Snake venom L-amino acid oxidases: trends in pharmacology and biochemistry.

L-amino acid oxidases are enzymes found in several organisms, including venoms of snakes, where they contribute to the toxicity of ophidian envenomation. Their toxicity is primarily due to enzymatic activity, but other mechanisms have been proposed recently which require further investigation. L-amino acid oxidases exert biological and pharmacological effects, including actions on platelet aggregation and the induction of apoptosis, hemorrhage, and cytotoxicity. These proteins present a high biotechnological potential for the development of antimicrobial, antitumor, and antiprotozoan agents. This review provides an overview of the biochemical properties and pharmacological effects of snake venom L-amino acid oxidases, their structure/activity relationship, and supposed mechanisms of action described so far.

BnSP-7 toxin, a basic phospholipase A2 from Bothrops pauloensis snake venom, interferes with proliferation, ultrastructure and infectivity of Leishmania (Leishmania) amazonensis.

This paper reports the effects of BnSP-7 toxin, a catalytically inactive phospholipase A2 from Bothrops pauloensis snake venom, on Leishmania (Leishmania) amazonensis. BnSP-7 presented activity against promastigote parasite forms both in the MTT assay, with IC50 of 58.7 µg mL(-1) of toxin, and a growth curve, inhibiting parasite proliferation 60-70% at concentrations of 50-200 µg mL(-1) of toxin 96 h after treatment. Also, the toxin presented effects on amastigotes, reducing parasite viability by 50% at 28.1 µg mL(-1) and delaying the amastigote-promastigote differentiation process. Ultrastructural studies showed that BnSP-7 caused severe morphological changes in promastigotes such as mitochondrial swelling, nuclear alteration, vacuolization, acidocalcisomes, multiflagellar aspects and a blebbing effect in the plasma membrane. Finally, BnSP-7 interfered with the infective capacity of promastigotes in murine peritoneal macrophages, causing statistically significant infectivity-index reductions (P < 0.05) of 20-35%. These data suggest that the BnSP-7 toxin is an important tool for the discovery of new parasite targets that can be exploited to develop new drugs for treating leishmaniasis.

Molecular characterization of an acidic phospholipase A(2) from Bothrops pirajai snake venom: synthetic C-terminal peptide identifies its antiplatelet region.

This paper describes a biochemical and pharmacological characterization of BpirPLA(2)-I, the first acidic Asp49-PLA(2) isolated from Bothrops pirajai. BpirPLA(2)-I caused hypotension in vivo, presented phospholipolytic activity upon artificial substrates and inhibitory effects on platelet aggregation in vitro. Moreover, a synthetic peptide of BpirPLA(2)-I, comprising residues of the C-terminal region, reproduced the antiplatelet activity of the intact protein. A cDNA fragment of 366 bp encompassing the mature form of BpirPLA(2)-I was cloned by reverse transcriptase-PCR of B. pirajai venom gland total RNA. A Bayesian phylogenetic analysis indicated that BpirPLA(2)-I forms a clade with other acid Asp49-PLA(2) enzymes from the Bothrops genus, which are characterized by the high catalytic activity associated with anticoagulant or hypotensive activity or both. Comparison of the electrostatic potential (EP) on the molecular surfaces calculated from a BpirPLA(2)-I homology model and from the crystallographic models of a group of close homologues revealed that the greatest number of charge inversions occurred on the face opposite to the active site entrance, particularly in the Ca(2+) ion binding loop. This observation suggests a possible relationship between the basic or acid character of PLA(2) enzymes and the functionality of the Ca(2+) ion binding loop.

Acute toxicity of Schizolobium parahyba aqueous extract in mice.

