Snake Venom Phospholipase A2 Inhibitors: Medicinal Chemistry and Therapeutic Potential

Current Topics in Medicinal Chemistry, 2007, 7, 000-000 1

1568-0266/07 $50.00+.00 © 2007 Bentham Science Publishers Ltd.

Snake Venom Phospholipase A2 Inhibitors: Medicinal Chemistry and Therapeutic Potential

Silvana Marcussia,b, Carolina D. Sant’Anaa, Clayton Z. Oliveiraa, Aristides Quintero Ruedaa,c, Danilo L. Menaldoa, Rene O. Belebonid, Rodrigo G. Stabelie, José R. Gigliob, Marcos R. M. Fontesf and Andreimar M. Soaresa,*

aDepartamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de

Ribeirão Preto, FCFRP, Universidade de São Paulo, USP-RP, Ribeirão Preto-SP, Brazil; bDepartamento de

Bioquímica e Imunologia, Faculdade de Ciências Médicas de Ribeirão Preto, FMRP, Universidade de São Paulo, USP-

RP, Ribeirão Preto-SP, Brazil; cDepartamento de Química, Faculdade de Ciencias Naturaes e Exactas, Universidade

Autónoma de Chiriquí, UNACHI, Chiriquí, Panamá; dDepartamento de Biotecnologia, Universidade de Ribeirão Preto,

UNAERP-SP, Ribeirão Preto, Brazil; eInstituto de Pesquisas em Patologias Tropical, IPEPATRO, Universidade

Federal de Rondônia, UNIR, Rondônia-RO, Brazil; fDepartamento de Física e Biofísica, Instituto de Biociências,

Universidade Estadual Paulista, UNESP, Botucatu-SP, Brazil.

Abstract: 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.

Keywords: Phospholipases A2, phospholipase A2 inhibitors, natural and artificial inhibitors, snake venoms.

1. INTRODUCTION

Global health statistics for incidence of snake bite envenomations and their severity remain unknown or misun-derstood. In spite of the lack of data, a global estimation of the number of ophidian accidents reaches one million cases per year, accounting for 20,000 deaths, especially along rural areas in Asia, South America and Africa. In addition to mortality, these envenomations are also a public health concern as a result of the chronic morbidity associated with them (e.g. amputations, deformations and renal failure), which causes significant social and economic impact [1, 2].

Snake venoms induce shock, proteolysis, blood clotting, release of bioactive substances such as histamine and bradykinin, hemorrhage, necrosis and several other effects [3]. Necrosis may result from a direct action of myotoxins or myotoxic PLA2s on muscle cell plasma memb-ranes, or indirectly, as a consequence of blood vessel dege-neration and schemia caused by hemorrhagins. Disorga-nization of membrane phospholipid components promotes release of intracellular creatine kinase, which may be used as a biomarker for myotoxic activity evaluation [4, 5].

The high medical-scientific concern evoked by the involvement of these proteins in different physiopathological processes promoted an increasing search for natural or *Address correspondence to this author at the FCFRP-USP, Ribeirão Preto-SP, Brasil; Fax: 55-16-3602-4725; E-mail: andreims@fcfrp.usp.br

artificial inhibitors, aiming at PLA2s neutralization, and a better understanding of their mechanism of action and their structure-function relationship. Different venom toxins can be recognized and inhibited by poly or monoclonal antibodies, as well as by some diversified agents including chemicals (EDTA, BPB, and others), as well as animal and vegetal compounds [6-9], such as heparin.

Low molecular weight heparin, an anionic natural polysaccharide, showed to be able to inhibit, at least in part, the myotoxic, cytotoxic and edema inducing effects of B. moojeni MjTX-II and B. neuwiedi BnSP-7 venoms and able to abolish the neuromuscular effect of the latter. These data confirmed the hypothesis that the 115-129 C-terminal region of B. asper myotoxin II, named heparin binding region, would be responsible for PLA2 cytotoxicity and at least in part for its myotoxicity, since this polyanion interacts with this region of the enzyme [9-13].

Some PLA2 inhibitors may be found in different orga-nisms. Manoalide (A) is a non-steroidal sesquiterpenoid from the marine sponge Luffariella variabilis, while manoalide (B) was artificially synthesized based on its natural analogue. These terpenoids compounds promote irreversible inhibitory effects upon several PLA2s from snakes, bees and mammals [14, 15]

Aside from traditional knowledge or the popular use, several scientific evidences have pointed to the efficacy of crude extracts or fractions from plants, such as Eclipta

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prostata, Tabernaemontana catharinensis, Casearia sylvestris, Mandevilla velutina, Sapindus sapindus and Cordia verbenacea [6, 7, 16-18], in the treatment of ophidian accidents. A short description is made of WSG, an antitoxic-PLA2 glycoprotein isolated from Withania somnifera, a medicinal plant whose aqueous extract neutralizes the PLA2 activity of Naja naja venom [19, 20].

Snake blood-derived inhibitors have been grouped into three major classes ( , and ), based on common structural motifs found in other proteins with diverse physiological properties. In mammals, DM64, an antimyotoxic protein isolated from Didelphis marsupialis opossum serum, belongs to the super-immunoglobulin gene family and it is homologous to human -1 -glycoprotein, and DM43 is a metalloprotease inhibitor from the same organism. Examples of inhibitors from animal serum are the antibothropic complex (ABC) from the marsupial Didelphis [21, 22] and PLA2 inhibitors (PLIs) from snake blood Agkistrodon, Trimeresurus, Bothrops, Crotalus, Naja, Laticaudata and Elaphe [23-27].

Several reviews dealing with inhibitors of snake venom hemorrhagins [28-32], myotoxins and neurotoxins [28, 32-34] have been published along the past two decades. Lizano et al. [35] reviewed the current knowledge regarding PLA2s inhibitor proteins derived from both snake and mammalian blood, with particular emphasis on the classification, mole-cular and functional characterization of myotoxic PLA2 inhibitors. A special mention was made to a newly described glycoprotein PLA2 inhibitor from the medicinal plant Withania somnifera, which exhibits antimyotoxic and antiedema properties against a multitoxic PLA2 from the Indian cobra venom (NNXIa-PLA).

In this review, we will broadly discuss the implications of the PLA2 toxin inhibitor groups on current understanding of snake biology, as well as in the development of new therapeutic drugs for treatment of snake envenomations.

1.1. SNAKE VENOM PHOSPHOLIPASES A2 (PLA2S)

Intra and extracellular PLA2s are largely distributed, being found in pancreatic secretions, inflammatory exudates, arthropod and snake venoms [36]. These enzymes play important roles in the dietary lipid catabolism, in the cell membrane methabolism and signal transduction in diverse organisms. PLA2s are involved with several human inflammatory diseases, in addition to their pharmacological and pathological effects on living organisms and snake or bee envenomation [27, 37, 38].

PLA2s (EC 3.1.1.4) catalyze the hydrolysis of 2-acyl ester bonds of 3-sn-phospholipids producing fatty acids and lysophospholipids [39]. The Ca2+ ion, an essential cofactor, and an Asp residue at position 49 are required for catalysis on artificial substrates [39]. Snake venoms constitute a rich source of PLA2 enzymes, which show remarkable functional diversity.

Independently of their primary catalytic function, snake venom PLA2s can induce several additional effects such as pre or postsynaptic neurotoxicity, cardiotoxicity, myotoxi-city, platelet aggregation inhibition, edema, hemolysis, anticoagulation, convulsion and hypotension [40, 41]. Other

researchers also reported the following effects: bactericidal, anti-Plasmodium, anti-HIV, anti-Leishmania, antitumoral, anti-Schistosoma and others [40-46].

PLA2s were recently divided into 11 groups based on biochemical and structural criteria, considering their molecular weight, disulfide bonds profile, phospholipid substrates, amino acid sequence, sensibility to Ca2+ ions, catalytic activity and genic structure [47-49]. Snake venoms are especially rich in I and II PLA2 groups, found in the Elapidae or Viperidae families, respectively. Their catalytic activity upon cell membranes of specific tissues suggests an important role of these enzymes in venoms toxicity. Group II PLA2s can be further subdivided into two main types commonly referred to as Asp49 and Lys49 isoforms [50].

The disarrangement of phospholipids components can result in severe alterations of the structural and functional membrane integrity, with subsequent influx of Ca2+ ions, causing sarcomer contraction, activation of Ca2+ ion dependent proteases and endogenous PLA2s, besides the envenoming of mitochondria [51-54]. Cell death is a possible result of the sum of all these alterations.

The amino acid sequence of more than 200 venom PLA2s were elucidated, while some of their tridimentional struc-tures were evidenced by X-ray crystallography and by NMR spectroscopy [55].

Group II PLA2s display a binding site for Ca2+, which is highly conserved (X28CGXGG33) in addition to the highly conserved catalytic site (D42XCCXXHD49) previously reported [56]. The Ca2+ binding site is formed by the ß-carboxyl group of Asp49 and the carbonyl groups of Tyr28, Gly30 and Gly32 [57]. Even a discrete change of the amino acid sequence in the Ca2+ binding site can cause a drastic drop of their catalytic activity as shown in the PLA2 from Trimeresurus gramineus venom [58].

His48 residue is similarly relevant, being involved with catalysis through its N-1 group oriented toward the solvent. Thus, it is suggested that one water molecule at about 3Å from N-1 would play a nucleophilic role in the ester hydrolysis [59]. When this water molecule attacks the carbonyl C of the substrate, the imidazol ring of His48 catches the proton of the water molecule making the reaction easier. This proton is further released by the imidazol ring to the oxygen of Asp99 carboxyl group. Studies showed that the integrity of His48 is an additional requirement and the alkylation of this residue by p-bromophenacyl bromide (BPB) causes a loss of enzymatic and toxic activities [60-62].

Functional and structural features of toxic basic PLA2s from several snake venoms have been investigated by site directed mutagenesis, spectrophotometry, crystallography, NMR, sequencing of amino acid residues and cloning of these enzymes.

Up to the 90’s, only Asp49-PLA2s had been described. Since then, the myotoxic basic PLA2s isoforms from Bothrops snakes were isolated and classified into group II as well, where the Asp49 residue was replaced by Lys49, resulting thus two classes of this enzyme: (a) Asp49 myotoxins showing moderate catalytic activity and (b)

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Lys49 myoto-xins with low or no enzymatic activity upon artificial substrates [40, 50].

During the last years, myonecrotic venom components were extensively studied. Several myotoxic Bothrops PLA2s were then characterized, such as B. asper PLA2s, B. moojeni myotoxins I and II, and B. jararacussu bothropstoxins I (Lys49) and II (Asp49), responsible for several biological effects including myonecrosis, edema, irreversible neuro-muscular blockage and in vitro cell lysis [51, 63-66].

