Screening for target toxins of the antiophidic protein DM64 through a gel-based interactomics approach.

DM64 is a glycosylated protein with antivenom activity isolated from the serum of the opossum Didelphis aurita. It binds non-covalently to myotoxins I (Asp49) and II (Lys49) from Bothrops asper venom and inhibits their myotoxic effect. In this study, an affinity column with immobilized DM64 as bait was used to fish potential target toxins. All ten isolated myotoxins tested were able to effectively bind to the DM64 column. To better access the specificity of the inhibitor, crude venoms from Bothrops (8 species), Crotalus (2 species) and Naja naja atra were submitted to the affinity purification. Venom fractions bound and nonbound to the DM64 column were analyzed by two-dimensional gel electrophoresis and MALDI-TOF/TOF MS. Although venom fractions bound to the column were mainly composed of basic PLA2, a few spots corresponding to acidic PLA2 were also observed. Some unexpected protein spots were also identified: C-type lectins and CRISP may represent putative new targets for DM64, whereas the presence of serine peptidases in the venom bound fraction is likely a consequence of nonspecific binding to the column matrix. The present results contribute to better delineate the inhibitory potential of DM64, providing a framework for the development of more specific antivenom therapies. BIOLOGICAL SIGNIFICANCE: Local tissue damage induced by myotoxic PLA2 remains a serious consequence of snake envenomation, since it is only partially neutralized by traditional antivenom serotherapy. Myotoxin inhibition by highly specific molecules offers great promise in the treatment of snakebites, a health problem largely neglected by governments and pharmaceutical industries. Bioactive compounds such as DM64 can represent a valuable source of scaffolds for drug development in this area. The present study has systematically profiled the binding specificity of DM64 toward a variety of snake venom toxin classes and therefore can lead to a better understanding of the structure-function relationship of this important antivenom protein.

Structural and functional evidence for membrane docking and disruption sites on phospholipase A2-like proteins revealed by complexation with the inhibitor suramin.

Local myonecrosis resulting from snakebite envenomation is not efficiently neutralized by regular antivenom administration. This limitation is considered to be a significant health problem by the World Health Organization. Phospholipase A2-like (PLA2-like) proteins are among the most important proteins related to the muscle damage resulting from several snake venoms. However, despite their conserved tertiary structure compared with PLA2s, their biological mechanism remains incompletely understood. Different oligomeric conformations and binding sites have been identified or proposed, leading to contradictory data in the literature. In the last few years, a comprehensive hypothesis has been proposed based on fatty-acid binding, allosteric changes and the presence of two different interaction sites. In the present study, a combination of techniques were used to fully understand the structural-functional characteristics of the interaction between suramin and MjTX-II (a PLA2-like toxin). In vitro neuromuscular studies were performed to characterize the biological effects of the protein-ligand interaction and demonstrated that suramin neutralizes the myotoxic activity of MjTX-II. The high-resolution structure of the complex identified the toxin-ligand interaction sites. Calorimetric assays showed two different binding events between the protein and the inhibitor. It is demonstrated for the first time that the inhibitor binds to the surface of the toxin, obstructing the sites involved in membrane docking and disruption according to the proposed myotoxic mechanism. Furthermore, higher-order oligomeric formation by interaction with interfacial suramins was observed, which may also aid the inhibitory process. These results further substantiate the current myotoxic mechanism and shed light on the search for efficient inhibitors of the local myonecrosis phenomenon.

Correlação e associação de renda e escolaridade com condições de saúde e nutrição em obesos graves / Correlation and association of income and educational level with health and nutritional conditions among the morbidly obese

O objetivo deste artigo é investigar relações entre renda e escolaridade com condições de saúde e nutrição em obesos graves. Estudo transversal ambulatorial com 79 pacientes de primeira consulta, com Índice de Massa Corporal (IMC) ≥ 35 kg/m2 e idade ≥ 20 anos. Coletaram-se dados: sociodemográficos, antropométricos, estilo de vida, exames bioquímicos e consumo alimentar. O IMC médio foi 48,3 ± 6,9 kg/m2. Observou-se correlação negativa significante de escolaridade com variáveis peso (r = -0,234) e IMC (r = -0,364) e de renda familiar per capita com consumo diário de vegetal A (r = -0,263). Após análise multivariada maior renda familiar per capita se associou à ausência de cardiopatia (RP: 0,51, IC95%: 0,32-0,81), maior consumo diário de vegetal A (RP: 1,79, IC95%: 1,16-2,75) e doces (RP: 3,12, IC95%: 1,21-8,04). Em obesos graves a maior renda familiar per capita se associou à ausência de cardiopatia e maior consumo de vegetais folhosos e doces. Já a escolaridade não se manteve associada às condições de saúde e nutrição.

