Evaluation of the Inhibitory Potential of Synthetic Peptides Homologous to CDR3 Regions of a Monoclonal Antibody against Bothropic Venom Serine Proteases.

Snakebite accidents, neglected tropical diseases per the WHO, pose a significant public health threat due to their severity and frequency. Envenomation by Bothrops genus snakes leads to severe manifestations due to proteolytic enzymes. While the antibothropic serum produced by the Butantan Institute saves lives, its efficacy is limited as it fails to neutralize certain serine proteases. Hence, developing new-generation antivenoms, like monoclonal antibodies, is crucial. This study aimed to explore the inhibitory potential of synthetic peptides homologous to the CDR3 regions of a monoclonal antibody targeting a snake venom thrombin-like enzyme (SVTLE) from B. atrox venom. Five synthetic peptides were studied, all stable against hydrolysis by venoms and serine proteases. Impressively, four peptides demonstrated uncompetitive SVTLE inhibition, with Ki values ranging from 10-6 to 10-7 M. These findings underscore the potential of short peptides homologous to CDR3 regions in blocking snake venom toxins, suggesting their promise as the basis for new-generation antivenoms. Thus, this study offers potential advancements in combatting snakebites, addressing a critical public health challenge in tropical and subtropical regions.

Isolation and Functional Characterization of Erythrofibrase: An Alfa-Fibrinogenase Enzyme from Trimeresurus erythrurus Venom of North-East India.

Green pit viper bites induce mild toxicity with painful local swelling, blistering, cellulitis, necrosis, ecchymosis and consumptive coagulopathy. Several bite cases of green pit vipers have been reported in several south-east Asian countries including the north-eastern region of India. The present study describes isolation and characterization of a haemostatically active protein from Trimeresurus erythrurus venom responsible for coagulopathy. Using a two-step chromatographic method, a snake venom serine protease erythrofibrase was purified to homogeneity. SDS-PAGE of erythrofibrase showed a single band of ~30 kDa in both reducing and non-reducing conditions. The primary structure of erythrofibrase was determined by ESI LC-MS/MS, and the partial sequence obtained showed 77% sequence similarity with other snake venom thrombin-like enzymes (SVTLEs). The partial sequence obtained had the typical 12 conserved cysteine residues, as well as the active site residues (His57, Asp102 and Ser195). Functionally, erythrofibrase showed direct fibrinogenolytic activity by degrading the Aα chain of bovine fibrinogen at a slow rate, which might be responsible for causing hypofibrinogenemia and incoagulable blood for several days in envenomated patients. Moreover, the inability of Indian polyvalent antivenom (manufactured by Premium Serum Pvt. Ltd., Maharashtra, India) to neutralize the thrombin-like and plasmin-like activity of erythrofibrase can be correlated with the clinical inefficacy of antivenom therapy. This is the first study reporting an α-fibrinogenase enzyme erythrofibrase from T. erythrurus venom, which is crucial for the pathophysiological manifestations observed in envenomated victims.

Examination of the Efficacy and Cross-Reactivity of a Novel Polyclonal Antibody Targeting the Disintegrin Domain in SVMPs to Neutralize Snake Venom.

Snake envenomation can result in hemorrhage, local necrosis, swelling, and if not treated properly can lead to adverse systemic effects such as coagulopathy, nephrotoxicity, neurotoxicity, and cardiotoxicity, which can result in death. As such, snake venom metalloproteinases (SVMPs) and disintegrins are two toxic components that contribute to hemorrhage and interfere with the hemostatic system. Administration of a commercial antivenom is the common antidote to treat snake envenomation, but the high-cost, lack of efficacy, side effects, and limited availability, necessitates the development of new strategies and approaches for therapeutic treatments. Herein, we describe the neutralization ability of anti-disintegrin polyclonal antibody on the activities of isolated disintegrins, P-II/P-III SVMPs, and crude venoms. Our results show disintegrin activity on platelet aggregation in whole blood and the migration of the SK-Mel-28 cells that can be neutralized with anti-disintegrin polyclonal antibody. We characterized a SVMP and found that anti-disintegrin was also able to inhibit its activity in an in vitro proteolytic assay. Moreover, we found that anti-disintegrin could neutralize the proteolytic and hemorrhagic activities from crude Crotalus atrox venom. Our results suggest that anti-disintegrin polyclonal antibodies have the potential for a targeted approach to neutralize SVMPs in the treatment of snakebite envenomations.

Repurposing Cancer Drugs Batimastat and Marimastat to Inhibit the Activity of a Group I Metalloprotease from the Venom of the Western Diamondback Rattlesnake, Crotalus atrox.

