Quercetin as an inhibitor of snake venom secretory phospholipase A2

As polyphenolic compounds isolated from plants extracts, flavonoids have been applied to various pharmaceutical uses in recent decades due to their anti-inflammatory, cancer preventive, and cardiovascular protective activities. In this study, we evaluated the effects of the flavonoid quercetin on Crotalus durissus terrificus secretory phospholipase A2 (sPLA2), an important protein involved in the release of arachidonic acid from phospholipid membranes. The protein was chemically modified by treatment with quercetin, which resulted in modifications in the secondary structure as evidenced through circular dichroism. In addition, quercetin was able to inhibit the enzymatic activity and some pharmacological activities of sPLA2, including its antibacterial activity, its ability to induce platelet aggregation, and its myotoxicity by approximately 40%, but was not able to reduce the inflammatory and neurotoxic activities of sPLA2. These results suggest the existence of two pharmacological sites in the protein, one that is correlated with the enzymatic site and another that is distinct from it. We also performed molecular docking to better understand the possible interactions between quercetin and sPLA2. Our docking data showed the existence of hydrogen-bonded, polar interactions and hydrophobic interactions, suggesting that other flavonoids with similar structures could bind to sPLA2. Further research is warranted to investigate the potential use of flavonoids as sPLA2 inhibitors. (C) 2010 Elsevier B.V. All rights reserved.

Crystal structure of Bn IV in complex with myristic acid: A Lys49 myotoxic phospholipase A(2) from Bothrops neuwiedi venom

The LY549-PLA(2)s myotoxins have attracted attention as models for the induction of myonecrosis by a catalytically independent mechanism of action. Structural studies and biological activities have demonstrated that the myotoxic activity of LYS49-PLA(2) is independent of the catalytic activity site. The myotoxic effect is conventionally thought to be to due to the C-terminal region 111-121, which plays an effective role in membrane damage. In the present study, Bn IV LYS49-PLA(2) was isolated from Bothrops neuwiedi snake venom in complex with myristic acid (CH3(CH2)(12)COOH) and its overall structure was refined at 2.2 angstrom resolution. The Bn IV crystals belong to monoclinic space group P2(1) and contain a dimer in the asymmetric unit. The unit cell parameters are a = 38.8, b = 70.4, c = 44.0 angstrom. The biological assembly is a "conventional dimer" and the results confirm that dimer formation is not relevant to the myotoxic activity. Electron density map analysis of the Bn IV structure shows clearly the presence of myristic acid in catalytic site. The relevant structural features for myotoxic activity are located in the C-terminal region and the Bn IV C-terminal residues NKKYRY are a probable heparin binding domain. These findings indicate that the mechanism of interaction between Bn IV and muscle cell membranes is through some kind of cell signal transduction mediated by heparin complexes. (C) 2010 Elsevier Masson SAS. All rights reserved.

Crystallization and preliminary X-ray diffraction analysis of a eumenine mastoparan toxin: a new class of mast-cell degranulating peptide in the wasp venom

Mastoparans are tetradecapeptides found to be the major component of vespid venoms. A mastoparan toxin isolated from the venom of Anterhynchium flavomarginatum micado has been crystallized and X-ray diffraction data collected to 2.7 Angstrom resolution using a synchrotron-radiation source. Crystals were determined to belong to the space group P6(2)22 (P6(4)22). This is the first mastoparan to be crystallized and will provide further insights into the conformational significance of mastoparan toxins with respect to their potency and activity in G-protein regulation.

Preliminary cryocrystallography analysis of an eumenine mastoparan toxin isolated from the venom of the wasp Anterhynchium flavomarginatum micado

Mastoparans are tetradecapeptides found to be the major component of vespid venoms. These peptides present a wide spectrum of biological activities, such as mast cell degranulation, hemolytic activity and also reveals antimicrobial activity. A mastoparan toxin isolated from the venom of Anterhynchium flavomarginatum micado has been crystallized. At room temperature these crystals diffracted to 2.8 Angstrom resolution. However, upon cooling to cryogenic temperature around 85 K, the original resolution limit could be improved to 2.0 Angstrom. Crystals were determined to belong to the space group P3(1) (P3(2)). This is the first mastoparan to be crystallized and it will provide further insights in the conformational significance of mastoparan toxins, with respect to their potency and activity in G protein regulation. (C) 3001 Elsevier B.V. B.V. All rights reserved.