This article seeks to investigate the relationship between income and educational level and health and nutritional conditions among the morbidly obese. A cross-sectional study was conducted with 79 patients at first appointment, with Body Mass Index (BMI) ≥ 35 kg/m2 and age ≥ 20 years. The following data was collected: demographic, socioeconomic, anthropometric, lifestyle, biochemical and food intake data. Average BMI was 48.3 ± 6.9 kg/m2. There was a significant negative correlation between education level and the variables of weight (r = -0.234) and BMI (r = -0.364) and per capita family income with daily consumption of leafy vegetables (r = -0.263). After multivariate analysis, higher per capita family income was associated with the absence of heart disease (PR: 0.51, CI95%: 0.32-0.81), higher daily consumption of leafy vegetables (PR: 1.79, CI95%: 1.16-2.75) and candy (PR: 3.12, CI95%: 1.21-8.04). In the morbidly obese, per capita household income was associated with absence of heart disease and higher consumption of leafy vegetables and candy. On the other hand, education level was not associated with health and nutrition conditions.

Inhibition of snake venoms and phospholipases A(2) by extracts from native and genetically modified Eclipta alba: isolation of active coumestans.

We genetically modified Eclipta alba using Agrobacterium rhizogenes LBA 9402, with the aim of producing secondary metabolites with pharmacological properties against phospholipase A(2) and the myotoxic activities of snake venom. Extracts from in natura aerial parts and roots, both native and genetically modified (in vitro), were prepared and analysed by high-performance liquid chromatography. In natura materials showed the coumestan wedelolactone at higher concentration in the aerial parts, while demethylwedelolactone appeared at higher concentration in roots. Among the modified roots, clone 19 showed higher concentrations of these coumestans. Our results show that the in natura extracts of plants collected from Botucatu and Ribeirão Preto were efficient in inhibiting snake venom phospholipase A(2) activity. Regarding in vitro material, the best effect against Crotalus durissus terrificus venom was that of clone 19. Clone 19 and isolated coumestans (wedelolactone and demethylwedelolactone) inhibited the myotoxic activity induced by basic phospholipases A(2) isolated from the venoms of Crotalus durissus terrificus (CB) and Bothrops jararacussu (BthTX-I and II). The search for antivenom is justified by the need of finding active principles that are more efficient in neutralizing snake venoms and also as an attempt to complement serum therapy.

Isolation and characterization of ellagic acid derivatives isolated from Casearia sylvestris SW aqueous extract with anti-PLA(2) activity.

The Casearia sylvestris SW (Flacourtiaceae) is utilized in folk medicine (Brazil and all Latin American) to treat several pathologic processes as inflammation, cancer, microbial infection and snake bites. Studies showed that C. sylvestris aqueous extract can inhibit many toxic effects caused by snake venoms (or caused by phospholipase A(2) isolated) from different species, mainly of Bothrops genus. Inhibition of enzymatic and myotoxic activities, decrease of edema formation and increase of the survival rate of rats injected with lethal doses of bothropic venoms are some toxic effects inhibited by C. sylvestris. In this study, four ellagic acid derivatives from aqueous extracts of C. sylvestris were isolated, characterized, and tested against effects from both total venom and PLA(2) (Asp 49 BthTX-II) from the venom of Bothrops jararacussu. The isolated compounds were as follows: ellagic acid (A), 3'-O-methyl ellagic acid (B), 3,3'-di-O-methyl ellagic acid (C), 3-O-methyl-3',4'-methylenedioxy ellagic acid (D). The inhibition constant values (Ki) for enzymatic activity, as well the IC(50) values found in the edematogenic and myotoxic activities, indicate that the ellagic acid is the best inhibitor of these activities, while compounds C and D are the substances with lowest capacity on inhibiting these same effects. Our results show that the presence of hydroxyls at position 3 or 3' (compounds A and B) increases the capacity of these derivatives on inhibiting these toxic effects. However, the presence of methoxyl groups at position 3 or 3' reduced, but did not completely inhibit the capacity of compounds C and D on inhibiting all the toxic effects studied.