Many acidic PLA2s have been isolated from Agkistrodon halys, Pseudechis papuanus, Bothrops neuwiedi, Bothrops lanceolatus, Trimeresurus jerdonii, Heloderma horridum, Ophiophagus hannah, Lachesis muta, B. jararaca, B. moojeni and B. jararacussu [12, 37, 67-76]. Toxicity and pharmacological effects differ in acidic isoforms. For instance, the acidic PLA2 isolated from Lachesis muta venom is myonecrotic, proteolytic, anticoagulant and platelet aggregation inhibitor. Another myotoxic PLA2 from the same venom did not show anticoagulant or lethal activity [67-69].

Considering their function, origin, regulation, action mechanism, structure and role of divalent cations, it has been suggested that PLA2s represent a class of versatile enzymes and, as multifunctional proteins, they are extre-mely relevant as mediators of several inflammatory diseases and promising agents for use in biotechnological areas [12, 37]. An increasing search for use of these enzymes is therefore not surprising, including their general anesthetic action, treat-ment of rheumatoid arthritis, bactericidal action, novel class of antiparasitary agents, HIV inhibitors and others [45, 77-80]. Fig. (1) shows the most conventional structural repre-sentation of a typical Bothrops svPLA2.

Fig. (1). Schematic representation of a typical Asp49-PLA2 monomer

from snake venom. In detail, amino acid side chains essential for Ca2+

binding (Y28; G30; G32; D49) and catalytic site (H48; D99; Y73).

2. PLANT EXTRACTS AND THEIR ISOLATED PLA2S INHIBITORS

Several plants have been reported to show antiophidian activity due to the presence of biomolecules able to

neutralize many local and systemic toxic effects. The use of medicinal plants for treatment of ophidian envenomation is very difused among some populations, mainly in native communities in several parts of the world. The use of plant extracts in ophidian accidents is common in regions where access to serum therapy is lengthy or almost inviable. A large number of antiophidian plants have been investigated in the search of new biologically active substances able to inhibit snake venom effects [81-84]. Many studies however were not successful to elucidate the mechanisms involved with this neutralizing activity. The active components, named secondary metabolites, generally act as enzymatic inhibitors, chemical inactivators or immunomodulators able to interact with the venom target macromolecules [81].

The therapeutic potential of medicinal plants is traditio-nally attributed to classes of active constituents, including flavonoids, alkaloids, sesquiterpenes, lignins and others. These natural inhibitors can represent an additional aid in the traditional serum therapy and can be useful as molecular models for new drugs at a lower price, with lower side effects and easily distributed to communities far from medi-cal access, or even animals, since for them the traditional serum therapy is not completely suitable [83].

Aside from a wide array of well-documented non-protein chemical compounds with anti-PLA2 properties from plants and even marine organisms, the antitoxic protein of W. somnifera represents the first known plant protein to inhibit toxic PLA2s. Early studies have showed that aqueous extracts of this plant neutralize the PLA2 activity of Naja naja venom and, recently, the active anti-PLA2 glycoprotein named WSG was isolated from this extract. WSG is a 27 kDa glycoprotein that inhibits in vitro cytotoxic effects as well as the in vivo myotoxicity and edema inducing potential of the multitoxic PLA2 NNXIa-PLA2. However, its neuro-toxicity is not abolished by WSG [19, 20, 85, 86]. As established to snake PLIs, WSG remains pharmacologically active after removal of the glycosylated moiety. No structural or inhibitory spectrum information on this protein is available to date.

Other examples are wedelolactone, isolated from Eclipta prostata and the alkaloid 12-methoxy-4methyvoachalotine, isolated from Tabernaemontana catharinensis. Recently, wedelolactone was assayed against Calloselasma rhodos-toma venom, neutralizing the toxic effects of isolated PLA2s and the crude venom lethality [16, 17, 87-90]. From Baccharis trimera (Asteraceae) a diterpenoid was isolated, neo-clerodane inhibitor of snake venom metalloproteases, with antiproteolytic and antihemorrhagic properties [91]. Several authors listed 578 plants with therapeutic potential in ophidian accidents, belonging to 94 families, mainly Asteraceae (9%), Leguminosae (7.8%) and Euphorbiaceae (4.5%) [92, 93]. Recently, 77 antivenom plants from Colombia were listed, with the help of witch doctors, with their popular names, indicated uses, administration via and localization [94]. In most cases, extracts are prepared by infusion, decoction or maceration and administered orally. Sometimes, they are used directly on the bite site when it is washed or sprayed with the plant extract or even as cataplasm by use of wet compresses on the affected site.

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Mandevilla velutina and illustris, Brazilian plants, should be outstood due to its known antiophidian properties, such as inhibition of PLA2s and phospholipase C from Naja naja venom, crotoxin and other toxins isolated from C. d. terrificus and Bothrops venoms [18, 95, 96].

Inhibition of the hemorrhagic effect of B. asper venom by extracts of several plants from Costa Rica was reported [97]. Ethanolic extracts from 74 plants from Colombia showed to be active against B. atrox venom lethality, while seven of them were able to abolish this activity (the stem barks of Brownea rosade-monte – Caesalpiniaceae and Tabebuia rosea - Bignoniaceae; rhizomes of Renealmia alpinia – Zingiberaceae and Heliconia curtispatha – Helico-niaceae; the whole plant of Pleopeltis percussa – Poly-podiaceae and Trichomanes elegans - Hymenophyllaceae; and the ripe fruits of Citrus limon – Rutaceae). Some of them could also inhibit the PLA2 activity of this venom.

In 2004, Hung et al. [98] reported the inhibitory effect of melanin from Thea sinensis Linn., against Agkistrodon contortrix laticinctus, Agkistrodon halys blomhoffii and Crotalus atrox venoms lethality, in addition to an isolated PLA2. Moreover, phenylated and benzylated pterocarpans were also described as inhibitors of the myotoxic PLA2 and the proteolytic activity of B. jararacussu venom [99].

Almeida et al. [100] showed that fractions of the aqueous extract from Tabernaemontana catharinensis abolished the lethal action of C. d. terrificus venom and crotoxin. The antiophidian effect of extracts from roots, stems and leaves of Mikania glomerata was described [101], and a protein (mikagin) from the same plant showed to be a potent inhibitor of several snake venoms and isolated toxins [unpublished data].

Mors et al. [81] quoted 104 plants referred to as antio-phidian by folk medicine, in addition to several compounds with this same action, especially from species of the Apocynaceae family. Inhibition of the lethality and myotoxic activities of Crotalus d. terrificus venom by other Apocynaceae, Tabernaemontana catharinensis, was reported [17], showing that this family is a promising source of bioactive compounds that neutralize animal venoms.

Borges et al. [7] obtained interesting results with Casearia sylvestris, popularly known as “guaçatonga”, which significantly inhibited the activity of PLA2s isolated from Bothrops, Crotalus and Micrurus snake venoms. This plant species also showed inhibitory effects against the hemorrhagic activity induced by several Bothrops venoms [8]. Belonging to the same genus, the aqueous extract from Casearia mariquitensis also showed neutralizing activity on hemorrhagic and hemostatic effects induced by neuwiedase, a metalloprotease isolated from B. neuwiedi [102].

Several other plants with antiphopholipase A2 and antivenom properties were described: (i) the root extract from Mimosa pudica neutralized myotoxins, toxic enzymes and lethality from Naja kaouthia venom; (ii) methanolic extracts of roots from Hemidesmus indicus and H. pluchea inhibited the hemorrhagic and lethal effects of Vipera ruselli venom; (iii) the methanolic extract from Eryngium creticum inhibited the hemolytic activity of Leiurus quinquestriatus http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Search&d

b=pubmed&term=leiurus+quinquestriatus&tool=fuzzy&ot=Leiurus+quinquesteiartus> scorpion venom; (iv) decrease of the lethal effect of C. d. terrificus venom by the extract from Peschiera fuchsiaefolia was also reported; (v) the venoms from Naja nigricollis and Echis ocellatus had their neuro-toxic and myotoxic activities inhibited by the methanolic extract from Parkia biglobosa bark; (vi) the methanolic extract from other Indian plants (Vitex negundo and Emblica officinalis) neutralized the hemorr-hagic, coagulant and inflammatory effects of Vipera russellii and Naja kaouthia venoms; (vii) preliminary studies showed the inhibitory activity of the extract from Marsypianthes chamaedrys against the clotting activity of B. jararaca venom; (ix) Musa paradisiaca, an important food all over the world, also showed antiophidian properties against PLA2, myotoxic, hemorrhagic and lethal activities of Crotalidae venoms; (x) inhibitory effects of the compound arturmerone isolated from Curcuma longa, able to neutralize the hemorrhagic activity of B. jararaca venom and the lethal effect of C. d. terrificus venom was also showed [88, 103-111]

da Silva et al. [99] isolated edunol, a pterocarpan from Harpalyce brasiliana, with antimyotoxic and antiproteolytic activities against B. jararacussu venom, besides expressive PLA2s inhibitory properties. Recently, rosmarinic acid, isolated from Cordia verbenacea, known as “erva baleeira” (Waler herb), was shown to neutralize the PLA2s BthTX-I and II from B. jararacussu snake venom [6, 112] (Fig. (2)). Also, antiophidian properties of the aqueous extracts from Pentaclethra macroloba and Bauhinia forficata, inhibiting the toxic and enzymatic activities of crude venoms and isolated toxins, were described [113, 114].

Angulo and Lomonte [115] showed the antivenom activity of fucoidan, a polyssacharide sulphate from the brown marine alga Fucus vesiculosus. Several myotoxic PLA2s from B. asper, Cerrophidion godman, Atropoides nummifer and Bothriechis schlegelii venoms were assayed and inhibition of the cytolytic activity and of tissue damage was observed when fucoidan was administered.

Núnëz et al. [116] isolated 4-neolidylcatechol from extracts of Piper umbelatum and Piper peltatum, which effectively neutralized toxic effects of Bothrops myotoxins, inhibiting their enzymatic, myotoxic and edema inducing activities. Results from mass spectrometry suggested a covalent modification of the myotoxin by 4-neolidyl-catechol.

Aristolochic acid (9-hydroxy-8-methoxy-6-nitrophe-nanthro (3,4-d)-1,3-dioxole-5-carboxylic acid) and the alkaloid from Aristolochia sp. interact with some venom toxins and enzymes. Chandra et al. [117] showed, by crys-tallography, the aristolochic acid-alkaloid-Vipera russelli PLA2s complex. Some natural pterocarpans have been isolated from plants, showing inhibitory activity against venom PLA2s. da Silva et al. [99] produced synthetic pterocarpans able to inhibit the myotoxic and PLA2 activities of B. jararacussu.

