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

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Antibacterial and antiparasitic effects of Bothrops marajoensis venom and its fractions: Phospholipase A(2) and L-amino acid oxidase

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Neutralization of snake venom phospholipase A(2) toxins by aqueous extract of Casearia sylvestris (Flacourtiaceae) in mouse neuromuscular preparation

Aqueous extract of Casearia sylvestris (Flacourtiaceae) has been shown to inhibit enzymatic and biological properties of some Bothrops and Crotalus venoms and their purified phospholipase A(2) (PLA(2)) toxins. In this work we evaluated the influence of C sylvestris aqueous extract upon neuromuscular blocking and muscle damaging activities of some PLA(2)S (crotoxin from C. durissus terrificus, bothropstoxin-I from B.jararacussu, piratoxin-I from B. pirajai and myotoxin-II from B. moojeni) in mouse phrenic-diaphragm preparations. Crotoxin (0.5 mu M) and all other PLA2 toxins (1.0 mu M) induced irreversible and time-dependent blockade of twitches. Except for crotoxin, all PLA2 toxins induced significant muscle damage indices, assessed by microscopic analysis. Preincubation of bothropstoxin-I, piratoxin-I or myotoxin-II with C. sylvestris extract (1:5 (w/w), 30 min, 37 degrees C significantly prevented the neuromuscular blockade of preparations exposed to the mixtures for 90 min; the extent of protection ranged from 93% to 97%. The vegetal extract also neutralized the muscle damage (protection of 80-95%). Higher concentration of the C. sylvestris extract (1: 10, w/w) was necessary to neutralize by 90% the neuromuscular blockade induced by crotoxin. These findings expanded the spectrum of C. sylvestris antivenom activities, evidencing that it may be a good source of potentially useful PLA2 inhibitors. (c) 2007 Elsevier B.V.. All rights reserved.

Crystallization and preliminary X-ray diffraction analysis of an acidic phospholipase A(2) complexed with p-bromophenacyl bromide and alpha-tocopherol inhibitors at 1.9-and 1.45-A resolution

An acidic phospholipase A(2) (PLA(2)) isolated from Bothrops jararacussu snake venom was crystallized with two inhibitors: alpha-tocopherol (vitamin E) and p-bromophenacyl bromide (BPB). The crystals diffracted at 1.45- and 1.85-Angstrom resolution, respectively, for the complexes with alpha-tocopherol and p-bromophenacyl bromide. The crystals are not isomorphous with those of the native protein, suggesting the inhibitors binding was successful and changes in the quaternary structure may have occurred. (C) 2004 Elsevier B.V. All rights reserved.

Snake venom phospholipase A(2) inhibitors: Medicinal chemistry and therapeutic potential

Phospholipases A(2) (PLA(2)s) 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 PLA(2)s. These inhibitors act on PLA(2)S 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 PLA(2)s and their role in envenomations caused by snake bite. Furthermore, the biotechnological potential of PLA(2) inhibitors may provide therapeutic molecular models with antiophidian activity to supplement the conventional serum therapy against these multifunctional enzymes.

Cloning and Identification of a Complete cDNA Coding for a Bactericidal and Antitumoral Acidic Phospholipase A(2) from Bothrops jararacussu Venom

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Insights into the role of oligomeric state on the biological activities of crotoxin: crystal structure of a tetrameric phospholipase A(2) formed by two isoforms of crotoxin B from Cratalus durissus terrificus venom

Crotoxin B (CB or Cdt PLA(2)) is a basic Asp49-PLA(2) found in the venom of Crotalus durissus terrificus and it is one of the subunits that constitute the crotoxin (Cro). This heterodimeric toxin, main component of the C. d. terrificus venom, is completed by an acidic, nontoxic, and nonenzymatic component (crotoxin A, CA or crotapotin), and it is related to important envenomation effects such as neurological disorders, myotoxicity, and renal failure. Although Cro has been crystallized since 1938, no crystal structure of this toxin or its subunits is currently available. In this work, the authors present the crystal structure of novel tetrameric complex formed by two dimers of crotoxin B isoforms (CB1 and CB2). The results suggest that these assemblies are stable in solution and show that Ser1 and Glu92 of CB1 and CB2, respectively, play an important role in the oligomerization. The tetrameric and dimeric conformations resulting from the association of the isoforms may increase the neurotoxicity of the toxin CB by the creation of new binding sites, which could improve the affinity of the molecular complexes to the presynaptic membrane.

Crystallization and preliminary X-ray diffraction studies of BmooPLA(2)-I, a platelet-aggregation inhibitor and hypotensive phospholipase A(2) from Bothrops moojeni venom

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Comparative biochemical studies of myotoxic phospholipase A(2) from Bothrops venom

Venoms from Bothrops jararacussu, Bothrops asper, Bothrops atrox, Bothrops pirajai, Bothrops moojeni, Bothrops alternatus and Bothrops (Bothriopsis) bilineata were fractionated using a simplified procedure based on ion-exchange chromatography on CM-Sepharose at pH 8.0 or reverse phase HPLC. The resulting elution profiles showed important differences in the myotoxin content of these venoms. The venoms from B. alternatus, B. atrox and Bothriopsis bilineata did not contain the major myotoxin found in the other venoms. The amino acid sequence of the first 50 residues of the N-terminal region of the PLA(2)-like myotoxins showed a homology of 90-96% with other bothropic myotoxins. All of the myotoxins isolated induced rat paw edema, increased the level of plasma creatine kinase and produced myonecrosis together with polymorphonuclear cell infiltration.