Triterpenoid saponins, new metalloprotease snake venom inhibitors isolated from Pentaclethra macroloba.

We report here the antiproteolytic and antihemorrhagic properties of triterpenoid saponin inhibitors, named macrolobin-A and B, from Pentaclethra macroloba, against Bothrops snake venoms. The inhibitors were able to neutralize the hemorrhagic, fibrin(ogen)olytic, and proteolytic activities of class P-I and P-III metalloproteases isolated from B. neuwiedi and B. jararacussu venoms. Clotting and fibrinogenolytic activities induced by snake venoms and isolated thrombin-like enzymes were partially inhibited. Furthermore, the potential use of these inhibitors to complement antivenom therapy as an alternative treatment and/or used as molecular models for development of new therapeutical agents in the treatment of snake bite envenomations needs to be evaluated in future studies.

Snake venom phospholipase A2 inhibitors: medicinal chemistry and therapeutic potential.

Phospholipases A2 (PLA2s) are commonly found in snake venoms from Viperidae, Hydrophidae and Elaphidae families and have been extensively studied due to their pharmacological and physiopathological effects in living organisms. This article reports a review on natural and artificial inhibitors of enzymatic, toxic and pharmacological effects induced by snake venom PLA2s. These inhibitors act on PLA2s through different mechanisms, most of them still not completely understood, including binding to specific domains, denaturation, modification of specific amino acid residues and others. Several substances have been evaluated regarding their effects against snake venoms and isolated toxins, including plant extracts and compounds from marine animals, mammals and snakes serum plasma, in addition to poly or monoclonal antibodies and several synthetic molecules. Research involving these inhibitors may be useful to understand the mechanism of action of PLA2s and their role in envenomations caused by snake bite. Furthermore, the biotechnological potential of PLA2 inhibitors may provide therapeutic molecular models with antiophidian activity to supplement the conventional serum therapy against these multifunctional enzymes.

Inhibition of phospholipase A2 reduces neurite outgrowth and neuronal viability.

Phospholipase A2 (PLA(2)) has been implicated in neurodevelopmental processes and in the early development of the nervous system. We investigated the effects of the inhibition of calcium-dependent and calcium-independent subtypes of cytosolic PLA2 (cPLA2 and iPLA2) on the development and viability of primary cultures of cortical and hippocampal neurons. PLA2 in these cultures was continuously inhibited with methylarachidonyl-fluorophosphonate (MAFP), an irreversible inhibitor of cPLA2 and iPLA2, or with bromoenol lactone (BEL), an irreversible selective iPLA2 inhibitor. The effect of PLA2 inhibitors on the development of neuronal cultures was ascertained by total cell count and morphological characterisation. Neuronal viability was quantified with MTT assays. Inhibition of PLA2 resulted in reduction of neuritogenesis and neuronal viability, disrupting neuronal homeostasis and leading to neuronal death. We conclude that the functional integrity of both calcium-dependent and calcium-independent cytosolic PLA2 is necessary for the in vitro development of cortical and hippocampal neurons.

Requirement of hippocampal phospholipase A2 activity for long-term memory retrieval in rats.

In rats, the inhibition of phospholipase A(2) (PLA(2)) in hippocampus was reported to impair memory acquisition. In the present study we investigated in rats whether PLA(2) inhibition in hippocampus is also related to impairment of memory retrieval. Rats were bilaterally implanted with cannulae in hippocampal CA1 region. After recovery, animals were submitted to one-trial step-down inhibitory avoidance task and tested for long-term memory (LTM) 24 h later. Before test session, animals received infusions of vehicle or the PLA(2) inhibitor PACOCF(3). Inhibition of PLA(2) activity impaired LTM retrieval. Memory impairment was fully reversed once PLA(2) activity was recovered. Moreover, LTM retrieval per se increased PLA(2) activity. To our knowledge, we demonstrated for the first time that PLA(2) activity is required for memory retrieval. Because reduced PLA(2) activity has been found in Alzheimer's disease brains, the present results may be relevant to clarify at least part of the biology of this disorder.