Recently, Soares et al. [82] listed 152 plant species used in folk medicine in Brazil as antivenoms (Fig. (3)). From those, only 18 (12%) have scientific validity, showing that only a small fraction of the reported antiophidian plants were

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scientifically documented. Further investigations on the isolation and characterization of these bioactive inhibitors of snake venom components are therefore needed before its use as therapeutic agents [83]. However, it is important to note that the presence of PLA2 inhibitory proteins and other compounds in plants opens the possibility to search for natural inhibitors snake venoms for therapeutic pur-poses, which could have a significant social and economic impact on developing countries. Some typical Brazilian plant species well established as antiophidian are represented in Fig. (3).

3. ARTIFICIAL INHIBITORS OF svPLA2s

Most of the described inhibitors are derived from plants or marine animals, used as such or representing molecular models for development of synthetic analogues. An example is manoalide (MLD), a natural marine product, and its synthetic analogue manoalogue (MLG), both able to cause an irreversible inhibition of snake venom PLA2s [14, 118]. Comparative studies on the action of both agents on snake and bee venom PLA2s have been reported [15], showing similar kinetic effects in all cases. The most significant difference was found in the inactivation time among PLA2s, being shorter for bee PLA2s when compared with snake PLA2s. In the presence of Ca2+, a faster inactivation occurs with rattlesnake PLA2s when compared with bee or other snake PLA2s.

Other experiments occurred with type II enzymes from Crotalus durissus and Crotalus atrox, which have different amino acid sequences at the amino terminal as well as different 70–74 region. These experiments demonstrated a new method for specific inhibition of phospholipase A2 by synthetic peptides derived from the primary sequence [119].

Other types of PLA2 inhibitors as BMS-229724 (4-[4-[2-[2-[bis(4-chlorophenyl)methoxy]ethyl-sulfonyl]ethoxy] phenyl]-1,1,1-trifluoro-2-butanone) are now known. BMS-229724 represents a novel inhibitor of cPLA2, promoting a potent inhibition of the enzyme translated into anti-inflammatory activity [120].

Chacur et al. [121] showed that Asp49 PLA2s from Bothrops asper venom cause a considerable hyperalgesic effect. After chemical modification by BPB, its enzymatic activity was diminished to 1% and the hyperalgesic effect was considerably reduced.

Basappa et al. [122] described the synthesized imidazolyl substituted d2-isoxazolines. The crystal structure of the compound 2-butyl-5-chloro-3H-imidazolyl-4-carbalde-hyde-oxime 2, an intermediate for the chemical synthesis of isoxa-zolines, was also reported. These compounds exerted a significant inhibitory effect against group II PLA2s from snake venoms.

The neuromuscular blockade caused by B. jararacussu venom and BthTX-I was also neutralized by the antiserum specific to Crotalus d. colillineatus(cdc)-crotoxin and cdc-PLA2 at a venom/ toxin:antiserum ratio of 1:10 for both. For example, the commercial equine antivenom raised against C. d. terrificus venom was effective in preventing the typical neuromuscular blockade caused by B. jararacussu venom and BthTX-I. These results showed that antiserum produced against PLA2, the major toxin in C. durissus cascavella venom, efficiently neutralized the neurotoxicity of C. d. terrificus and B. jararacussu venoms and their PLA2 toxins [123].

PLA2s can undergo chemical modifications at different amino acid residues according to the used reagent. His48 is

Fig. (2). Molecular bonding between a typical Lys49-PLA2 (BthTX-I) and rosmarinic acid, a phospholipase inhibitor from C. verbenacea. (A) Molecular interaction between the monomer BthTX-I (Lys49 PLA2) with rosmarinic acid (Cv-RA). (B) In detail, catalytic amino acid side chains probably involved on Cv-RA interaction and its bonding distances.

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alkylated by BPB or methylated by methyl-p-nitroben-zenesulfonate. Lys residues can be carbamilated by KCN, guanidinated by o-methylisourea, trinitrophenylated by TNBS or acethylated by acetic anhydride. Tyr can be sulphonated by NBSF, while Trp is sulphonated by NPSC or alkylated by 2-hydroxy-5-nitrobenzyl bromide. Met can be oxidized by chloramide or carboxymethylated by iodoacetic acid. CNBr is responsible for cleavage of PLA2s releasing the N-terminal octapeptide as previously described for piratoxins I and II from Bothrops pirajai [61].

The enzyme BthA-I-PLA2, from Bothrops jararacussu venom, was treated with BPB (Fig. (4)), undergoing confor-mational changes in its terciary and quaternary structures and becoming more stable [61]. Magro et al. [124] suggested that structural modifications caused by BPB can indirectly inhibit the anticoagulant effect and other pharmacological activities, such as hypotension and platelet aggregation, through oligomeric changes caused in regions or simple residues,

which are fundamental for these activities, as the C-terminal, His48 and other regions.

Previous studies on Micropechis ikaheka snake venom have indicated the presence of neurotoxins and myotoxins. The in vitro myotoxic effects of the venom and the efficacy of a polyvalent antivenom in neutralizing these effects were studied. Also, the venom PLA2 activity was inhibited by

alkylation with BPB [125].

Benzoylation of phenyl methanone 2a–g to benzoyl phenyl benzoates 4a–g, a benzophenone analogue, was achieved in good yield. All the newly synthesized com-pounds were evaluated for their PLA2 and hyaluronidase enzyme inhibitory activity and for their structure–activity relationship respecting different groups. The in vitro PLA2 inhibitory activity of benzoyl phenyl benzoates was also illustrated [126].

Georgieva et al. [127] studied vipoxin, a PLA2 isolated from Vipera ammodytes meridionalis venom, composed by

Fig. (3). Typical Brazilian medicinal plants used as antivenom on snake bite. Leaves of Casearia sylvestris (A) and Mikania glomerata (B). Tissue damage caused by crude venom of Bothrops moojeni (C) and Lys49 MjTX-II from the same venom (D). Note in (C and D): extensive muscle necrosis (N), hemorrhage (H)*, edema and inflammatory infiltrate (I). (E): Neutralizing tissue damage effects by application of Casearia and Mikania plant extracts. (*except for D).

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two subunits non covalently linked. One subunit is basic and highly toxic (Asp49-PLA2), while the other is acidic, non-toxic and with no catalytic activity, probably a chaperone molecule (Gln48-PLA2). Its enzymatic activity was almost abolished by the synthetic inhibitor elaidoylamide (the amide of trans-9-octadecenoic acid). For the first time, crystalli-zation and preliminary studies on X-ray diffraction of a subunit of PLA2 complexed with a synthetic inhibitor, elaidoyl-amide, was carried out. The subunit used for this study was the toxic part of the heterodimer (Asp49-PLA2).

4. PLA2S INHIBITORS ISOLATED FROM SNAKES AND OTHER ANIMALS

For a long time it is known that snakes and some mammals are resistant to snake venom envenomations [128, 129]. Venomous and non-venomous snakes display PLA2 inhibitory proteins, named PLIs, in their blood plasma, in order to protect themselves against toxins from their own venom, which, eventually, could reach the circulatory system [33, 34]. The presence of these inhibitors has been associated with the tolerance of snakes to the damaging effects of PLA2s in their venoms, although additional physiologic roles may exist as well [28, 30, 32, 129].

Nowadays, description of several inhibitors in the blood of different animals can be found, including hemorrhage, neurotoxicity, myotoxicity and PLA2 inhibitors, among others [31, 130, 131]. All PLIs isolated from snake blood plasma or serum are acidic oligomeric proteins with molecular mass from 18 to 75 kDa, formed by 3-6 subunits of 20-50 kDa, linked by non-covalent bonds [35].

Many studies have been carried out in search for these natural snake PLA2 inhibitors, which may be isolated from mammal plasmas, as that from Didelphis albiventris [22, 132] and Didelphis marsupialis [21, 133]. Also, from oviparous animals as that in the aqueous extract from Pavo cristatus plumes, inhibitor of Naja Naja and Vipera russelii venom PLA2s [134], and from snake plasmas [35, 135].

DM64, an antimyotoxic protein isolated from Didelphis marsupialis serum, belongs to the super-immunoglobulin gene family and is homologous to human 1B-glycoprotein and DM43, a metalloprotease inhibitor from the same organism. Studies on molecular cloning have also been per-formed with Lachesis muta muta and Bothrops jararacussu [136, 137]. Independently of their antitoxic activity, these blood inhibitors are oligomeric globular glycoproteins which migrate electrophoretically in the -globulin fraction of blood plasma. They are acidic (pI = 4-5), not antibodies and are present in the blood of these animals before exposition to venom components [28, 30, 129].

These natural neutralizing factors are classified based on their structures [138] and sequential homology with other proteins [139-141]. According to Lizano et al. [35], these PLA2 protein inhibitors have been isolated and characterized from Agkistrodon, Trimeresurus, Bothrops, Crotalus, Naja, Laticaudata and Elaphe (Table (1)).

PLA2 inhibitors are classified into types , and , according to structural features, based on common motifs found in other proteins with diverse physiological properties [142]. Type inhibitors (PLI s), isolated from Crotalinae

Fig. (4). Acidic snake PLA2 bound to the chemical inhibitor BPB. (A): Acid Asp49-PLA2 monomer from B. jararacussu covalent bound to BPB. (B): In detail, electron density map in the catalytic site region showing the His48 residue bound to BPB.

8 Current Topics in Medicinal Chemistry, 2007, Vol. 7, No. ? Soares et al.

snakes as Trimeresurus flavoviridis [143], Agkistrodon blomhoffii [23], Bothrops asper [131], Bothrops moojeni [27] are acidic glycoproteins constituted by several subunits, with sequential similarity with the carbohydrate recognizing domain (CRD) of type-C lectins [139].

Type inhibitors are typically globular proteins composed of two subunits of 20-25 kDa with 147 amino acid residues and a constitutive N-glycosylation [35]. A PLI isolated from A. blomhoffii inhibited acidic PLA2s from group II, while B. asper BaMIP and B. moojeni BmjMIP inhibited basic PLA2s from the same group. The inhibitor BaMIP shows the structural domain (CRD) similar to that of Ca2+ dependent lectins (type-C), able to bind to carbohydrate residues containing mannose. In macrophage mannose receptors, the domain CRD may play an important role in endocytosis of glycoproteins and in the recognition of mannose residues on the surface of microorganisms before

phagocytosis [144]. Type CRD domains are also structural elements of type M PLA2 receptors, which bind to pan-creatic PLA2s from group I and those secreted from group II. Similarity among inhibitors and snake or mammal PLA2 receptors, respectively, might explain, in part, the physiolo-gical function as a soluble antitoxin present in snake bloods [48, 145].