Snakebite envenomation causes over 140,000 deaths every year, predominantly in developing countries. As a result, it is one of the most lethal neglected tropical diseases. It is associated with incredibly complex pathophysiology due to the vast number of unique toxins/proteins present in the venoms of diverse snake species found worldwide. Here, we report the purification and functional characteristics of a Group I (PI) metalloprotease (CAMP-2) from the venom of the western diamondback rattlesnake, Crotalus atrox. Its sensitivity to matrix metalloprotease inhibitors (batimastat and marimastat) was established using specific in vitro experiments and in silico molecular docking analysis. CAMP-2 shows high sequence homology to atroxase from the venom of Crotalus atrox and exhibits collagenolytic, fibrinogenolytic and mild haemolytic activities. It exerts a mild inhibitory effect on agonist-induced platelet aggregation in the absence of plasma proteins. Its collagenolytic activity is completely inhibited by batimastat and marimastat. Zinc chloride also inhibits the collagenolytic activity of CAMP-2 by around 75% at 50 µM, while it is partially potentiated by calcium chloride. Molecular docking studies have demonstrated that batimastat and marimastat are able to bind strongly to the active site residues of CAMP-2. This study demonstrates the impact of matrix metalloprotease inhibitors in the modulation of a purified, Group I metalloprotease activities in comparison to the whole venom. By improving our understanding of snake venom metalloproteases and their sensitivity to small molecule inhibitors, we can begin to develop novel and improved treatment strategies for snakebites.

Delineating the venom toxin arsenal of Malabar pit viper (Trimeresurus malabaricus) from the Western Ghats of India and evaluating its immunological cross-reactivity and in vitro cytotoxicity.

The venom protein components of Malabar pit viper (Trimeresurus malabaricus) were identified by combining SDS-PAGE and ion-exchange chromatography pre-fractionation techniques with LC-MS/MS incorporating Novor and PEAKS-assisted de novo sequencing strategies. Total 97 proteins that belong to 16 protein families such as L-amino acid oxidase, metalloprotease, serine protease, phospholipase A2, 5'-nucleotidase, C-type lectins/snaclecs and disintegrin were recognized from the venom of a single exemplar species. Of the 97 proteins, eighteen were identified through de novo approaches. Immunological cross-reactivity assessed through ELISA and western blot indicate that the Indian antivenoms binds less effectively to Malabar pit viper venom components compared to that of Russell's viper venom. The in vitro cell viability assays suggest that compared to the normal cells, MPV venom induces concentration dependent cell death in various cancer cells. Moreover, crude venom resulted in chromatin condensation and apoptotic bodies implying the induction of apoptosis. Taken together, the present study enabled in dissecting the venom proteome of Trimeresurus malabaricus and revealed the immuno-cross-reactivity profiles of commercially available Indian polyvalent antivenoms that, in turn, is expected to provide valuable insights on the need in improving antivenom preparations against its bite.

Atroxlysin-III, A Metalloproteinase from the Venom of the Peruvian Pit Viper Snake Bothrops atrox (Jergón) Induces Glycoprotein VI Shedding and Impairs Platelet Function.

Atroxlysin-III (Atr-III) was purified from the venom of Bothrops atrox. This 56-kDa protein bears N-linked glycoconjugates and is a P-III hemorrhagic metalloproteinase. Its cDNA-deduced amino acid sequence reveals a multidomain structure including a proprotein, a metalloproteinase, a disintegrin-like and a cysteine-rich domain. Its identity with bothropasin and jararhagin from Bothrops jararaca is 97% and 95%, respectively. Its enzymatic activity is metal ion-dependent. The divalent cations, Mg2+ and Ca2+, enhance its activity, whereas excess Zn2+ inhibits it. Chemical modification of the Zn2+-complexing histidine residues within the active site by using diethylpyrocarbonate (DEPC) inactivates it. Atr-III degrades plasma fibronectin, type I-collagen, and mainly the α-chains of fibrinogen and fibrin. The von Willebrand factor (vWF) A1-domain, which harbors the binding site for GPIb, is not hydrolyzed. Platelets interact with collagen via receptors for collagen, glycoprotein VI (GPVI), and α2ß1 integrin. Neither the α2ß1 integrin nor its collagen-binding A-domain is fragmented by Atr-III. In contrast, Atr-III cleaves glycoprotein VI (GPVI) into a soluble ~55-kDa fragment (sGPVI). Thereby, it inhibits aggregation of platelets which had been stimulated by convulxin, a GPVI agonist. Selectively, Atr-III targets GPVI antagonistically and thus contributes to the antithrombotic effect of envenomation by Bothrops atrox.