COX-2 and sPLA2 inhibitory activity of aqueous extract and polyphenols of Rhizophora mangle (red mangrove).

The aqueous extract of Rhizophora mangle bark and its polyphenolic fractions showed remarkable in vitro antiinflammatory activity in a preliminary study. The low molecular weight fraction exhibited cyclooxygenase-2 inhibitory activity while the total aqueous extract and the low molecular weight fraction showed secretory phospholipase A(2) inhibitory activity.

The action of extracellular NAD+ on gluconeogenesis in the perfused rat liver.

In the rat liver NAD+ infusion produces increases in portal perfusion pressure and glycogenolysis and transient inhibition of oxygen consumption. The aim of the present work was to investigate the possible action of this agent on gluconeogenesis using lactate as a gluconeogenic precursor. Hemoglobin-free rat liver perfusion in antegrade and retrograde modes was used with enzymatic determination of glucose production and polarographic assay of oxygen uptake. NAD+ infusion into the portal vein (antegrade perfusion) produced a concentration-dependent (25-100 microM) transient inhibition of oxygen uptake and gluconeogenesis. For both parameters inhibition was followed by stimulation. NAD+ infusion into the hepatic vein (retrograde perfusion) produced only transient stimulations. During Ca2+-free perfusion the action of NAD+ was restricted to small transient stimulations. Inhibitors of eicosanoid synthesis with different specificities (indo-methacin, nordihydroguaiaretic acid, bromophenacyl bromide) either inhibited or changed the action of NAD+. The action of NAD+ on gluconeogenesis is probably mediated by eicosanoids synthesized in non-parenchymal cells. As in the fed state, in the fasted condition extracellular NAD+ is also able to exert two opposite effects, inhibition and stimulation. Since inhibition did not manifest significantly in retrograde perfusion it is likely that the generating signal is located in pre-sinusoidal regions.

Apoptosis-inducing factor of a cytotoxic T cell line: involvement of a secretory phospholipase A2.

Natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) kill target cells by the granule-exocytosis pathway and by the engagement of molecules belonging to the tumor necrosis factor family. The involvement of secretory phospholipase A(2) (sPLA(2)) in the cytotoxic process has been proposed in NK cells. However, its molecular identity and intracellular localization remain unknown, and its mechanism of action is poorly understood. Here, we have readdressed this issue by studying the cytotoxic activity of whole cell extracts of a CTL line. We observed that inactivation of the perforin-granzyme pathway at 37 degrees C in the presence of 1 mM Ca(2+) enhanced the ability of CTL extracts to induce apoptosis. This potentiation of cell death was Ca(2+)-dependent, thermo-resistant, and inhibited by 4-bromophenacyl bromide and scalaradial (two inhibitors of sPLA(2)). The involvement of an sPLA(2) was confirmed by blocking the pro-apoptotic activity of the Ca(2+)-treated cell extract with an anti-sPLA(2) polyclonal antibody. By cell fractionation assays, we showed that the pro-apoptotic sPLA(2) was localized in the cytoplasmic fraction but not in perforin-rich granules or plasma membrane fractions. Western blotting analysis revealed the presence of four distinct bands of 56, 29.5, 21, and 15 kDa. The highest molecular weight band was consistent with the expression of a group III sPLA2. Taken together, these data indicate that an apoptosis-inducing sPLA(2) is expressed in the cytosol of a CTL cell line and suggest that it plays an effector role in CTL-mediated cytotoxicity.

Blockade of arachidonic acid pathway induces sprouting in the adult but not in the neonatal uncrossed retinotectal projection.