Type inhibitors (PLI ) show multiple repetitions of successive Leu rich domains and also show 33% homology with human 2-glycoprotein, a Leu rich serum protein of unknown function [146]. This type of inhibitor has been studied in the plasma of Agkistrodon blomhoffi siniticus (Crotalinae) [141].

Finally, type inhibitors (PLI ) display several protein subunits showing similarity with mammal proteins as members of the superfamily of tridactile neurotoxins [26].

Table 1. List of Inhibitors Isolated from Snake Plasma

Origin/Species Inhibitor Mr N° subunits Reference

Trimeresurus flavoviridis PLI 75,000 3 [138]

Agkistrodon b. siniticus PLI 75,000 3 [23]

Trimeresurus flavoviridis PLI 100,000 4 [143]

Bothrops moojeni BmjMIP(PLI ) 120,000 5.6 [27]

Bothrops asper BaMIP (PLI ) 120,000 5 [131]

Agkistrodon b. siniticus PLI 160,000 3 [146]

Elaphe quadrivirgata Eq PLI 130,000 2 [1II51]

Agkistrodon b. siniticus PLI 100,000 4-5 [141]

CNF (PLI ) 140,000 6-8 [24] Crotalus d. terrificus

CICS (PLI ) 130,000 6 [25]

Naja n. kaouthia PLI 90,000 2 [152, 147]

Laticauda semifasciata LsPLI 100,000 2 [26]

Elaphe quadrivirgata Eq PLI nd nd [149]

Cerrophidion godmani CgMIP-I(PLI )

CgMIP-II

110,000

180,000

2

2

[153]

Python reticulates PIP 140,000 nd [154]

Notechis ater NAI 110,000 2 [155]

Notechis ater serventyi NAsI nd nd [156]

Notechis scutatus NSI nd nd [157]

Oxyuranus scutellatus OSI nd nd [156]

Oxyuranus microlepidotus OMI nd nd [156]

Pseudonaja textilis PTI nd nd [156]

Naja Naja NND-IV-PLA2 13,262 nd [135]

Crotalus durissus terrificus CNF 23,600 nd [148]

Snake Venom PLA2s Inhibitors Current Topics in Medicinal Chemistry, 2007, Vol. 7, No. ? 9

These inhibitor types may occur in a single snake species, but may also show different distributions between Elapidae and Viperidae families. Type inhibitors have intramolecular repetitions of Cys rich domains in common, which are structurally related with those shown by urokinase type receptor of plasminogen activator (u-PAR) [26, 147]. These PLI proteins were studied in plasma from Trimeresurus flavoviridis (Viperidae) [143], and Agkistrodon blomhoffi siniticus (Viperidae) [141], in serum from Crotalus durissus terrifus (Viperidae) [24, 25, 148], from Naja naja kaouthia (Elapidae) [152], plasma from Laticauda semisfaciata (Hidrophiidae) [26] and serum from Elaphe quadrivirgata (non venomous snake) [149].

More recently, following a division into two subclasses of type inhibitors according to their heteromeric or homomeric characteristics [35], a potent neurotoxin from Crotalus durissus terrificus venom (crotoxin), present in its blood, was classified as homomeric due to its structural characteristics [150].

5. CONCLUDING REMARKS

Continuous efforts toward the study of new natural antivenoms are relevant for the understanding of snake venom biology as well as the physiopatology involving snake bite effects in different organisms. Regarding PLA2s,

these studies could serve as a valuable input on compre-hension of its mode of action, classification and their structure-function relationship, so extending the protein science field.

Since snake bite envenomations are a public health con-cern as a result of their high mortality and chronic morbidity, such type of investigation could have a biotechnological application with social and economic impact on developing regions around the world. It could contribute to development of molecular models for new drugs probably at a lower price, with lower side effects and easily distributed to communities far from medical access. Furthermore, a better input could be raised in treatment of envenomations of human and domestic animals, since for the latter the traditional serum therapy is not completely suitable. Therefore, natural antivenoms research could represent an additional aid in the traditional approach with significant impact on developing nations.

Popular culture is usually wise and can help to guide scientific studies. In addition, biotechnological application of these inhibitors, as helpful alternative remedies and for supplemental treatment to serum therapy, as well as important models for synthesis of new therapeutic drugs of medical interest, need to be better oriented and scientifically explored.

ACKNOWLEDGEMENTS

The authors express their gratitude to Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and CAPES for financial support. We thank Dr. Angelo J. Magro (UNESP/ Botucatu, Brasil) for their relevant contribution in the molecular simulation figures and discussion of this article.

ABBREVIATIONS

AA = Aristolochic acid from Aristolochia sp

ABC = Antibothropic complex from Didelphis sp

BPB = p-bromophenacyl bromide

Bt-CD = 7 -hydroxy-3, 13-clerodadiene-16, 15:18, 19-diolide from Baccharis trimera.

BthA-I-PLA2 = Acidic phospholipase A2 from Bothrops jararacussu venom

BthTX-I = Myotoxin I Lys49 phospholipase A2-like from Bothrops jararacussu venom

BthTX-II = Myotoxin II Asp49 phospholipase A2 from Bothrops jararacussu venom

CB = Crotoxin B, a Asp49 phospholipase A2 from Crotalus durissus terrificus venom

CK = Creatine kinase

Cv-RA = Cordia verbenaceae rosmarinic acid

EDTA = Ethylenediaminetetraacetic acid

EGTA = Ethylene glycol-bis N, N, N’, N’- tetraacetic acid

MMV = 12-Methoxy-4-methylvoachalotine from Tabernaemontana chatarinensis

MTx = Myotoxins

NBSF = 2-Nitrobenzenesulphonyl fluoride

NPSC = o-Nitrophenylsulphenyl chloride

svPLA2s = Snake venom phospholipases A2

PLA2s = Phospholipases A2

PLIs = Phospholipase A2 inhibitors

PMSF = Alpha-toluenesulfonyl fluoride

WSG = Whitania somnifera glycoprotein inhibitor.

REFERENCES

[1] Chippaux J. P. Snake-bites: appraisal of the global situation. Bull

World Health Organ 1998, 76, 515-524. [2] Chippaux J. P. The development and use of immunotherapy in

Africa. Toxicon 1998, 36, 1503-1506. [3] Vital Brazil, O. Peçonhas. In: Farmacodinâmica; Corbett, C. E. Ed.;

Editora Guanabara-Koogan: Rio de Janeiro, Brazil, 1982. [4] Gutiérrez, J. M.; Romero, M.; Nunez, J.; Chaves, F.; Borkow, G.;

Ovadia, M. Skeletal muscle necrosis and regeneration after injection of BaH1, a hemorrhagic metalloproteinase isolated from the venom of the snake Bothrops asper (Terciopelo). Exp. Mol.

Pathol. 1995, 62, 28-41. [5] Rucavado, A.; Gutiérrez, J. M. Snake venom metalloproteinases:

their role in the pathogenesis of local tissue damage. Biochimie 2000, 82, 841-850.

[6] Ticli, T. K. Propriedades antiofídicas do ácido rosmarínico isolado do extrato vegetal de Cordia verbenacea sobre as ações tóxicas e farmacológicas do veneno de Bothrops jararacussu e suas fosfolipases A2 miotóxicas. Master Thesis 2003, USP, Ribeirão Preto, 1-100.

[7] Borges, M. H.; Soares, A. M.; Rodrigues, V. M.; Andrião-Escarso, S. H.; Diniz, H.; Hamaguchi, A.; Quintero, A.; Lizano, S.; Gutiérrez, J. M.; Giglio, J. R.; Homsi-Brandeburgo, M. I. Effects of aqueous extract of Casearia sylvestris (Fracourtiaceae) on actions of snake and bee venoms and on activity of phospholipases A2. Comp. Biochem. Physiol. 2000, 127, 21-30.

10 Current Topics in Medicinal Chemistry, 2007, Vol. 7, No. ? Soares et al.

[8] Borges, M. H.; Soares, A. M.; Rodrigues, V. M.; Oliveira, F.; Fransheschi, A. M.; Rucavado, A.; Giglio, J. R.; Homsi-Brandeburgo, M. I. Neutralization of proteases from Bothrops snake venoms by the aqueous extract from Casearia sylvestris (Flacourtiaceae). Toxicon 2001, 39, 1863-1869.

[9] Stabeli, R.G.; Magalhaes, L. M.; Selistre-de-Araujo, H. S.; Oliveira, E. B. Antibodies to a fragment of the Bothrops moojeni l-amino acid oxidase cross-react with snake venom components unrelated to the parent protein. Toxicon 2005, 46, 308-317.

[10] Stabeli, R.G.; Amui, S. F.; Sant'ana, C. D.; Pires, M.G.; Nomizo, A.; Monteiro, M.C.; Romão, P. R.; Guerra-Sa, R.; Vieira, C. A.; Giglio, J. R.; Fontes, M. R.; Soares, A. M. Bothrops moojeni myotoxin-II, a Lys49-phospholipase A2 homologue: an example of function versatility of snake venom proteins. Comp. Biochem.

Physiol. 2006, 142, 371-381. [11] Lomonte, B.; Tarkowishi, A.; Bagge, U.; Hanson, L. A. Neutrali-

zation of the cytolytic and myotoxic activities of phospholipase A2

from Bothrops asper snake venom by glycosaminoglycans of the heparin/heparan sulfate family. Biochem. Pharmacol. 1994, 47,

1509-1518. [12] Soares, A. M.; Guerra-Sá, R.; Borja-Oliveira, C. R.; Rodrigues, V.

M.; Rodrigues-Simioni, L.; Rodrigues, V.; Fontes, M. R. M.; Lomonte, B.; Gutiérrez, J. M.; Giglio, J. R. Structural and functional characterization of BnSP-7, a Lys49 myotoxic phospholipase A2 homologue from Bothrops neuwiedi pauloensis venom. Arch.

Biochem. Biophys. 2000, 378, 201-209. [13] Calderon, L.; Lomonte, B. Immunochemical characterization and

role in toxic activities of region 115-129 of myotoxin II, a Lys49 phospholipase A2 from Bothrops asper snake venom. Arch.

Biochem. Biophys. 1998, 358, 343-350. [14] Lombardo, D.; Dennis, E. A. Cobra venom phospholipase A2

inhibition by manoalide. A novel type of phospholipase inhibitor. J.