Differential Macrophage Subsets in Muscle Damage Induced by a K49-PLA2 from Bothrops jararacussu Venom Modulate the Time Course of the Regeneration Process.

Bothrops snakes cause around 80% of snakebites in Brazil, with muscle tissue damage as an important consequence, which may cause dysfunction on the affected limb. Bothropstoxin-I (BthTX-I) from Bothrops jararacussu is a K49-phospholipase A2, involved in the injury and envenomation's inflammatory response. Immune system components act in the resolution of tissue damage and regeneration. Thus, macrophages exert a crucial role in the elimination of dead tissue and muscle repair. Here, we studied the cellular influx and presence of classical and alternative macrophages (M1 and M2) during muscle injury induced by BthTX-I and the regeneration process. BthTX-I elicited intense inflammatory response characterized by neutrophil migration, then increased influx of M1 macrophages followed by M2 population that declined, resulting in tissue regeneration. The high expressions of TNF-α and IL6 were changed by increased TGF-ß expression after BthTX-I injection, coinciding with the iNOs and arginase expression and the peaks of M1 and M2 macrophages in muscle tissue. A coordinated sequence of PAX7, MyoD, and myogenin expression involved in muscle regenerative process appeared after BthTX-I injection. Together, these results demonstrate a direct correlation between the macrophage subsets, cytokine microenvironment, and the myogenesis process. This information may be useful for new envenomation and muscular dysfunction therapies.

Identification of a peptide derived from a Bothrops moojeni metalloprotease with in vitro inhibitory action on the Plasmodium falciparum purine nucleoside phosphorylase enzyme (PfPNP).

There is a growing need for research on new antimalarial agents against Plasmodium falciparum infection, especially in regards to planning molecular architecture for specific molecular targets of the parasite. Thus, a metalloprotease from Bothrops moojeni, known as BmooMPα-I, was explored in this study, through in silico assays, aiming at the development of a peptide generated from this molecule with potential inhibitory action on PfPNP, an enzyme necessary for the survival of the parasite. In order to isolate BmooMPα-I, cation exchange and reverse phase chromatographies were performed, followed by in vitro assays of antiparasitic activity against the W2 strain of P. falciparum. The interactions between BmooMPα-I and PfPNP were evaluated via docking, and the resulting peptide, described as Pep1 BM, was selected according to the BmooMPα-I region demonstrating the best interaction score with the target of interest. The values for the specific activities of the PfPNP reaction were measured using the inorganic phosphate substrate and MESG. The fraction corresponding to BmooMPα-I was identified as fraction 4 in the cation exchange chromatography step, due to proteolytic activity on casein and the presence of a major band at ≅ 23 kDa. BmooMPα-I was able to inhibit in vitro growth of W2 P. falciparum, with an IC50 value of 16.14 µg/mL. Virtual screening with Pep1 BM demonstrated two PfPNP target binding regions, with ΔG values at the interaction interface of -10.75 kcal/mol and -11.74 kcal/mol. A significant reduction in the enzymatic activity of PfPNP was observed in the presence of Pep 1 BM when compared to the assay in the absence of this possible inhibitor. BmooMPα-I showed activity in vitro against W2 P. falciparum. By means of in silico techniques, the Pep 1 BM was identified as having potential binding affinity to the catalytic site of PfPNP and of inhibiting its catalytic activity in vitro.

Proteomic profiling, functional characterization, and immunoneutralization of the venom of Porthidium porrasi, a pitviper endemic to Costa Rica.

The genus Porthidium includes nine pitviper species inhabiting Mexico, Central America, and northern South America. Porthidium porrasi is a species endemic to the Southwest of Costa Rica, for which no information on its venom was available. In this study, the proteomic composition and functional activities of P. porrasi venom are described. The most abundant venom proteins were identified as metalloproteinases (36.5%). In descending order of abundance, proteins belonging to the disintegrin, phospholipase A2, serine proteinase, C-type lectin/lectin-like, vascular endothelial growth factor, Cysteine-rich secretory protein, L-amino acid oxidase, phospholipase B, and phosphodiesterase families were also identified. P. porrasi venom showed a weak lethal potency in mice (10 µg/g body weight by intraperitoneal route), induced marked hemorrhage and edema, and weak myotoxic effect. These in vivo activities, as well as those assayed in vitro (proteolytic and phospholipase A2 activities) correlated with compositional data. A comparison of P. porrasi venom with those of three other Porthidium species studied to date reveals a generally conserved compositional and functional pattern in this pitviper genus. Importantly, the lethal effect of P. porrasi venom in mice was adequately cross-neutralized by a heterospecific polyvalent antivenom, supporting its use in the treatment of eventual envenomings by this species.

cDNA cloning, heterologous expression, protein folding and immunogenic properties of a phospholipase A2 from Bothrops ammodytoides venom.