The uncrossed retinotectal projection of rats undergoes extensive axonal elimination and subsequent growth of axonal arbors in topographically appropriate territories within the first two/three postnatal weeks. Nitric oxide has been implicated in development and stabilization of synapses in the retinotectal pathway since blockade of nitric oxide synthesis disrupts the normal pattern of retinal innervation in subcortical nuclei. The present work investigated the role of arachidonic acid pathway in the development and maintenance of ipsilateral retinotectal axons. We also investigated the role of this retrograde messenger in the modulation of plasticity that follows retinal lesions in the opposite eye. Pigmented rats received systemic treatment with quinacrine, a phospholipase A2 inhibitor, indomethacin, a cyclooxygenase inhibitor, nordihydroguaiaretic acid, a 5-lipoxygenase inhibitor or vehicle during 4-8 days at various postnatal ages. Rats given a unilateral temporal retinal lesion were treated with either quinacrine or vehicle during the same period. For anterograde tracing of ipsilateral retinal projections, animals received intraocular injections of horseradish peroxidase. Before the third postnatal week no difference was observed in the laminar or topographic organization of the ipsilateral retinotectal projection between vehicle and treated rats in either normal or lesion conditions. After the third postnatal week, however, systemic blockade of phospholipase A2 or 5-lipoxygenase, but not cyclooxygenase induced sprouting of uncrossed axons throughout the collicular visual layers in unoperated rats. In retinal lesion groups, phospholipase A2 blockade increased the sprouting of uncrossed intact axons to the collicular surface in the same period. The results suggest that arachidonic acid or lipoxygenase metabolites play a role in the maintenance of the retinotectal synapses after the critical period and that the blockade of the arachidonic acid pathway induces reactive sprouting of retinal axons late in development.

Structure of BthA-I complexed with p-bromophenacyl bromide: possible correlations with lack of pharmacological activity.

The crystal structure of an acidic phospholipase A(2) isolated from Bothrops jararacussu venom (BthA-I) chemically modified with p-bromophenacyl bromide (BPB) has been determined at 1.85 Angstroms resolution. The catalytic, platelet-aggregation inhibition, anticoagulant and hypotensive activities of BthA-I are abolished by ligand binding. Electron-density maps permitted unambiguous identification of inhibitor covalently bound to His48 in the substrate-binding cleft. The BthA-I-BPB complex contains three structural regions that are modified after inhibitor binding: the Ca(2+)-binding loop, beta-wing and C-terminal regions. Comparison of BthA-I-BPB with two other BPB-inhibited PLA(2) structures suggests that in the absence of Na(+) ions at the Ca(2+)-binding loop, this loop and other regions of the PLA(2)s undergo structural changes. The BthA-I-BPB structure reveals a novel oligomeric conformation. This conformation is more energetically and conformationally stable than the native structure and the abolition of pharmacological activities by the ligand may be related to the oligomeric structural changes. A residue of the ;pancreatic' loop (Lys69), which is usually attributed as providing the anticoagulant effect, is in the dimeric interface of BthA-I-BPB, leading to a new hypothesis regarding the abolition of this activity by BPB.

Eicosanoid-mediated proinflammatory activity of Pseudomonas aeruginosa ExoU.

As Pseudomonas aeruginosa ExoU possesses two functional blocks of homology to calcium-independent (iPLA(2)) and cytosolic phospholipase A(2) (cPLA(2)), we addressed the question whether it would exhibit a proinflammatory activity by enhancing the synthesis of eicosanoids by host organisms. Endothelial cells from the HMEC-1 line infected with the ExoU-producing PA103 strain exhibited a potent release of arachidonic acid (AA) that could be significantly inhibited by methyl arachidonyl fluorophosphonate (MAFP), a specific PLA(2) inhibitor, as well as significant amounts of the cyclooxygenase (COX)-derived prostaglandins PGE(2) and PGI(2). Cells infected with an isogenic mutant defective in ExoU synthesis did not differ from non-infected cells in the AA release and produced prostanoids in significantly lower concentrations. Infection by PA103 induced a marked inflammatory response in two different in vivo experimental models. Inoculation of the parental bacteria into mice footpads led to an early increase in the infected limb volume that could be significantly reduced by inhibitors of both COX and lipoxygenase (ibuprofen and NDGA respectively). In an experimental respiratory infection model, bronchoalveolar lavage (BAL) from mice instilled with 10(4) cfu of PA103 exhibited a marked influx of inflammatory cells and PGE(2) release that could be significantly reduced by indomethacin, a non-selective COX inhibitor. Our results suggest that ExoU may contribute to P. aeruginosa pathogenesis by inducing an eicosanoid-mediated inflammatory response of host organisms.

Antiophidian properties of the aqueous extract of Mikania glomerata.