Biol. Chem. 1985, 260, 7234-7240. [15] Reynolds, L. J.; Mihelich, E. D.; Dennis, E. A. Inhibition of venom

phospholipases A2 by manoalide and manoalogue. Stoichiometry of incorporation. J. Biol. Chem. 1991, 266, 16512-16517.

[16] Melo, P. A.; Nascimento, M. C.; Mors, W. B.; Suarez-Kurtz, G. Inhibition of the myotoxic and hemorrhagic activities of crotalid venoms by Eclipa prostrata extracts and constituents. Toxicon 1994, 32, 595-603.

[17] Batina, M. F. C.; Cintra, A. C. O.; Veronese, E. L. G.; Lavrador, M. A. E.; Giglio, J. R.; Pereira, P. S.; Dias, D. A.; França, S. C.; Sampaio, S. V. Inhibition of the lethal and myotoxic activities of Crotalus durissus terríficus venom by Tabernaemontana catharinensis: Identification of one of the active components. Planta Medica 2000, 66, 424-428.

[18] Biondo, R.; Pereira, A. M. S.; Marcussi, S.; Pereira, P. S.; França, S. C.; Soares, A. M. Inhibition of enzymatic and pharmacological activities of some snake venoms and toxins by Mandevilla velutina (Apocynaceae) aqueous extract. Biochimie 2003, 85, 1017-1025.

[19] Mishra, L. C.; Singh, B. B.; Dagenais, S. Scientific basis for the therapeutic use of Withania somnifera (ashwagandha): a review. Altern. Med. Rev. 2000, 5, 334-346.

[20] Deepa, M.; Gowda, T. V. Purification and characterization of a glicoprotein inhibitor of toxic phospholipase from Withania somnifera. Arch. Biochem. Biophys. 2002, 408, 42-50.

[21] Neves-Ferreira, A. G.; Cardinale, N.; Rocha, S. L.; Perales, J.; Domont, G. B. Isolation and characterization of DM40 and DM43, two snake venom metalloproteinase inhibitors from Didelphis marsupials serum. Biochim. Biophys. Acta 2000, 1474, 309-320.

[22] Trento, E. P.; Garcia, O. S.; Rucavado, A.; França, S. C.; Batalini, C.; Arantes, E. C.; Giglio, J. R.; Soares, A. M. Inhibitory properties of the anti-bothropic complex from Didelphis albiventris serum on toxic and pharmacological actions of metalloproteases and myotoxins from Bothrops asper venom. Biochem. Pharmacol. 2001, 62, 1521-1529.

[23] Ohkura, N.; Inoue, S.; Ikeda, K.; Hayashi, K. Isolation and amino acid sequence of a phospholipase A2 inhibitor from the blood plasma of Agkistrodon blomhoffii siniticus. J. Biochem. 1993, 113,

413-419. [24] Fortes-Dias, C. L.; Diniz, C. R.; Liu, T. H.; Lin, Y. A

phospholipase A2 inhibitor from the plasma of the South American rattlesnake (Crotalus durissus terrificus). J. Biol. Chem. 1994, 269,

15646-15651.

[25] Perales, J.; Villela, C. G.; Domont, G. B.; Choumet, V.; Saliou, B.; Moussatché, H.; Bon, C.; Faure, G. Molecular structure and mechanism of action of the crotoxin inhibitor from Crotalus durisssus terrificus serum. Eur. J. Biochem. 1995, 227, 19-26.

[26] Ohkura, N.; Kitahara, Y.; Inoue, S.; Ikeda, K.; Hayashi, K. Isolation and amino acid sequence of a phospholipase A2 inhibitors from the blood plasma of the sea krait, Laticauda semifasciata. J.

Biochem. 1999, 125, 375-382. [27] Soares, A. M.; Marcussi, S.; Stábeli, R. G.; França, S. C.; Giglio, J.

R.; Ward, R. J.; Arantes, E. C. Structural and functional analysis of BmjMIP, a phospholipase A2 myotoxin inhibitor protein from Bothrops moojeni snake plasma. Biochem. Biophys. 2003, 302,

193-200. [28] Domont, G. B.; Perales, J.; Moussatche, H. Natural anti-snake

venom proteins. Toxicon 1991, 29, 1183-1194. [29] Bjarnason, J. B.; Fox, J. W. Hemorrhagic metalloproteinases from

snake venoms. Pharmacol. Ther. 1994, 62, 325-372. [30] Thwin, M. M.; Gopalakrishnakone, P. Snake envenomation and

protective natural endogenous proteins: a mini review of the recent developments (1991-1997). Toxicon 1998, 36, 1471-1482.

[31] Perez, J. C.; Sanchez, E. E. Natural protease inhibitors to hemorrhagins in snake venoms and their potential use in medicine. Toxicon 1999, 37, 703-728.

[32] Perales, J.; Domont, G. B. Are inhibitors of metalloproteinases, phospholipases A2 and myotoxins members of the innate immune system. In: Perspectives in Molecular toxinology, Ménez, A. Ed. Wiley: Chichester pp. 435-456, 2002.

[33] Faure, G. Natural inhibitors of toxic phospholipases A2. Biochimie 2000, 82, 833-840.

[34] Fortes-Dias, C. L. Endogenous inhibitors of snake venom phospholipase A2 in the blood plasma of snakes. Toxicon 2002, 40,

481-484. [35] Lizano, S.; Domont, G.B.; Perales, J. Natural phospholipase A2

myotoxin inhibitor proteins from snakes mammals and plants. Toxicon 2003, 42, 963-977.

[36] Dennis, E. A. Phospholipase A2 in eicosanoid generation. Am. J. Respir. Crit. Care. Med. 2000,161, 32-35.

[37] Soares, A. M.; Andrião-Escarso, S. H.; Angulo, Y.; Lomonte, B.; Gutiérrez, J. M.; Marangoni, S.; Toyama, M. H.; Arni, R. K.; Giglio, J. R. Structural and functional characterization of myotoxin I, a Lys49 phospholipase A2 homologue from Bothrops moojeni (Caissaca) snake venom. Arch. Biochem. Biophys. 2000, 373, 7-15.

[38] Gutiérrez, J. M. Understanding snake venoms: 50 years of research in Latin America. Rev. Biol. Trop. 2002, 50, 377-394.

[39] Yu, B. Z.; Berg, O. G.; Jain, M. K. The divalent cation is obrigatory for the binding of ligands to the catalytic site of secreted PLA2. Biochemistry 1993, 32, 6484-6492.

[40] Gutiérrez, J. M.; Lomonte, B. Phospholipase A2 myotoxins from Bothrops snake venoms. Toxicon 1995, 33, 1405-1424.

[41] Ownby, C. L. Structure, function and biophysical aspects of the myotoxins from snake venoms. J. Toxicol.-Toxins Rev. 1998, 17,

213-238. [42] Chang, C. C.; Su, M. J.; Lee, J. D.; Eaker, D. Effects of Sr2+ and

Mg2+ on the phospholipase A and the presynaptic neuromuscular blocking actions of beta-bungarotoxin, crotoxin and taipoxin. Arch.

Pharmacol. 1977, 299, 155-161. [43] Bon, C.; Changeux, J. P.; Jeng, T. W.; Fraenkel-Conrat, H.

Postsynaptic effects of crotoxin and of its isolated subunits. Eur J

Biochem. 1979, 99, 471-481. [44] Fletcher, J. E.; Rapuano, B. E.; Condrea, E.; Yang, C. C.;

Rosenberg, P. Relationship between catalysis and toxicological properties of three phospholipases A2 from elapid snake venoms. Toxicol. Appl. Pharmacol. 1981, 59, 375-388.

[45] Fenard, D.; Lambeau, G.; Valentin, E.; Lefebvre, J. C.; Lazdunski, M.; Doglio, A. Secreted phospholipase A2, a new class of HIV inhibitors that block virus entry into host cells. J. Clin. Invest. 1999, 104, 611-618.

[46] Zieler, H.; Keister, D. B.; Dvorak, J. A.; Ribeiro, J. M. A snake venom phospholipase A2 blocks malaria parasite development in the mosquito midgut by inhibiting ookinete association with the midgut surface. J. Exp. Biol. 2001, 204, 4157-4167.

[47] Balsinde, J.; Shinohara, H.; Lefkowitz, L. J.; Johnson, C. A.; Balboa, M. A.; Dennis, E. A. Group V phospholipase A2-dependent

Snake Venom PLA2s Inhibitors Current Topics in Medicinal Chemistry, 2007, Vol. 7, No. ? 11

induction of cyclooxygenase-2 in macrophages. J. Biol. Chem. 1999, 274, 25967-25970.

[48] Lambeau, G.; Lazdunski, M. Receptors for a growing family of secreted phospholipases A2. Trends. Pharmacol. Sci. 1999, 20,

162-170. [49] Six, D. A.; Dennis, E. A. The expanding superfamily of

phospholipase A2 enzymes: classification and characterization. Biochim. Biophys. Acta 2000, 1488, 1-19.

[50] Ownby, C. L.; Selistre-de-Araújo, H. S.; White, S. P.; Fletcher, J. E. Lysine 49 phospholipase A2 proteins. Toxicon 1999, 37, 411-445.

[51] Gutiérrez, J. M.; Ownby, C. L.; Odell, G. V. Isolation of a myotoxin from Bothrops asper venom: partial characterization and action on skeletal muscle. Toxicon 1984, 22, 115-28.

[52] Gutiérrez, J. M.; Chaves, F.; Gene, J. A.; Lomonte, B.; Camacho, Z.; Schosinsky, K. Myonecrosis induced in mice by a basic myotoxin isolated from the venom of the snake Bothrops nummifer (jumping viper) from Costa Rica. Toxicon 1989, 27, 735-745.

[53] Duncan, C. J. Role of intracellular calcium in promoting muscle damage: a strategy for controlling the dystrophic condition. Experientia 1978, 34, 1531-1535.

[54] Gopalakrishnakone, P.; Dempster, D. W.; Hawgood, B. J.; Elder, H. Y. Cellular and mitochondrial changes induced in the structure of murine skeletal muscle by crotoxin, a neurotoxic phospholipase A2 complex. Toxicon 1984, 22, 85-98.

[55] Arni, R. K., Ward, R. J. Phospholipase A2 a structural review. Toxicon 1996, 34, 827-841.

[56] Murakami, M.; Kudo, I. Phospholipase A2. J. Biochem. 2002, 131,

285-292. [57] Fleer, E. A.; Verheij, H. M.; De Haas, G. H. Modification of

carboxylate groups in bovine pancreatic phospholipase A2. Identification of aspartate-49 as Ca2+-binding ligand. Eur. J.

