Biochemical and structural investigations of Bothropstoxin-II, a myotoxic Asp49 phospholipase A2 from Bothrops jararacussu venom.

Bothropstoxin-II a calcium-dependent enzyme from Bothrops jararacussu venom causes tissue damage and several haemostatic disorders including platelet aggregation. In order to elucidate the structural determinants of its multiple pharmacological activities, we have studied the effects of suramin on Bothropstoxin-II and present details concerning the mode of binding.

Mechanistic hypotheses for nonsynaptic epileptiform activity induction and its transition from the interictal to ictal state--computational simulation.

PURPOSE: The aim of this work is to study, by means of computational simulations, the induction and sustaining of nonsynaptic epileptiform activity. METHODS: The computational model consists of a network of cellular bodies of neurons and glial cells connected to a three-dimensional (3D) network of juxtaposed extracellular compartments. The extracellular electrodiffusion calculation was used to simulate the extracellular potential. Each cellular body was represented in terms of the transmembrane ionic transports (Na(+)/K(+) pumps, ionic channels, and cotransport mechanisms), the intercellular electrodiffusion through gap-junctions, and the neuronal interaction by electric field and the variation of cellular volume. RESULTS: The computational model allows simulating the nonsynaptic epileptiform activity and the extracellular potential captured the main feature of the experimental measurements. The simulations of the concomitant ionic fluxes and concentrations can be used to propose the basic mechanisms involved in the induction and sustaining of the activities. DISCUSSION: The simulations suggest: The bursting induction is mediated by the Cl(-) Nernst potential overcoming the transmembrane potential in response to the extracellular [K(+)] increase. The burst onset is characterized by a critical point defined by the instant when the Na(+) influx through its permeable ionic channels overcomes the Na(+)/K(+) pump electrogenic current. The burst finalization is defined by another critical point, when the electrogenic current of the Na(+)/K(+) pump overcomes its influx through the channels.

Ability of suramin to antagonize the cardiotoxic and some enzymatic activities of Bothrops jararacussu venom.

We have investigated the cardiotoxic effect of Bothrops jararacussu crude venom and the ability of suramin to antagonize this effect in the heart of rats, as well as the proteolytic and phospholipase A(2) (PLA(2)) venom activities. Continuous perfusion in an isolated heart of a rat on a Langendorff preparation with a Ringer's solution with B. jararacussu crude venom (2.5-10.0 microg/mL) induces stoppage and a decrease in the cardiac tension, which were time- and concentration dependent. The analysis of the heart perfusate solution showed an increase in the rate of creatine kinase induced by the venom. Pre-incubation with suramin (1.0-30.0 microM) protected against the venom cardiotoxic effect in a concentration-dependent way, reaching up to 90% with 30.0 microM, and prevent the heart stoppage and decrease the tension. These protective effects were increased by the association with polyvalent antibothropic antivenom, suggesting a synergic effect. The PLA(2) and proteolytic activities of B. jararacussu crude venom were also inhibited in a concentration-dependent way by suramin, showing that this polyanion antivenom activity has therapeutic potential to be used as an antivenom.

Evaluation of three Brazilian antivenom ability to antagonize myonecrosis and hemorrhage induced by Bothrops snake venoms in a mouse model.

Despite preventing death after snakebites, there is little evidence that polyvalent antivenoms (PAVs) protect against myotoxicity and local damages. We evaluated antibothropic Brazilian PAVs from three manufacturers against the myotoxicity and hemorrhagic activity of Bothrops jararacussu and B. jararaca venoms, respectively, by using two protocols: preincubation of PAVs with venom, and i.v. pretreatment with PAVs, prior to the venom inoculation. In this investigation, we used doses of PAVs ranging from 0.4 to 4.0mL/mg of venom equivalent up to 10 times the amount recommended by the producers for the clinical practice in Brazil. In our preincubation protocol in vivo, PAVs antagonized myotoxicity of B. jararacussu venom by 40-95%, while our pretreatment protocol antagonized myotoxic activity by 0-60%. Preincubation of antivenoms with B. jararaca venom antagonized its hemorrhagic activity by 70-95%, while pretreatment antagonized hemorrhagic activity by 10-50%. Although all PAVs demonstrated partial antagonism against both venoms, the magnitude of these effects varied greatly among the manufactures. The results suggest that the current clinical doses of these PAVs may have negligible antimyotoxic effect.