Aqueous extracts, prepared from dried or fresh roots, stems or leaves of Mikania glomerata, a plant found in Mata Atlântica in Southeastern Brazil, were able to efficiently neutralize different toxic, pharmacological, and enzymatic effects induced by venoms from Bothrops and Crotalus snakes. Phospholipase A(2) activity and the edema induced by Crotalus durissus terrificus venom were inhibited around 100 and approximately 40%, respectively, although this inhibition was only partial for Bothrops venoms. The hemorrhagic activity of Bothrops venoms (Bothrops altenatus, Bothrops moojeni, Bothrops neuwiedi, and Bothrops jararacussu) was significantly inhibited by this vegetal species, while the clotting activity of Crotalus durissus terrificus, Bothrops jararacussu, and Bothrops neuwiedi venoms was totally inhibited. Although, the mechanism of action of Mikania glomerata extract is still unknown, the finding that no visible change was detected in the electrophoretic pattern of snake venom after incubation with the extract excludes proteolytic degradation as a potential mechanism. Since the extract of Mikania glomerata significantly inhibited the studied snake venoms, it may be used as an alternative treatment to serumtherapy and, in addition, as a rich source of potential inhibitors of PLA(2)s, metalloproteases and serineproteases, enzymes involved in several physiopathological human and animal diseases.

Renal and antibacterial effects induced by myotoxin I and II isolated from Bothrops jararacussu venom.

Bothrops jararacussu myotoxin I (BthTx-I; Lys 49) and II (BthTX-II; Asp 49) were purified by ion-exchange chromatography and reverse phase HPLC. In this work we used the isolated perfused rat kidney method to evaluate the renal effects of B. jararacussu myotoxins I (Lys49 PLA2) and II (Asp49 PLA2) and their possible blockage by indomethacin. BthTX-I (5 microg/ml) and BthTX-II (5 microg/ml) increased perfusion pressure (PP; ct120=110.28+/-3.70 mmHg; BthTX I=171.28+/-6.30*mmHg; BthTX II=175.50+/-7.20*mmHg), renal vascular resistance (RVR; ct120=5.49+/-0.54 mmHg/ml.g(-1)min(-1); BthTX I=8.62+/-0.37*mmHg/ml g(-1)min(-1); BthTX II=8.9+/-0.36*mmHg/ml g(-1)min(-1)), urinary flow (UF; ct(120)=0.14+/-0.01ml g(-1)min(-1); BthTX I=0.32+/-0.05*ml g(-1)min(-1); BthTX II=0.37+/-0.01*ml g(-1)min(-1)) and glomerular filtration rate (GFR; ct120=0.72+/-0.10 ml g(-1)min(-1); BthTX I=0.85+/-0.13*ml g(-1)min(-1); BthTX II=1.22+/-0.28*ml g(-1)min(-1)). In contrast decreased the percent of sodium tubular transport (%TNa(+); ct(120)=79,76+/-0.56; BthTX I=62.23+/-4.12*; BthTX II=70.96+/-2.93*) and percent of potassium tubular transport (%TK(+);ct120=66.80+/-3.69; BthTX I=55.76+/-5.57*; BthTX II=50.86+/-6.16*). Indomethacin antagonized the vascular, glomerular and tubular effects promoted by BthTX I and it's partially blocked the effects of BthTX II. In this work also evaluated the antibacterial effects of BthTx-I and BthTx-II against Xanthomonas axonopodis. pv. passiflorae (Gram-negative bacteria) and we observed that both PLA2 showed antibacterial activity. Also we observed that proteins Also we observed that proteins chemically modified with 4-bromophenacyl bromide (rho-BPB) decrease significantly the antibacterial effect of both PLA2. In conclusion, BthTx I and BthTX II caused renal alteration and presented activity antimicrobial. The indomethacin was able to antagonize totally the renal effects induced by BthTx I and partially the effects promoted by BthTx II, suggesting involvement of inflammatory mediators in the renal effects caused by myotoxins. In the other hand, other effects could be independently of the enzymatic activity of the BthTX II and the C-terminal domain could be involved in both effects promoted for PLA2.

Rosmarinic acid, a new snake venom phospholipase A2 inhibitor from Cordia verbenacea (Boraginaceae): antiserum action potentiation and molecular interaction.