A mRNA transcript that codes for a phospholipase (PLA2) was isolated from a single venom gland of the Bothrops ammodytoides viper. The PLA2 transcript was cloned onto a pCR®2.1-TOPO vector and subsequently expressed heterologously in the E. coli strain M15, using the pQE30 vector. The recombinant phospholipase was named rBamPLA2_1, and is composed of an N-terminal fusion protein of 16 residues, along with 122 residues from the mature protein that includes 14 cysteines that form 7 disulfide bonds. Following bacterial expression, rBamPLA2_1 was obtained from inclusion bodies and extracted using a chaotropic agent. rBamPLA2_1 had an experimental molecular mass of 15,692.5 Da that concurred with its theoretical molecular mass. rBamPLA2_1 was refolded in in vitro conditions and after refolding, three main protein fractions with similar molecular masses, were identified. Although, the three fractions were considered to represent different oxidized cystine isoforms, their secondary structures were comparable. All three recombinant isoforms were active on egg-yolk phospholipid and recognized similar cell membrane phospholipids to be native PLA2s, isolated from B. ammodytoides venom. A mixture of the three rBamPLA2_1 cystine isoforms was used to immunize a horse in order to produce serum antibodies (anti-rBamPLA2_1), which partially inhibited the indirect hemolytic activity of B. ammodytoides venom. Although, anti-rBamPLA2_1 antibodies were not able to recognize crotoxin, a PLA2 from the venom of a related but different viper genus, Crotalus durissus terrificus, they recognized PLA2s in other venoms from regional species of Bothrops.

Modeling and molecular dynamics indicate that snake venom phospholipase B-like enzymes are Ntn-hydrolases.

Phospholipase-B-like (SVPLB-like) enzymes are present in relatively small amounts in a number of venoms, however, their biological function and mechanisms of action are un-clear. A three-dimensional model of the SVPLB-like enzyme from Crotalus adamanteus was generated by homology modeling based on the crystal structures of bovine Ntn-hydrolyases and the modeled protein possesses conserved domains characteristic of Ntn-hydrolases. Molecular dynamics simulations indicate that activation by autocatalytic cleavage results in the removal of 25 amino acids which increases accessibility to the active site. SVPLB-like enzymes possess a highly reactive cysteine and are hence amidases that to belong to the N-terminal nucleophile (Ntn) hydrolase family. The Ntn-hydrolases (N-terminal nucleophile) form a superfamily of diverse enzymes that are activated autocatalytically; wherein the N-terminal catalytic nucleophile is implicated in the cleavage of the amide bond.

Cytotoxic, Anti-Proliferative and Apoptosis Activity of l-Amino Acid Oxidase from Malaysian Cryptelytrops purpureomaculatus (CP-LAAO) Venom on Human Colon Cancer Cells.

The aim of this study is to investigate the potential anti-cancer activity of l-amino acid oxidase (CP-LAAO) purified from the venom of Cryptelytrops purpureomaculatus on SW480 and SW620 human colon cancer cells. Mass spectrometry guided purification was able to identify and purify CP-LAAO. Amino acid variations identified from the partial protein sequence of CP-LAAO may suggest novel variants of these proteins. The activity of the purified CP-LAAO was confirmed with o-phenyldiamine (OPD)-based spectrophotometric assay. CP-LAAO demonstrated time- and dose-dependent cytotoxic activity and the EC50 value was determined at 13 µg/mL for both SW480 and SW620 cells. Significant increase of caspase-3 activity, reduction of Bcl-2 levels, as well as morphological changes consistent with apoptosis were demonstrated by CP-LAAO. Overall, these data provide evidence on the potential anti-cancer activity of CP-LAAO from the venom of Malaysian C. purpureomaculatus for therapeutic intervention of human colon cancer.

A novel pentameric phospholipase A2 myotoxin (PophPLA2) from the venom of the pit viper Porthidium ophryomegas.

The first toxin isolated from the venomous pit viper Porthidium ophryomegas is a basic pentameric phospholipase A2 (PophPLA2). Elucidation of its amino acid sequence showed that it belongs to the group IIA of secreted PLA2s, with the presence of all 14 conserved cysteine positions. The toxin displayed catalytic activity, in agreement with the presence of Asp49 in its sequence of 121 residues. SDS-PAGE analysis revealed that this toxin is pentameric in non-reducing conditions, a structural organization that has not been described for any viperid PLA2. PophPLA2 displayed moderate myotoxic (in vivo) and cytotoxic (in vitro) activities, as well as anticoagulant activity on human plasma (in vitro). PophPLA2 was not lethal, and did not induce signs of toxicity or distress in mice, when administered intravenously at a dose of up to 100 µg (5.9 µg/g). The toxin showed highest sequence identity with other PLA2s from the venoms of ancestral Asian pit viper species.