Suramin counteracts the haemostatic disturbances produced by Bothrops jararaca snake venom.

Snakebite accidents produced by Bothrops jararaca typically results in haemostatic changes including pro- and anticoagulant disturbs as well as interference with platelets. Suramin is a hexasulfonated naphthylurea derivative that was recently characterized as a thrombin inhibitor (Monteiro et al., 2004. Suramin interaction with human alpha-thrombin: inhibitory effects and binding studies. Int. J. Biochem. Cell Biol. 36(10), 2077-2085). Here, we evaluated the ability of suramin to counteract some of the haemostatic disturbs produced by B. jararaca venom. In vitro assays showed that suramin inhibited venom-induced hydrolysis of a number of synthetic substrates: S-2238, S-2266, S-2302 and S-2288, being this ability more prominent towards the thrombin substrate S-2238 (IC(50)=4.3 microM). It was also observed that suramin impaired the fibrinogen clotting induced by B. jararaca venom (IC(50)=124 microM). Accordingly, increasing concentrations of suramin progressively delayed venom-induced plasma clotting, with complete inhibition attained at concentrations above 1.0 mM. In addition, the platelet-aggregating properties of B. jararaca venom were inhibited by suramin in a dose-dependent fashion (IC(50)=127 microM). Suramin showed no effect in the in vivo hemorrhagic effect of venom in mouse skin. The in vivo effect of suramin was further tested using a previously established venous thrombosis model in rats induced by intravenous administration of B. jararaca venom combined with stasis. Venom doses of 100 microg/kg produced 100% of thrombus incidence (10.6+/-1.7 mg). On the other hand, previous administration of suramin partially inhibited thrombus formation. Thus, 12.5 or 25 mg/kg of suramin decreased thrombus weight by 24% and 40%, respectively. Remarkably, co-administration of 3 microL/kg of antibothropic serum (which has no effect on thrombus formation) and 12.5 mg/kg of suramin decreased thrombus weight by 75%, suggesting a synergic effect. Altogether, we demonstrate here that suramin inhibits in vitro and in vivo haemostatic changes caused by B. jararaca venom. At this point, this drug could be of potential interest for association with conventional antiserum therapy.

Interfacial surface charge and free accessibility to the PLA2-active site-like region are essential requirements for the activity of Lys49 PLA2 homologues.

Lys49 phospholipase A2 homologues are highly myotoxic and cause extensive tissue damage but do not display hydrolytic activity towards natural phospholipids. The binding of heparin, heparin derivatives and polyanionic compounds such as suramin result in partial inhibition (up to 60%) of the myotoxic effects due to a change in the overall charge of the interfacial surface. In vivo experiments demonstrate that polyethylene glycol inhibits more than 90% of the myotoxic effects without exhibiting secondary toxic effects. The crystal structure of bothropstoxin-I complexed with polyethylene glycol reveals that this inhibition is due to steric hindrance of the access to the PLA2-active site-like region. These two inhibitory pathways indicate the roles of the overall surface charge and free accessibility to the PLA2-active site-like region in the functioning of Lys49 phospholipases A2 homologues. Molecular dynamics simulations, small angle X-ray scattering and structural analysis indicate that the oligomeric states both in solution and in the crystalline states of Lys49 phospholipases A2 are principally mediated by hydrophobic contacts formed between the interfacial surfaces. These results provide the framework for the potential application of both clinically approved drugs for the treatment of Viperidae snakebites.

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.

Crystallization and preliminary X-ray diffraction analysis of suramin, a highly charged polysulfonated napthylurea, complexed with a myotoxic PLA2 from Bothrops asper venom.