Phospholipase A(2) - A structural review

Phospholipases A(2) (PLA(2)) are widely distributed in nature and are well characterized proteins with respect to their catalytic and pharmacological activities, A wealth of structural information has recently become available both from X-ray diffraction and NMR studies, and although a detailed model of the catalytic mechanism of PLA(2) has been proposed, the structural bases of other aspects of PLA(2) function, such as interfacial activation and venom PLA(2) pharmacological activities, are still under debate. An appreciation of the PLA(2) protein structure will yield new insights with regard to these activities, the salient structural features of the class I, II and III PLA(2) are discussed with respect to their functional roles. Copyright (C) 1996 Published by Elsevier B.V. Ltd

Isolation, characterization and biological activity of acidic phospholipase A(2) isoforms from Bothrops jararacussu snake venom

Acidic phospholipase A(2) (PLA(2)) isoforms in snake venoms, particularly those from Bothrops jararacussu, have not been characterized. This article reports the isolation and partial biochemical, functional and structural characterization of four acidic PLA(2)s (designated SIIISPIIA, SIIISPIIB, SIIISPIIIA and SIIISPIIIB) from this venom. The single chain purified proteins contained 122 amino acid residues and seven disulfide bonds with approximate molecular masses of 15 kDa and isoelectric points of 5.3. The respective N-terminal sequences were: SIIISPIIA-SLWQFGKMIDYVMGEEGAKS; SIIISPIIB-SLWQFGKMIFYTGKNEPVLS; SIIISPIIIA-SLWQFGKMILYVMGGEGVKQ and SIIISPIIIB-SLWQFGKMIFYEMTGEGVL. Crystals of the acidic protein SIIISPIIIB diffracted beyond 1.8 Angstrom resolution. These crystals are monoclinic with unit cell dimensions of a = 40.1 Angstrom, b = 54.2 Angstrom and c = 90.7 Angstrom. The crystal structure has been refined to a crystallographic residual of 16.1% (R-free = 22.9%). Specific catalytic activity (U/mg) of the isolated acidic PLA(2)s were SIIISPIIA = 290.3 U/mg; SIIISPIIB = 279.0 U/mg; SIIISPIIIA = 270.7 U/mg and SIIISPIIIB = 96.5 U/mg. Although their myotoxic activity was low, SIIISPIIA, SIIISPIIIB and SIIISPIIIA showed significant anticoagulant activity. However, there was no indirect hemolytic activity. SIIISPIIIB revealed no anticoagulant, but presented indirect hemolytic activity. With the exception of SIIISPIIIB, which inhibited platelet aggregation, all the others were capable of inducing time-independent edema. Chemical modification with 4-bromophenacyl bromide did not inhibit the induction of edema, but did suppress other activities. (C) 2003 Editions scientifiques et medicales Elsevier SAS. All rights reserved.

Inhibition of inducible nitric oxide synthase-derived nitric oxide as a therapeutical target for acute pancreatitis induced by secretory phospholipase A(2)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Insights on the structure of native CNF, an endogenous phospholipase A(2) inhibitor from Crotalus durissus terrificus, the South American rattlesnake

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Agelotoxin: a phospholipase A(2) from the venom of the neotropical social wasp cassununga (Agelaia pallipes pallipes) (Hymenoptera-Vespidae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Active site mutants of human secreted Group IIA Phospholipase A(2) lacking hydrolytic activity retain their bactericidal effect

The Human Secreted Group IIA Phospholipase A(2) (hsPIA2GIIA) presents potent bactericidal activity, and is considered to contribute to the acute-phase immune response. Hydrolysis of inner membrane phospholipids is suggested to underlie the bactericidal activity, and we have evaluated this proposal by comparing catalytic activity with bactericidal and liposome membrane damaging effects of the G30S, H48Q and D49K h5PLA2GIIA mutants. All mutants showed severely impaired hydrolytic activities against mixed DOPC:DOPG liposome membranes, however the bactericidal effect against Micrococcus luteus was less affected, with 50% killing at concentrations of 1, 3, 7 and 9 mu g/mL for the wild-type, D49K, H48Q and G30S mutants respectively. Furthermore, all proteins showed Ca2+-independent damaging activity against Liposome membranes demonstrating that in addition to the hydrolysis-dependent membrane damage, the hsPLA2GIIA presents a mechanism for permeabilization of phospholipid bilayers that is independent of catalytic activity, which may play a role in the bactericidal function of the protein (C) 2011 Elsevier Masson SAS. All rights reserved.