Biochem. 1981, 113, 283-288. [58] Fukagawa, T.; Nose, T.; Shimohigashi, Y.; Ogawa, T.; Oda, N.;

Nakashima, K.; Chang, C. C.; Ohno, M. Purification, sequencing and characterization of single amino acid-substituted phospholipase A2 isozymes from Trimeresurus gramineus (green habu snake) venom. Toxicon 1993, 31, 957-967.

[59] Verheij, H. M.; Volwerk, J. J.; Jansen, E. H.; Puyk, W. C.; Dijkstra, B. W.; Drenth, J.; De Haas, G. H. Methylation of histidine-48 in pancreatic phospholipase A2. Role of histidine and calcium ion in the catalytic mechanism. Biochemistry 1980, 19, 743-750.

[60] Rodrigues, V. M.; Soares, A. M.; Mancin, A. C.; Fontes, M. R. M.; Homsi-Brandeburgo, M. I.; Giglio, J. R. Geographic variations in the composition of myotoxins from Bothrops neuwiedi snake venoms: biochemical characterization and biological activity. Comp. Biochem. Physiol. 1998, 121, 215-222.

[61] Soares, A. M.; Andrião-Escarso, S. H.; Rodrigues-Simioni, L.; Arni, R. K.; Bortoleto, R. K.; Ward, R. J.; Gutiérrez, J. M.; Giglio, J. R. Dissociation of enzymatic and pharmacological properties of piratoxins-I and -III, two myotoxic phospholipases A2 from Bothrops pirajai snake venom. Arch. Biochem. Biophys. 2001, 387, 188-196.

[62] Soares, A. M.; Giglio, J. R. Chemical modifications of phospholipases A2 from snake venoms: effects on catalytic and pharmacological properties. Toxicon 2003, 42, 855-868.

[63] Homsi-Brandeburgo, M. I.; Queiroz, L. S.; Santo-Neto, H.; Rodrigues-Simioni, L.; Giglio, J. R. Fractionation of Bothrops jararacussu snake venom: partial chemical characterization and biological activity of bothropstoxin. Toxicon 1988, 26, 615-627.

[64] Soares, A. M.; Rodrigues, V. M.; Homsi-Brandeburgo, M. I.; Toyama, M. H.; Lombardi, F. R.; Arni, R. K.; Giglio, J. R. A rapid procedure for the isolation of the Lys-49 myotoxin II from Bothrops moojeni (caissaca) venom: biochemical characterization, crystallization, myotoxic and edematogenic activity. Toxicon 1998, 36, 503-514.

[65] Angulo, Y.; Gutierrez, J.M.; Soares, A.M.; Cho, W.; Lomonte, B. Myotoxic and cytolytic activities of dimeric Lys49 phospholipase A2 homologues are reduced, but not abolished, by a pH-induced dissociation. Toxicon 2005, 46, 291-296.

[66] Andrião-Escarso, S. H.; Soares, A. M.; Rodrigues, V. M.; Angulo, Y.; Diaz, C.; Lomonte, B.; Gutiérrez, J. M.; Giglio, J. R. Myotoxic phospholipases A2 in Bothrops snake venom: effect of chemical modifications on the enzymatic and pharmacological properties of

bothropstoxins from Bothrops jararacussu. Biochimie 2000, 82,

755-763. [67] Fuly, A. L.; Machado, O. L.; Alves, E. W.; Carlini, C. R.

Mechanism of inhibitory action on platelet activation of a phospholipase A2 isolate from Lachesis muta (bushmaster) snake venom. Thromb. Haemost. 1997, 78, 1372-1380.

[68] Fuly, A. L.; Calil-Elias, S.; Zingali, R. B.; Guimaraes, J. A.; Melo, P. A. Myotoxic activity of an acidic phospholipase A2 isolated from Lachesis muta (Bushmaster) snake venom. Toxicon 2000, 38, 961-972.

[69] Fuly, A. L.; Calil-Elias, S.; Martinez, A. M.; Melo, P. A.; Guimaraes, J. A. Myotoxicity induced by an acidic Asp-49 phos-pholipase A2 isolated from Lachesis muta snake venom. Comparison with lysophosphatidylcholine. Int. J. Biochem. Cell.

Biol. 2003, 35, 1470-1478. [70] Ouyang, C.; Yeh, H. I.; Huang, T. F. A potent platelet aggregation

inhibitor purified from Agkistrodon halys (mamushi) snake venom. Toxicon 1983, 21, 797-804.

[71] Kamiguti, A. S.; Laing, G. D.; Lowe, G. M.; Zuzel, M.; Warrell, D. A.; Theakston, R. D. Biological properties of the venom of the Papuan black snake (Pseudechis papuanus): presence of a phospholipase A2 platelet inhibitor. Toxicon 1994, 32, 915-925.

[72] de Araujo, A. L.; Radvanyif,; Bon, C. Purification of an acidic phospholipase A2 from Bothrops lanceolatus (fer de lance) venom: molecular and enzymatic properties. Toxicon 1994, 32, 1069-1081.

[73] Daniele, J. J.; Bianco, I. D.; Fidelio, G. D. Kinetic and pharmacologic characterization of phospholipases A2 from Bothrops neuwiedii venom. Arch Biochem Biophys. 1995, 318, 65-70.

[74] Daniele, J. J.; Bianco, I. D.; Delgado, C.; Carrillo, D. B.; Fidelio, G. D. A new phospholipase A2 isoform isolated from Bothrops neuwiedii (Yarara chica) venom with novel kinetic and chromatographic properties. Toxicon 1997, 35, 1205-1215.

[75] Lu, Q. M.; Jin, Y.; Wei, J. F.; Li, D. S.; Zhu, S. W.; Wang, W. Y.; Xiong, Y. L. Characterization and cloning of a novel phospholipase A2 from the venom of Trimeresurus jerdonii snake. Toxicon 2002, 40, 1313-1319.

[76] Andrião-Escarso, S. H.; Soares, A. M.; Fontes, M. R. M.; Fuly, A. L.; Corrêa, F. M. A.; Rosa, J. C.; Greene, L. J.; Giglio, J. R. Structural and functional characterization of an acidic platelet aggregation inhibitor and hypotensive phospholipase A2 from Bothrops jararacussu snake venom. Biochem. Pharmacol. 2002, 64, 723-732.

[77] Denson, D. D.; Worrell, R. T.; Eaton, D. C. A possible role for phospholipase A2 in the action of general anesthetics. Am. J. Physiol. 1996, 270, 636-644.

[78] Winkler, J. D.; Marshall, L. A. Phospholipase A2 in arthritis. In: Phospholipase A2 basic and clinical aspects in inflammatory diseases. Uhl, W.; Nevalainen, T. J.; Buchler, M. W.; Eds. Karger Ed. pp. 214-224, 1997.

[79] Elsbach, P. Determinants of the anti-microbial action of 14-kDa phospholipase A2. In: Phospholipase A2 basic and clinical aspects in inflammatory diseases. Uhl, W.; Nevalainen, T. J.; Buchler, M. W.; Eds. Karger Ed. pp. 17-22, 1997.

[80] Santamaría, C.; Larios, S.; Angulo, Y.; Pizarro-Cerda, J.; Gorvel, J.-P.; Moreno. E.; Lomonte, B. Antimicrobial activity of myotoxic phospholipases A2 from crotalid snake venoms and synthetic peptide variants derived from their C-terminal region. Toxicon 2005, 45, 807-815.

[81] Mors, W. B.; Nascimento, M. C.; Pereira, B. M. R.; Pereira, N. A. Plant natural products active against snake bite: the molecular approach. Phytochemistry 2000, 55, 627-642.

[82] Soares, A. M.; Sestito, W. P.; Marcusssi, S.; Stabeli, R. G.; Andrião-Escarso, S. H.; Cunha, O. A. B.; Vieira, C. A.; Giglio, J. R. Alkylation of myotoxic phospholipases A2 in Bothrops moojeni venom: a promising approach to an enhanced antivenom production. Int. J. Biochem. Cell. Biol. 2004, 36, 258-270.

[83] Soares, A. M.; Marcussi, S.; Lourenço, M. V.; Januário, A. H.; Sampaio, S. V.; Giglio, J. R.; Lomonte, B.; Pereira, P. S. Medicinal plants with inhibitory properties against snake venoms. Curr. Med. Chem. 2005, 12, 2625-2641.

[84] Soares, A. M.; Januário, A. H.; Lourenço, M. V.; Pereira, A. M. S.; Pereira, P. S. Neutralizing effects of Brazilian plants against snake venoms. Drugs Future 2004, 29, 1105-1117.

12 Current Topics in Medicinal Chemistry, 2007, Vol. 7, No. ? Soares et al.

[85] Potts, B. C.; Faulkner, D. J.; Jacobs, R. S. Phospholipase A2 inhibitors from marine organisms. J. Nat. Prod. 1992, 55, 1701-1717.

[86] Middleton, E. J. Effect of plant flavonoids on immune and inflammatory cell function. Adv. Exp. Med. Biol. 1998, 439, 175-182.

[87] Melo, P. A.; Ownby, C. L. Ability of wedelolactone, heparin and p-bromophenacyl bromide to antagonize the myotoxic effects of two crotalide venoms and their PLA2 myotoxins. Toxicon 1999, 37,

199-215. [88] Batina, M. F. C.; Giglio, J. R.; Sampaio, S. V. Methodologic care

in the evaluation of the L.D.50 (i.m.) and of the neutralization of the lethal effects of Crotalus durissus terrificus venom by the plant Peschiera fuchsiaefolia (Apocynaceae). J. Venom. Animals Toxins 1997, 3, 23-31.

[89] Mors, W. B.; Nascimento, M. C.; Parente, J. P.; Da Silva, M. H.; Melo, P. A.; Suarez-Kurtz, G. Neutralization of lethal and myotoxic activities of South American rattlesnake venom by extracts and constituents of the plant Eclipta prostata (Asteraceae). Toxicon 1989, 27, 1003-1009.

[90] Pithayanukul, P.; Laovachirasuwan, S.; Bavovada, R.; Pakmanee, N.; Suttisri, R. Anti-venom potential of butanolic extract of Eclipta prostata against Malayan pit viper venom. J. Ethnopharmacol. 2003, 90, 347-352.

[91] Januário, A. H.; Santos, S. L.; Marcussi, S.; Mazzi, M. V.; Pietro, R. C. L.; Sato, D. N.; Sampaio, S. V.; França, S. C.; Soares, A. M. Neo-clerodane diterpenoid, a new metalloprotease snake venom inhibitor from Baccharis trimera (Asteraceae): anti-proteolytic and anti-hemorrhagic properties. Chem.-Biol. Interactions 2004, 150,

243-251. [92] Mors, W. B. Plants active against snake bite. Econ. Med. Plant Res.