The herbal extract of Schizolobium parahyba leaves is used commonly in the Brazil central region to treat snakebites. This study evaluates the acute toxicological effects of Schizolobium parahyba aqueous extract in mice 24 h after intraperitoneal administration. Acute toxicity was evaluated using biochemical, hematological and histopathological assays. Alterations in the levels of transaminases, bilirubin, albumin and prothrombrin time were observed, and these are likely to occur due to hepatic injury, which was confirmed by light microscopy. Liver histopathological analysis revealed the presence of lymph plasmocitary inflammatory infiltrate, but no other histopathological alterations were observed in any of the other organs analysed. The data confirm the low toxicity of the extract of Schizolobium parahyba and provide a model for the selection of a dose that does not cause injuries in the organism.

Snake venom phospholipases A2: a new class of antitumor agents.

Phospholipases A(2) (PLA(2)) are enzymes of high medical scientific interest due to their involvement in a large number of human inflammatory diseases. PLA(2) constitute a diverse family of enzymes which catalyses the hydrolysis of the sn-2 ester bond in glycerophospholipids and exhibit a wide range of physiological and pathological effects. The ubiquitous nature of PLA(2) highlights the important role they play in many biological processes, as cell signaling and cell growth, including the generation of proinflammatory lipid mediators such as prostaglandin and leukotrienes, regulation of lipid mediators. The activity and expression of several PLA(2) isoforms are increased in several human cancers, suggesting that these enzymes have a central role in both tumor development and progression and can be targets for anti-cancer drugs. On the other hand, some PLA(2) isolated from Viperidae venoms are capable to induce antitumoral activity. In summary PLA(2) from snake venoms can be a new class of anticancer agents and provide new molecular and biological insights of cancer development.

Anti-snake venom properties of Schizolobium parahyba (Caesalpinoideae) aqueous leaves extract.

Many medicinal plants have been recommended for the treatment of snakebites. The aqueous extracts prepared from the leaves of Schizolobium parahyba (a plant found in Mata Atlantica in Southeastern Brazil) were assayed for their ability to inhibit some enzymatic and biological activities induced by Bothrops pauloensis and Crotalus durissus terrificus venoms as well as by their isolated toxins neuwiedase (metalloproteinase), BnSP-7 (basic Lys49 PLA(2)) and CB (PLA(2) from crotoxin complex). Phospholipase A(2), coagulant, fibrinogenolytic, hemorrhagic and myotoxic activities induced by B. pauloensis and C. d. terrificus venoms, as well as by their isolated toxins were significantly inhibited when different amounts of S. parahyba were incubated previously with these venoms and toxins before assays. However, when S. parahyba was administered at the same route as the venoms or toxins injections, the tissue local damage, such as hemorrhage and myotoxicity was only partially inhibited. The study also evaluated the inhibitory effect of S. parahyba upon the spreading of venom proteins from the injected area into the systemic circulation. The neutralization of systemic alterations induced by i.m. injection of B. pauloensis venom was evaluated by measuring platelet and plasma fibrinogen levels which were significantly maintained when S. parahyba extract inoculation occurred at the same route after B. pauloensis venom injection. In conclusion, the observations confirmed that the aqueous extract of S. parahyba possesses potent snake venom neutralizing properties. It may be used as an alternative treatment to serum therapy and as a rich source of potential inhibitors of toxins involved in several physiopathological human and animal diseases.

Molecular characterization of BjussuSP-I, a new thrombin-like enzyme with procoagulant and kallikrein-like activity isolated from Bothrops jararacussu snake venom.

A thrombin-like enzyme, named BjussuSP-I, isolated from Bothrops jararacussu snake venom, is an acidic single-chain glycoprotein with M(r)=61,000, pI approximately 3.8 and 6% sugar. BjussuSP-I shows high proteolytic activity upon synthetic substrates, such as S-2238 and S-2288. It also shows procoagulant and kallikrein-like activity, but is unable to act on platelets and plasmin. These activities are inhibited by specific inhibitors of this class of enzymes. The complete cDNA sequence of BjussuSP-I with 696bp encodes open reading frames of 232 amino acid residues, which conserve the common domains of thrombin-like serine proteases. BjussuSP-I shows a high structural homology with other thrombin-like enzymes from snake venoms where common amino acid residues are identified as those corresponding to the catalytic site and subsites S1, S2 and S3 already reported. In this study, we also demonstrated the importance of N-linked glycans to improve thrombin-like activity of BjussuSP-I toxin.