Structure-function relationship of new crotamine isoform from the Crotalus durissus cascavella

In this work we isolated a novel crotamine like protein from the Crotalus durissus cascavella venom by combination of molecular exclusion and analytical reverse phase HPLC. Its primary structure was:YKRCHKKGGHCFPKEKICLPPSSDLGKMDCRWKRK-CCKKGS GK. This protein showed a molecular mass of 4892.89 da that was determined by Matrix Assisted Laser Desorption Ionization Time-of-flight (MALDI-TOF) mass spectrometry. The approximately pI value of this protein was determined in 9.9 by two-dimensional electrophoresis. This crotamine-like protein isolated here and that named as Cro 2 produced skeletal muscle spasm and spastic paralysis in mice similarly to other crotamines like proteins. Cro 2 did not modify the insulin secretion at low glucose concentration (2.8 and 5.6 mM), but at high glucose concentration (16.7 mM) we observed an insulin secretion increasing of 2.7-3.0-fold than to control. The Na+ channel antagonist tetrodoxin (6 mM) decreased glucose and Cro 2-induced insulin secretion. These results suggested that Na+ channel are involved in the insulin secretion. In this article, we also purified some peptide fragment from the treatment of reduced and carboxymethylated Cro 2 (RC-Cro 2) with cyanogen bromide and protease V8 from Staphylococcus aureus. The isolated pancreatic beta-cells were then treated with peptides only at high glucose concentration (16.7 mM), in this condition only two peptides induced insulin secretion. The amino acid sequence homology analysis of the whole crotamine as well as the biologically-active peptide allowed determining the consensus region of the biologically-active crotamine responsible for insulin secretion was KGGHCFPKE and DCRWKWKCCKKGSG.

Antiophidic Solanidane Steroidal Alkaloids from Solanum campaniforme

Three new solanidane alkaloids bearing a 22,23-epoxy ring (1-3) and four known compounds were isolated from leaves of Solanum campaniforme. The structures were determined using spectroscopic techniques, including ID and 2D NMR, and HRESIMS experiments. The antiophidic activity of the alkaloids was tested against Bothrops pauloensis venom. Compounds 1-3 completely inhibited myotoxicity without inhibiting phospholipase A(2) activity of the venom, while hemorrhage and skin necrosis were significantly reduced in the presence of alkaloids 1 and 2.

Purification and biological effects of a C-type lectin isolated from Bothrops moojeni

Snake venom proteins from the C-type lectin family have very distinct biological activities despite their highly conserved primary structure, which is homologous to the carbohydrate recognition region of true C-type lectins. We purified a lectin-like protein (BmLec) from Bothrops moojeni venom and investigated its effect on platelet aggregation, insulin secretion, antibacterial activity, and isolated kidney cells. The BmLec was purified using two chromatographic steps: affinity chromatography and reverse phase high performance liquid chromatography (HPLC). BmLec showed a dose-dependent platelet aggregation and significantly decreased the bacterial growth rate in approximately 15%. During scanning electron microscopy, the profile of Xanthomonas axonopodis pv. passiflorae treated with lectin disclosed a high vesiculation and membrane rupture. BmLec induced a strong and significant increase in insulin secretion at 2.8 and 16.7 mM glucose concentrations, and this effect was seen in the presence of EGTA in both experiments. BmLec (10 mu g/mL) increased the perfusion pressure, renal vascular resistance and urinary flow. The glomerular filtration rate and percentages of sodium, potassium and chloride tubular transport were reduced at 60 minutes of perfusion. Renal alterations caused by BmLec were completely inhibited by indomethacin in all evaluated parameters. In conclusion, the C-type lectin isolated from Bothrops moojeni affected platelet aggregation, insulin secretion, antibacterial activity and isolated kidney function.

Abdominal hyperalgesia in secretory phospholipase A(2)-induced rat pancreatitis: Distinct roles of NK1 receptors