1991, 5, 352-382. [93] Martz, W. Plant with a reputation against snakebite. Toxicon 1992,

30, 1131-1142. [94] Otero, R.; Núnez, V.; Jiménez, S. L.; Fonnegra, R.; Osorio, R. G.;

Garcia, M. E.; Diaz, A. Snakebites and ethnobotany in the nortwest region of Colombia. Part II: Neutralization of lethal and enzymatic effects of Bothrops atrox venom. J. Ethnopharmacol. 2000, 71,

505-511. [95] Neves, P. C. A.; Neves, M. C. A.; Cruz, A. B.; Sant’ana, A. E. G.;

Yunes, R. A.; Calixto, J. B. Differential effects of Mandevilla velutina compounds on paw oedema induced by phospholipase A2

and phospholipase C. Eur. J. Pharmacol. 1993, 243, 213-219. [96] Biondo, R.; Soares, A. M.; Bertoni, B. W.; França, S. C.; Pereira,

A. M. Direct organogenesis of Mandevilla illustris (Vell) Woodson and effects of its aqueous extract on the enzymatic and toxic activities of Crotalus durissus terrificus snake venom. Plant Cell

Rep. 2004, 22, 549-552. [97] Castro, O.; Gutiérrez, J. M.; Barrios, M.; Castro, I.; Romero, M.;

Umana, E. Neutralization of the hemorrhagic effect induced by Bothrops asper (Serpentes: Viperidae) venom with tropical plant extracts. Rev. Biol. Trop. 1999, 47, 605-616.

[98] Hung, Y. C.; Sava, V.; Hong, M. Y.; Huang, G. S. Inhibitory effects on phospholipase A2 and antivenin activity of melanin extracted from Thea sinensis Linn. Lif Sci. 2004, 74, 2037-2047.

[99] da Silva, A. J.; Coelho, A. L.; Simas, A. B.; Moraes, R. A.; Pinheiro, D. A.; Fernandes, F. F.; Arruda, E. Z.; Costa, P. R.; Melo, P. A. Synthesis and pharmacological evaluation of prenylated and benzylated pterocarpans against snake venom. Bioorg. Med. Chem.

Lett. 2004, 14, 431-435. [100] Almeida, L.; Cintra, A. C.; Veronese, E. L.; Nomizo, A.; Franco, J.

J.; Arantes, E. C.; Giglio, J. R.; Sampaio, S. V. Anticrotalic and antitumoral activities of gel filtration fractions of aqueous extract from Tabernaemontana chatarinensis (Apocynaceae). Comp.

Biochem. Physiol. 2004, 137, 19-27. [101] Maiorano, V. A.; Marcussi, S.; Daher, M. A.; Oliveira, C. Z.;

Couto, L. B.; Gomes, O. A.; França, S. C.; Soares, A. M.; Pereira, P. S. Antiophidian properties of the aqueous extract of Mikania

glomerata. J. Ethnopharmacol. 2005, 102, 364-370. [102] Izidoro, L. F. M.; Rodrigues, V. M.; Rodrigues, R. S.; Ferro, E. V.;

Hamaguchi, A.; Giglio, J. R.; Homsi-Brandeburgo, M. I. Neutralization of some hematological and hemostatic alterations induced by neuwiedase, a metalloproteinase isolated from Bothrops neuwiedi pauloensis snake venom, by the aqueous extract from

Casearia mariquitensis (Flacourtiaceae). Biochimie 2003, 85, 669-675.

[103] Alam, M. I.; Gomes, A.; Auddy, B. Viper venom neutralization by Indian medicinal plant (Hemidesmus indicus and Pluchea indica) root extracts. Phytother. Res. 1996, 10, 58-61.

[104] Batina, M. F. C.; Giglio, J. R.; Sampaio, S. V. Methodological care in the evaluation of the LD50 and of the neutralization of the lethal effect of Crotalus durissus terrificus venom by the plant Peschiera fuchsiaefolia (Apocynaceae). J. Venom. Animal Toxins 1997, 3, 23-31.

[105] Mahanta, M.; Mukherjee, A. K. Neutralization of lethality, myotoxicity and toxic enzimes of Naja kaouthia venom by Mimosa pudica root extracts. J. Ethnopharmacol. 2001, 75, 55-60.

[106] Alam, M. I.; Gomes, A. Snake venom neutralization by Indian medicinal plants (Vitex negudo and Emblica officinalis) root extracts. J. Ethnopharmacol. 2003, 86, 75-80.

[107] Asuzu, I. U.; Harvey, A. L. The antisnake venom activities of Parkia biglobosa (Mimosaceae) stem bark extract. Toxicon 2003, 42, 763-768.

[108] Castro, E. M.; Pinto, J. E. B.; Alvarenga, A. A.; Júnior Lima, E. C.; Bertolucci, S. K. V.; Filho, J. L. S.; Vieira, C. V. Crescimento e anatomia foliar de plantas jovens de Mikania glomerata Sprengel (guaco) submetidas a diferentes fotoperíodos. Ciência e

Agrotecnologia 2003, 27, 1293-1300. [109] Borges, M. H.; Alves, D. L.; Raslan, D. S.; Pilo-Veloso, D.;

Rodríguez, V. M.; Homsi-Brandeburgo, M. I.; De Lima, M. E. Neutralizing properties of Musa paradisiaca L. (Musaceae) juice on phospholipase A2, myotoxic, hemorrhagic and lethal activities of crotalidae venoms. J. Ethnopharmacol. 2005, 98, 21-29.

[110] Ferreira, L. A. F.; Henriques, O. B.; Andreoni, A. A. S.; Vital, G. R. F.; Campos, M. M. C.; Habermehl, G. G.; Moraes, V. L. G. Antivenom and biological effects of ar-turmerone isolated from Curcuma longa (Zingiberiaceae). Toxicon 1992, 30, 1211-1218.

[111] Alam, M. I.; Gomes, A.; Auddy, B. Isolation, purafication and partial characterization of viper venom inhibiting factor from the root extract of the Indian medicinal plant sarsaparilla (Hemidesmus indicus R. Br.). Toxicon 1994, 32, 1551-1557.

[112] Ticli, F. K.; Hage, L. I.; Cambraia, R. S.; Pereira, P. S.; Magro, M. R.; Stabeli, R. G.; Giglio, J. R.; França, S. C.; Soares, A. M.; Sampaio, S. V. Rosmarinic acid, a new snake venom phospholipase A2 inhibitor from Cordia verbenacea (Boraginaceae): antiserum action potentiation and molecular interaction. Toxicon 2005, 46,

318-327. [113] da Silva, J. O.; Coppede, J. S.; Fernandes, V. C.; Sant’ana, C. D.;

Ticli, F. K.; Mazzi, M. V.; Giglio, J. R.; Pereira, P. S.; Soares, A. M.; Sampaio, S. V. Antihemorrhagic, antinucleolytic and other antiophidian properties of the aqueous extract from Pentaclethra macroloba. J. Ethnopharmacol. 2005, 100, 145-152.

[114] Oliveira, C. Z.; Maiorano, V. A.; Marcussi, S.; Sant'ana, C. D.; Januário, A. H.; Lourenço, M. V.; Sampaio, S. V.; França, S. C.; Pereira, P. S.; Soares, A. M. Anticoagulant and antifibrinogenolytic properties of the aqueous extract from Bauhinia forficata against snake venoms. J. Ethnopharmacol. 2005, 98, 213-216.

[115] Angulo, Y.; Lomonte, B. Inhibitory effect of fucoidan on the activities of crotaline snake venom myotoxic phospholipases A2. Biochem. Pharmacol. 2003, 66, 1993-2000.

[116] Nunez, V.; Castro, V.; Murillo, R.; Ponce-Soto, L. A.; Merfort, I.; Lomonte, B. Inhibitory effects of Piper umbellatum and Piper

peltatum extracts towards myotoxic phospholipases A2 from Bothrops snake venoms: isolation of 4-nerolidylcatechol as active principle. Phytochemistry 2005, 66, 1017-1025.

[117] Chandra, V.; Jasti, J.; Kaur, P.; Srinivasan, A.; Betzel, C. H.; Singh, T. P. Structural basis of phospholipase A2 inhibition for the synthesis of prostaglandins by the plant alkaloid aristolochic acid from a 1.7 A crystal structure. Biochemistry 2002, 41, 10914-10919.

[118] Reynolds, L. J.; Morgan, B.P.; Hite, G. A.; Mihelich, E. D.; Dennis, E. A. Phospholipase A2 inhibition and modification by manoalogue. Am. Chem. Soc. 1988, 110, 5172-5177.

[119] Tseng, A.; Inglis, A. S.; Scott, K. F. Native peptide inhibition. Specific inhibition of type II phospholipases A2 by synthetic peptides derived from the primary sequence. J. Biol. Chem. 1996, 271, 23992-23998.

Snake Venom PLA2s Inhibitors Current Topics in Medicinal Chemistry, 2007, Vol. 7, No. ? 13

[120] Burke, J. R.; Davern, L. B.; Stanley, P. L.; Gregor, K. R.; Banville, J.; Remillard, R.; Russell, J. W.; Brassil, P. J.; Witmer, M. R.; Johnson, G.; Tredup, J. A.; Tramposch, K. M. BMS-229724 is a tight-binding inhibitor of cytosolic phospholipase A2 that acts at the lipid/water interface and possesses anti-inflammatory activity in skin inflammation models. J. Pharmacol. Exp. Ther. 2001, 298,

376-385. [121] Chacur, M.; Longo, I.; Picolo, G.; Gutierrez, J. M.; Lomonte, B.;

Guerra, J. L.; Teixeira, C. F. P.; Cury, Y. Hiperalgesia induced by Asp49 and Lys49 phospholipases A2 from Bothrops asper snake venom: pharmacological mediation and molecular determinants. Toxicon 2003, 41, 667-678.

[122] Basappa,; Satish Kumar, M.; Nanjunda Swamy, S.; Mahendra, M.; Shashidhara Prasad, J.; Viswanath, B. S.; Rangappa, K. S. Novel delta2-isoxazolines as group II phospholipase A2 inhibitors. Bioorg. Med. Chem. Lett. 2004, 14, 3679-3681.

[123] Beghini, D. G.; Da Cruz-Hofling, M. A.; Randazzo-Moura, P.; Rodrigues-Simioni, L.; Novello, J. C.; Hyslop, S.; Marangoni, S. Cross-neutralization of the neurotoxicity of Crotalus durissus terrificus and Bothrops jararacussu venoms by antisera against crotoxin and phospholipase A2 from Crotalus durissus cascavella venom. Toxicon 2005, 46, 604-611.