Suramin is a highly charged polysulfonated napthylurea that interferes in a number of physiologically relevant processes such as myotoxicity, blood coagulation and several kinds of cancers. This synthetic compound was complexed with a myotoxic Lys49 PLA(2) from Bothrops asper venom and crystallized by the hanging-drop vapor diffusion method at 18 degrees C. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell parameters a=49.05, b=63.84 and c=85.67 angstroms. Diffraction data was collected to 1.78 angstroms.

Targeted deletion of the kynurenine aminotransferase ii gene reveals a critical role of endogenous kynurenic acid in the regulation of synaptic transmission via alpha7 nicotinic receptors in the hippocampus.

It has been postulated that endogenous kynurenic acid (KYNA) modulates alpha7* nicotinic acetylcholine receptor (nAChR) and NMDA receptor activities in the brain.a To test this hypothesis, alpha7* nAChR and NMDA receptor functions were studied in mice with a targeted null mutation in the gene encoding kynurenine aminotransferase II (mKat-2-/- mice), an enzyme responsible for brain KYNA synthesis. At 21 postnatal days, mKat-2-/- mice had lower hippocampal KYNA levels and higher spontaneous locomotor activity than wild-type (WT) mice. At this age, alpha7* nAChR activity induced by exogenous application of agonists to CA1 stratum radiatum interneurons was approximately 65% higher in mKat-2-/- than WT mice. Binding studies indicated that the enhanced receptor activity may not have resulted from an increase in alpha7* nAChR number. In 21-d-old mKat-2-/- mice, endogenous alpha7* nAChR activity in the hippocampus was also increased, leading to an enhancement of GABAergic activity impinging onto CA1 pyramidal neurons that could be reduced significantly by acute exposure to KYNA (100 nM). The activities of GABA(A) and NMDA receptors in the interneurons and of alpha3beta4* nAChRs regulating glutamate release onto these neurons were comparable between mKat-2-/- and WT mice. By 60 d of age, KYNA levels and GABAergic transmission in the hippocampus and locomotor activity were similar between mKat-2-/- and WT mice. Our findings that alpha7* nAChRs are major targets for KYNA in the brain may provide insights into the pathophysiology of schizophrenia and Alzheimer's disease, disorders in which brain KYNA levels are increased and alpha7* nAChR functions are impaired.

Synthesis and pharmacological evaluation of prenylated and benzylated pterocarpans against snake venom.

Edunol (3), a pterocarpan isolated from Harpalyce brasiliana, a plant used in the northeast of Brazil against snakebites, was obtained by synthesis and showed antimyotoxic, antiproteolytic and PLA2 inhibitor properties. These proprieties could be improved through the synthesis of a bioisoster (5), where the prenyl group was replaced by the benzyl group.

Antagonism of myotoxic and paralyzing activities of bothropstoxin-I by suramin.

Polyanionic substances are known to inhibit the myotoxic effects of some crotalide snake venoms. Bothropstoxin-I (BthTX-I), a basic Lys49 phospholipase (PLA2) homologue from Bothrops jararacussu venom, besides inducing muscle damage, also promotes the blockade of both directly and indirectly evoked contractions in mouse neuromuscular preparation. In this work, we evaluated the ability of suramin, a polysulfonated naphtylurea derivative, to antagonize the myotoxic and the paralyzing activities of BthTX-I on mice neuromuscular junction in vitro. Myotoxicity was assessed by light and electronic microscopic analysis of extensor digitorum longus (EDL) muscles; paralyzing activity was evaluated through the recording of both directly and indirectly evoked contractions of phrenic-diaphragm (PD) preparations. BthTX-I (1 microM) alone, or pre-incubated with suramin (10 microM) at 37 degrees C for 15 min was added to the preparations for 120 min. BthTX-I induced histological alterations typical of myonecrosis in 14.6 +/- 1.0% of EDL muscle fibers. In addition, BthTX-I blocked 50% of both directly and indirectly evoked contractions in PD preparations in 72.1 +/- 9.1 and 21.1 +/- 2.0 min, respectively. Pre-incubation with suramin abolished both the muscle-damaging and muscle-paralyzing activities of BthTX-I. Since suramin is a polyanionic substance, we suggested that its effects result from the formation of inactive acid-base complexes with BthTX-I.