the enzyme just through hydrophobic interactions. In addition, only PMP presents an intramolecular hydrogen bond that make difficult the formation of more efficient interactions with PLA2. These results help in the understanding of the experimental observations. Experimentally, the results showed that PLA2 from C. adamanteus present a typical Michaelian behavior. In addition, the calculated kinetic parameters showed that, in the presence of DIP or PMP, the maximum enzymatic velocity (VMAX) value was kept constant, whereas the Michaelis constant (KM) values increased and the inhibition constant (KI) decreased, indicating competitive inhibition. These results show that the phenyl‐pyrazolonic structures might help in the development and design of new drugs able to selectively inhibit PLA2. © 2008 Wiley Periodicals, Inc. Int J Quantum Chem, 2008