[124] Magro, A. J.; Takeda, A. A. S.; Soares, A. M.; Fontes, M. R. M. Structure of BthA-I complexed with p-bromophenacyl bromide: possible correlations with lack of pharmacological activity. Acta. Crystallogr. 2005, 61D, 1670-1677.

[125] Kuruppu, S.; Isbister, G. K.; Hodgson, W. C. Phospholipase A2-dependent effects of the venom from the New Guinean small-eyed snake Micropechis ikaheka. Muscle Nerve. 2005, 32, 81-87.

[126] Khanum, S. A.; Murari, S. K.; Vishwanth, B. S.; Shashikanth, S. Synthesis of benzoyl phenyl benzoates as effective inhibitors for phospholipase A2 and hyaluronidase enzymes. Bioorg. Med. Chem.

Lett. 2005, 15, 4100-4104. [127] Georgieva, D. N.; Rypnicwski, W.; Perbandt, M.; Jani, M.; Genov,

N.; Betzel, C. Crystallization and preliminary X-ray diffraction studies of a toxic phospholipase A2 from the venom of Vipera ammodytes meridionalis complexed to a synthetic inhibitor. Biochim. Biophys. Acta 2003, 1650, 1-3.

[128] Omori-Satoh, T.; Sadahiro, S.; Ohsaka, A.; Murata, R. Purification and characterization of an antihemorrhagic factor in the serum of Trimeresurus flavoviridis, a crotalid. Biochim. Biophys. Acta 1972, 285, 414-426.

[129] Ovadia, M.; Kochva, E. Neutralization of Viperidae and Elapidae snake venoms by sera of different animals. Toxicon 1977, 15, 541-547.

[130] Borkow, G.; Gutierrez, J. M.; Ovadia, M. Isolation, characteri-zation and mode of neutralization of a potent antihemorrhagic factor from the serum of the snake Bothrops asper. Biochim. Biophys. Acta 1995, 1245, 232-238.

[131] Lizano, S.; Lomonte, B.; Fox, J. W.; Gutiérrez, J. M. Biochemical characterization and pharmacological properties of an inhibitor of basic phospholipase A2 myotoxins from the plasma of the snake of Bothrops asper. Biochem. J. 1997, 326, 853-859.

[132] Soares, A. M.; Rodrigues, V. M.; Borges, M. H.; Andrião-Escarso, S. H.; Cunha, O. A. B.; Homsi-Brandesburgo, M. I.; Giglio, J. R. Inhibition of proteases, myotoxins and phospholipases A2 from Bothrops venoms by the heteromeric protein complex of Didelphys

albiventris opossum serum. Biochem. Mol. Biol. Int. 1997, 43,

1091-1099. [133] Rocha, S. L. G.; Lomonte, B.; Neves-Ferreira, A. G. C.; Domont,

G. B.; Gutiérrez, J. M.; Perales, J. Functional analysis of DM64 an antimyotoxic protein with immunoglobulin-like structure from Didelphis marsupialis serum. Eur. J. Biochem. 2002, 269, 6052-6062.

[134] Murari, S. K.; Frey, F. J.; Frey, B. M.; Gowda, T. V.; Vishwanath, B. S. Use of Pavo cristatus feather extract for the better management of snake-bites: Neutralization of inflammatory reactions. J. Ethnopharmacol. 2005, 99, 229-237.

[135] Satish, S.; Tejaswini, J.; Krishnakantha, T. P.; Gowda, T. V. Purification of a Class B1 platelet aggregation inhibitor phospholipaseA2 from Indian cobra (Naja Naja) venom. Biochimie 2004, 86, 203-210.

[136] Oliveira, C. Z.; Sant´Ana, C. D.; Amui, S. F.; Marcussi, S.; Stabeli, R. G.; Giglio, J. R.; Soares, A. M. Structural and functional

characterization of a phospholipase A2 inhibitors isolated from Bothrops jararacussu snake plasma. In preparation 2006.

[137] Forte-Dias, C. L.; Barcellos, C. J.; Estevao-Costa, M. I. Molecular cloning of a g-phospholipase A2 inhibitor from Lachesis muta muta (the bushmaster snake). Toxicon 2003, 41, 909-917.

[138] Nobuhisa, I.; Inamasu, S.; Nakai, M.; Tatsui, A.; Mimori, T.; Ogawa, T.; Shimohigashi, Y.; Fukumaki, Y.; Hattori, S.; Kihara, H.; Ohno, M. Characterization and evolution of a gene encoding a Trimeresurus flavoviridis serum protein that inhibits basic phospholipase A2 isozymes in the snake’s venom. Eur. J. Biochem. 1997, 249, 838-845.

[139] Inoue, S.; Kogaki, H.; Ikeda, K.; Samejima, Y.; Omori-Satoh, T. Amino acid sequences of the two subunits of a phospholipase A2 inhibitor from the blood plasma of Trimeresurus flavoviridis. Sequence homologies with pulmonary surfactant apoprotein and animal lectins. J. Biol. Chem. 1991, 266,1001-1007.

[140] Inoue, S.; Shimada, A.; Ohkura, N.; Ikeda, K.; Samejima, Y.; Omori-Satoh, T.; Hayashi, K. Specificity of two types of phospholipase A2 inhibitors from plasma of venomous snakes. Biochem. Mol. Biol. Int. 1997, 41, 529-537.

[141] Ohkura, N.; Okuhara, H.; Inoue, S.; Ikeda, K.; Hayashi, K. Purification and characterization of three distinct types of phospholipase A2 inhibitors from the blood plasma of the Chinese mamushi, Agkistrodon blomhoffii siniticus. J. Biochem. 1997, 325,

527-531. [142] Thwin, M. M.; Satish, R. L.; Chan, S. T. F.; Gopalakrishnakone, P.

Functional site of endogenous phospholipase A2 inhibitor from Python serum. Phospholipase A2 binding and anti-inflammatory activity. Eur. J. Biochem. 2002, 269, 719-727.

[143] Kogaki, H.; Inoue, S.; Ikeda, K.; Samejima, Y.; Omori-Satoh, T.; Hamaguchi, K. Isolation and fundamental properties of a phospholipase A2 inhibitor from the blood plasma of Trimeresurus flavoviridis. J. Biochem. 1989, 106, 966-971.

[144] Taylor, M. E.; Conary, J. T.; Lennartz, M. R.; Stahl, P. D.; Drickamer, K. Primary structure of the mannose receptor contains multiple motifs resembling carbohydrate-recognition domains. J.

Biol. Chem. 1990, 265, 12156-12162. [145] Dunn, R. D.; Broady, K. W. Snake inhibitors of phospholipase A2

enzymes. Biochim. Biophys. Acta 2001, 1533, 29-37. [146] Okumura, K.; Ohkura, N.; Inoue, S.; Ikeda, K.; Hayshi, K. A novel

phospholipase A2 inhibitor with leucine-rich repeats from the blood plasma of Agkistrodon blomhoffii siniticus: Sequence homologies with human leucine-rich 2-glycoprotein. J. Biol. Chem. 1998, 273,

19469-19475. [147] Ohkura, N.; Inoue, S.; Ikeda, K.; Hayashi, K. The two subunits of a

phospholipase A2 inhibitor from the plasma of Thailand cobra having structural similar to urokinase-type plasminogen activator receptor and LY-6 related proteins. Biochem. Biophys. Res.

Commun. 1994, 204, 1212-1218. [148] Fortes-Dias, C. L.; Fonseca, B. C.; Kochva, E.; Diniz, C. R.

Purification and properties of an antivenom factor from the plasma of the South American rattlesnake (Crotalus durissus terrificus). Toxicon 1991, 29, 997-1008.

[149] Okumura, K.; Masui, K.; Inoue, S.; Ikeda, K.; Hayshi, K. Purification, characterization and cDNA cloning of a phospholipase A2 inhibitor from the serum of the non-venomous snake Elaphe quadrivirgata. J. Biochem. 1999, 341, 165-171.

[150] Santos, R. M. M.; Oliveira, L. C.; Estevão-Costa, M. I.; Lima, M.E.; Santoro, M. M.; Dias, C. L. Inhibition of crotoxin binding to synaptosomes by a receptor-like protein from Crotalus durissus terrificus (the South American rattlesnake). Biochim. Biophys. Acta 2005, 1717, 27-33.

[151] Okumura, K.; Inoue, S.; Ikeda, K.; Hayashi, K. Identification of -Type phospholipase A2 inhibitor in a nonvenomous snake Elaphe quadrivirgata. Arch. Biochem. Biophys. 2002, 408, 124-130.

[152] Ohkura, N.; Inoue, S.; Ikeda, K.; Hayashi, K. Isolation and characterization of a phospholipase A2 inhibitor from the blood plasma of Thailand cobra, Naja naja kaothia. Biochem. Biophys.

Res. Commun. 1994, 200, 784-788. [153] Lizano, S.; Angulo, Y.; Lomonte, B.; Fox, J.W.; Lambeau, G.;

Lazdunski, M.; Guitiérrez, J. M. Two phospholipase A2 inhibitors from the plasma of Cerrophidian (Bothrops) godmani, which selectively inhibit two different group II phospholipase A2

14 Current Topics in Medicinal Chemistry, 2007, Vol. 7, No. ? Soares et al.

myotoxins from its own venom: isolation molecular cloning, and biological properties. Biochem. J. 2000, 346, 631-639.

[154] Thwin, M. M.; Gopalakrishanakone, P.; Kini, R. M.; Arugam, A.; Jeyaseelan, K. Recombinant antitoxic and anti-inflammatory factor from the nonvenomous snake Python reticulates: Phospholipase A2 inhibition and venom neutralizing potential. Biochem. 2000, 39,

9604-9611. [155] Hains, P. G.; Sung, K. L.; Tseng, A.; Broady, K. W. Functional

characteristics of a phospholipase A2 inhibitors from Notechis ater serum. J. Biol. Chem. 2000, 275, 983-991.

[156] Hains, P. G.; Broady, K. W. Purification and inhibitory profile of phospholipase A2 inhibitors from Australian elapid sera. Biochem.

J. 2000, 346, 139-146. [157] Hains, P. G.; Nield, B.; Sekuloski, S.; Dunn, R.; Broady, K.

Sequencing and two-dimensional structure prediction of a phospholipase A2 inhibitor from the serum of the common tiger snake (Notechis scutatus). J. Mol. Biol. 2001, 312, 875-884.

Received: July 28, 2006 Accepted: October 3, 2006