Many plants are used in traditional medicine as active agents against various effects induced by snakebite. The methanolic extract from Cordia verbenacea (Cv) significantly inhibited paw edema induced by Bothrops jararacussu snake venom and by its main basic phospholipase A2 homologs, namely bothropstoxins I and II (BthTXs). The active component was isolated by chromatography on Sephadex LH-20 and by RP-HPLC on a C18 column and identified as rosmarinic acid (Cv-RA). Rosmarinic acid is an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid [2-O-cafeoil-3-(3,4-di-hydroxy-phenyl)-R-lactic acid]. This is the first report of RA in the species C. verbenacea ('baleeira', 'whaler') and of its anti-inflammatory and antimyotoxic properties against snake venoms and isolated toxins. RA inhibited the edema and myotoxic activity induced by the basic PLA2s BthTX-I and BthTX-II. It was, however, less efficient to inhibit the PLA2 activity of BthTX-II and, still less, the PLA2 and edema-inducing activities of the acidic isoform BthA-I-PLA2 from the same venom, showing therefore a higher inhibitory activity upon basic PLA2s. RA also inhibited most of the myotoxic and partially the edema-inducing effects of both basic PLA2s, thus reinforcing the idea of dissociation between the catalytic and pharmacological domains. The pure compound potentiated the ability of the commercial equine polyvalent antivenom in neutralizing lethal and myotoxic effects of the crude venom and of isolated PLA2s in experimental models. CD data presented here suggest that, after binding, no significant conformation changes occur either in the Cv-RA or in the target PLA2. A possible model for the interaction of rosmarinic acid with Lys49-PLA2 BthTX-I is proposed.

Inhibition of myotoxic activity of Bothrops asper myotoxin II by the anti-trypanosomal drug suramin.

Suramin, a synthetic polysulfonated compound, developed initially for the treatment of African trypanosomiasis and onchocerciasis, is currently used for the treatment of several medically relevant disorders. Suramin, heparin, and other polyanions inhibit the myotoxic activity of Lys49 phospholipase A2 analogues both in vitro and in vivo, and are thus of potential importance as therapeutic agents in the treatment of viperid snake bites. Due to its conformational flexibility around the single bonds that link the central phenyl rings to the secondary amide backbone, the symmetrical suramin molecule binds by an induced-fit mechanism complementing the hydrophobic surfaces of the dimer and adopts a novel conformation that lacks C2 symmetry in the dimeric crystal structure of the suramin-Bothrops asper myotoxin II complex. The simultaneous binding of suramin at the surfaces of the two monomers partially restricts access to the nominal active sites and significantly changes the overall charge of the interfacial recognition face of the protein, resulting in the inhibition of myotoxicity.

Inhibitory effects of Piper umbellatum and Piper peltatum extracts towards myotoxic phospholipases A2 from Bothrops snake venoms: isolation of 4-nerolidylcatechol as active principle.

Phospholipases A(2) (PLA(2)) are important constituents of snake venoms, being responsible for several of their toxic actions. Extracts from plants used in folk medicine were screened for inhibition of the enzymatic activity of myotoxin I, a PLA(2) from Bothrops asper. Piper umbellatum and Piper peltatum extracts tested positive, and their fractionation resulted in the isolation of 4-nerolidylcatechol. Its inhibitory effects towards toxic activities of two Bothrops myotoxins, representing catalytically active (Asp49) and catalytically inactive (Lys49) types of group II PLA(2)s, respectively, were characterized. The enzyme activity of B. asper myotoxin I was completely inhibited by 4-nerolidylcatechol at an inhibitor:toxin ratio of 10:1 (wt/wt) with an IC50 of approximately 1mM. In addition, 4-nerolidylcatechol inhibited representatives of groups I and III of PLA(2)s. Its preincubation with Bothrops myotoxins significantly reduced their myotoxic and edema-inducing activities in animal experiments. However, when 4-nerolidylcatechol was administered in situ, immediately after toxin injection, its inhibitory ability was substantially lower or negligible. This might be explained by the rapid action of these toxins in vivo, together with the slow inactivation of PLA(2) activity observed in vitro. Electrophoretic and chromatographic analyses of myotoxins ruled out major changes in protein charge, hydrophobicity, or gross molecular mass being involved in the inhibition mechanism. Mass spectrometry determinations are consistent with the covalent modification of myotoxin by one molecule of 4-nerolidylcatechol. Finally, a novel compound was isolated from both Piper species, sharing the nerolidyl skeleton, but nevertheless not being inhibitory towards the PLA(2)s studied.