We investigated the potential of secretory phospholipase A(2) (sPLA(2))-induced pancreatitis to promote abdominal hyperalgesia, as well as to depolarize sensory fibres in vitro using a grease-gap technique. Pancreatitis was induced by the injection of sPLA(2) from Crotalus durissus terrificus (sPLA(2) Cdt, 300 mu g kg(-1)) venom into the common bile duct of rats. Pancreatic inflammatory signs, serum amylase levels and abdominal hyperalgesia were evaluated in rats treated or not with SR140333, a tachykinin NK1 receptor antagonist. Injection of sPLA(2) Cdt caused pancreatic oedema formation and increased pancreatic neutrophil infiltration and serum amylase at 4 h, which returned to normality by 24 h, except for the neutrophil infiltration, which was still increased at this time point. Animals injected with sPLA(2) exhibited a lower withdrawal threshold to electronic von Frey stimulation in the upper abdominal region at 4 h, but not 24 h, post-injection when compared with saline-injected rats. Pre-treatment of animals with SR140333 significantly reduced the sPLA(2) Cdt-induced abdominal hyperalgesia, without affecting the other parameters. Neither sPLA(2) Cdt nor sPLA(2) from Naja mocambique mocambique venom depolarized capsaicin-sensitive sensory fibres from rat vagus nerve, but they decreased the propagated compound action potentials in both A and C fibres. These data show for the first time that NK1 receptors play an important role in the early abdominal hyperalgesia in a rat model of sPLA(2)-induced pancreatitis, suggesting that these receptors are of importance in the development of pain in the pancreatitis condition. We also provide evidence that sPLA(2)s do not directly depolarize sensory fibres in vitro. (C) 2011 European Federation of International Association for the Study of Pain Chapters. Published by Elsevier Ltd. All rights reserved.

Umbelliferone induces changes in the structure and pharmacological activities of Bn IV, a phospholipase A(2) isoform isolated from Bothrops neuwiedi

In this paper was demonstrated that umbelliferone induces changes in structure and pharmacological activities of Bn IV, a lysine 49 secretory phospholipase A(2) (sPLA2) from Both tops neuwiedi. Incubation of Bn IV with umbelliferone virtually abolished platelet aggregation, edema, and myotoxicity induced by native Bn IV. The amino acid sequence of Bn IV showed high sequence similarities with other Lys49 sPLA2s from B. jararacussu (BthTx-I), B. pirajai (PrTx-I), and B. neuwiedi pauloensis (Bn SP6 and Bn SP7). This sPLA2 also has a highly conserved C-terminal amino acid sequence, which has been shown as important for the pharmacological activities of Lys49 sPLA2. Sequencing of Bn IV previously treated with umbelliferone revealed modification of S(1) and S(20). Fluorescent spectral analysis and circular dichroism (CD) studies showed that umbelliferone modified the secondary structure of this protein. Moreover, the pharmacological activity of Bn IV is driven by synergism of the C-terminal region with the a-helix motifs, which are involved in substrate binding of the Asp49 and Lys49 residues of 5PLA2 and have a direct effect on the Ca2+-independent membrane damage of some secretory snake venom PLA2. For Bn IV, these interactions are potentially important for triggering the pharmacological activity of this 5PLA2. (C) 2011 Elsevier Ltd. All rights reserved.

Effect of the synthetic coumarin, ethyl 2-oxo-2H-chromene-3-carboxylate, on activity of Crotalus durissus ruruima sPLA2 as well as on edema and platelet aggregation induced by this factor

We show that ethyl 2-oxo-2H-chromene-3-carboxylate (EOCC), a synthetic coumarin, irreversibly inhibits phospholipase A(2) (sPLA2) from Crotalus durissus ruruima venom (sPLA2r) with an IC(50) of 3.1 +/- 0.06 nmol. EOCC strongly decreased the V(max) and K(m), and it virtually abolished the enzyme activity of sPLA2r as well as sPLA2s from other sources. The edema induced by 5PLA2r + EOCC was less than that induced by 5PLA2r treated with p-bromophenacyl bromide, which was more efficient at neutralizing the platelet aggregation activity of native 5PLA2r. Native 5PLA2r induced platelet aggregation of 91.54 +/- 9.3%, and sPLA2r +/- EOCC induced a platelet aggregation of 18.56 +/- 6.5%. EOCC treatment also decreased the myotoxic effect of sPLA2r. Mass spectrometry showed that EOCC formed a stable complex with sPLA2r, which increased the mass of native 5PLA2r from 14,299.34 da to 14,736.22 Da. Moreover, the formation of this complex appeared to be involved in the loss of 5PLA2r activity. Our results strongly suggest that EOCC can be used as a pharmacological agent against the 5PLA2 in Crotalus durissus sp. venom as well as other sPLA2s. (C) 2010 Elsevier Ltd. All rights reserved.