Molecular and functional characterization of a new non-hemorrhagic metalloprotease from Bothrops jararacussu snake venom with antiplatelet activity.

BjussuMP-II is an acidic low molecular weight metalloprotease (Mr approximately 24,000 and pI approximately 6.5), isolated from Bothrops jararacussu snake venom. The chromatographic profile in RP-HPLC and its N-terminal sequence confirmed its high purity level. Its complete cDNA was obtained by RT-PCR and the 615bp codified for a mature protein of 205 amino acid residues. The multiple alignment of its deduced amino acid sequence and those of other snake venom metalloproteases showed a high structural similarity, mainly among class P-I proteases. The molecular modeling analysis of BjussuMP-II showed also conserved structural features with other SVMPs. BjussuMP-II did not induce hemorrhage, myotoxicity and lethality, but displayed dose-dependent proteolytic activity on fibrinogen, collagen, fibrin, casein and gelatin, keeping stable at different pHs, temperatures and presence of several divalent ions. BjussuMP-II did not show any clotting or anticoagulant activity on human citrated plasma, in contrast to its inhibitory effects on platelet aggregation. The aspects broached, in this work, provide data on the relationship between structure and function, in order to better understand the effects elicited by snake venom metalloproteases.

Isolation and functional characterization of a new myotoxic acidic phospholipase A(2) from Bothrops pauloensis snake venom.

This article reports the purification procedure and the biochemical/functional characterization of Bp-PLA(2), a new myotoxic acidic phospholipase A(2) from Bothrops pauloensis snake venom. It was highly purified through three chromatographic steps (ion-exchange on CM-Sepharose, hydrophobic chromatography on Phenyl-Sepharose and RP-HPLC on a C8 column). Bp-PLA(2) is a single-chain protein of 15.8kDa and pI 4.3. Its N-terminal sequence revealed a high homology with other Asp49 acidic PLA(2)s from snake venoms. Its specific activity was 585.3U/mg. It displayed a high indirect hemolytic activity and inhibited platelet aggregation induced by collagen or ADP. It also induced in vivo edema and myotoxicity. Pretreatment of Bp-PLA(2) with BPB reduced the enzymatic activity, the inhibitory action on platelet aggregation and myotoxicity in vitro. Morphological analyses indicated that Bp-PLA(2) induced an intense edema, with visible leukocyte infiltrate and damaged muscle cells 24h after injection. Acidic myotoxic PLA(2)s from Bothrops snake venoms are still not extensively explored and knowledge of their structural and functional features will contribute for a better understanding of their action mechanism regarding enzymatic and toxic activities.

Biochemical and functional characterization of an L-amino acid oxidase isolated from Bothrops pirajai snake venom.

In this work we describe the isolation of a new l-amino acid oxidase (LAAO) referred to as BpirLAAO-I from Bothrops pirajai snake venom, which was highly purified using a combination of molecular exclusion, affinity, and hydrophobic chromatography steps. BpirLAAO-I homodimeric acid glycoprotein (approximate Mr and pI of 130,000 and 4.9, respectively) displays high specificity toward hydrophobic/aromatic amino acids, while deglycosylation does not alter its enzymatic activity. The N-terminal LAAO sequence of its first 49 amino acids presented a high similarity between a amino acid sequence with other LAAOs from: Bothrops spp., Crotalus spp., Calloselasma rhodostoma, Agkistrodon spp., Trimeresurus spp., Pseudechis australis, Oxyuranus scutellatus, and Notechis scutatus. BpirLAAO-I induces time-dependent platelet aggregation, mouse paw edema, cytotoxic activity against Escherichia coli, Pseudomonas aeruginosa, Leishmania sp., and tumor cells, and also a typical fago (M13mp18) DNA fragmentation. Platelet aggregation, leishmanicidal and antitumoral activities were reduced by catalase. Thus, BpirLAAO-I is a multifunctional protein with promising biotechnological and medical applications.