Evaluation of anti-inflammatory activity of derivatives from aerial parts of Baccharis uncinella

Context: Species of Baccharis exhibit antibiotic, antiseptic, and wound-healing properties, and have been used in the traditional medicine of South America for the treatment of inflammation, headaches, diabetes, and hepatobiliary disorders.Objective: To investigate the anti-inflammatory activity of organic phases from EtOH extract of the aerial parts of Baccharis uncinella DC (Asteraceae).Materials and methods: The crude EtOH extract from the aerial parts of B. uncinella was subjected to partition procedures and the corresponding CH(2)Cl(2) and EtOAc phases were subjected to several chromatographic separation procedures. Thus, these phases and their purified compounds were assayed for evaluation of anti-inflammatory activity.Results: The CH(2)Cl(2) phase from EtOH extract from B. uncinella contained two triterpenoids (oleanolic and ursolic acids) and one flavonoid (pectolinaringenin), whereas the respective EtOAc phase showed to be composed mainly by two phenylpropanoid derivatives (caffeic and ferulic acids). The CH(2)Cl(2) and EtOAc phases as well as their isolated compounds exhibited anti-inflammatory effects against inflammatory reactions induced by phospholipase A2 (from Crotalus durissus terrificus venom) and by carrageenan.Discussion and conclusion: The results suggested that the components obtained from partition phases of EtOH extract of B. uncinella could represent lead molecules for the development of anti-inflammatory agents. Additionally, the results confirmed the use of Baccharis genus in the traditional medicine of South America for the treatment of inflammation and other heath disorders. To date, the present work describes for the first time the anti-inflammatory effects of compounds isolated from B. uncinella.

Inhibition of Neurotoxic Secretory Phospholipases A(2) Enzymatic, Edematogenic, and Myotoxic Activities by Harpalycin 2, an Isoflavone Isolated from Harpalyce brasiliana Benth

Secretory phospholipases A(2) (sPLA(2)) exert proinflammatory actions through lipid mediators. These enzymes have been found to be elevated in many inflammatory disorders such as rheumatoid arthritis, sepsis, and atherosclerosis. The aim of this study was to evaluate the effect of harpalycin 2 (Har2), an isoflavone isolated from Harpalyce brasiliana Benth., in the enzymatic, edematogenic, and myotoxic activities of sPLA2 from Bothrops pirajai, Crotalus durissus terrificus, Apis mellifera, and Naja naja venoms. Har2 inhibits all sPLA(2) tested. PrTX-III (B. pirajai venom) was inhibited at about 58.7%, Cdt F15 (C. d. terrificus venom) at 78.8%, Apis (from bee venom) at 87.7%, and Naja (N. naja venom) at 88.1%. Edema induced by exogenous sPLA(2) administration performed in mice paws showed significant inhibition by Har2 at the initial step. In addition, Har2 also inhibited the myotoxic activity of these sPLA(2)s. In order to understand how Har2 interacts with these enzymes, docking calculations were made, indicating that the residues His48 and Asp49 in the active site of these enzymes interacted powerfully with Har2 through hydrogen bonds. These data pointed to a possible anti-inflammatory activity of Har2 through sPLA(2) inhibition.

Purification and preliminary crystallographic analysis of a new Lys49-PLA2 from B-jararacussu

BjVIII is a new myotoxic Lys49-PLA2 isolated from Bothrops jararacussu venom that exhibits atypical effects on human platelet aggregation. To better understand the mode of action of BjVIII, crystallographic studies were initiated. Two crystal forms were obtained, both containing two molecules in the asymmetric unit (ASU). Synchrotron radiation diffraction data were collected to 2.0 angstrom resolution and 1.9 angstrom resolution for crystals belonging to the space group P2(1)2(1)2(1) (a = 48.4 angstrom, b = 65.3 angstrom, c = 84.3 angstrom) and space group P3(1)21 (a = b = 55.7 angstrom, c = 127.9 angstrom), respectively. Refinement is currently in progress and the refined structures are expected to shed light on the unusual platelet aggregation activity observed for BjVIII.

BIOCHEMICAL VARIABILITY BETWEEN VENOMS FROM DIFFERENT HONEYBEE (APIS-MELLIFERA) RACES

1. The comparison of molecular exclusion cromatography profiles of venoms from sting apparatuses of Apis mellifera ligustica, Apis mellifera adansonii and Africanized honey-bees in Sephadex G-100 revealed both qualitative and quantitative differences.2. The venoms from A.m. ligustica and A.m. adansonii presented, respectively, three and two peaks characteristic of each sub-species, while Africanized honey-bee was characterized by the absence of eight peaks common to the former.3. The polypeptides with M(r) in the range from 100,000 to 7500 da correspond respectively to 62.0%, 66.6% and 68.7% of total proteins from the venon of A.m. ligustica, A.m. adansonii and Africanized honey-bees, while the peptidic fraction with M(r) range from 4100 to 2000 da corresponds to 11.4%, 32.4% and 10.2% of venom protein, respectively.