Signaling pathways involved in zymosan phagocytosis induced by two secreted phospholipases A2 isolated from Bothrops asper snake venom in macrophages.

Phagocytosis, a process involved in host defense, requires coordination of a variety of signaling reactions. MT-II, a catalytically-inactive Lys49-PLA2¸ and MT-III, an active Asp49-PLA2 isolated from Bothrops asper snake venom, activate phagocytosis in macrophages. In this study the signal pathways mediating zymosan phagocytosis, focusing in lipidic second messengers, were investigated. Macrophages collected from male Swiss mouse peritoneum were obtained 96h after i.p. injection of thioglycollate. Phagocytosis was evaluated with non-opsonized zymosan in the presence or absence of specific inhibitors. Data showed that both venom PLA2s increased phagocytosis. Zileuton, Etoricoxib, PACOCF3 (5-LO, COX-2 and iPLA2 inhibitors, respectively), as well as WEB2170 (PAF receptor antagonist) significantly reduced phagocytosis induced by both venom PLA2s. However, Indomethacin (COX-1/COX-2 inhibitor) and Montelukast (CysL receptor antagonist) did not affect the toxins-induced phagocytosis. Moreover, while PACOCF3 (iPLA2 inhibitor), reduced the phagocytosis induced by MT-II and MT-III, AACOCF3 (cPLA2 inhibitor) significantly reduced the MT-II, but not MT-III-induced phagocytosis. These data suggest the effect of both sPLA2s depends on iPLA2 and that the effect of MT-II depends on activation of cPLA2. COX-2 and 5-LO-derived metabolites as well as PAF are involved in the signaling events required for phagocytosis induced by both venom sPLA2s.

Identification of the Molecular Determinants of the Antibacterial Activity of LmutTX, a Lys49 Phospholipase A2 Homologue Isolated from Lachesis muta muta Snake Venom (Linnaeus, 1766).

Snake venom phospholipases A2 (PLA2 s) are responsible for numerous pathophysiological effects in snakebites; however, their biochemical properties favour antimicrobial actions against different pathogens, thus constituting a true source of potential microbicidal agents. This study describes the isolation of a Lys49 PLA2 homologue from Lachesis muta muta venom using two chromatographic steps: size exclusion and reverse phase. The protein showed a molecular mass of 13,889 Da and was devoid of phospholipase activity on an artificial substrate. The primary structure made it possible to identify an unpublished protein from L. m. muta venom, named LmutTX, that presented high identity with other Lys49 PLA2 s from bothropic venoms. Synthetic peptides designed from LmutTX were evaluated for their cytotoxic and antimicrobial activities. LmutTX was cytotoxic against C2C12 myotubes at concentrations of at least 200 µg/mL, whereas the peptides showed a low cytolytic effect. LmutTX showed antibacterial activity against Gram-positive and Gram-negative bacteria; however, S. aureusATCC 29213 and MRSA strains were more sensitive to the toxin's action. Synthetic peptides were tested on S. aureus, MRSA and P. aeruginosaATCC 27853 strains, showing promising results. This study describes for the first time the isolation of a Lys49 PLA2 from Lachesis snake venom and shows that peptides from specific regions of the sequence may constitute new sources of molecules with biotechnological potential.

Betulinic, oleanolic and ursolic acids inhibit the enzymatic and biological effects induced by a P-I snake venom metalloproteinase.

Betulinic acid (BA), Oleanolic acid (OA) and Ursolic acid (UA), are pentacyclic triterpenoids with widespread occurrence throughout the plant kingdom, these compounds are widely recognized by their pharmacological and biological properties, such as, anti-tumoral, anti-inflammatory, anti-microbial and hepatoprotective activity. In this work we determined the inhibitory ability of these compounds on the enzymatic, hemorrhagic, myotoxic and edema-inducing activities of Batx-I, a P-I metalloproteinase isolated from Bothrops atrox venom. BA, UA and OA inhibited the proteolytic activity of Batx-I on gelatin with IC50 values of 115.3, 223.0 and 357.3 µM, respectively. Additionally, these compounds showed inhibition of the hemorrhagic activity of Batx-I in skin with IC50 345.7, 643.5 and 1077.0 µM for BA, UA and OA in preincubation experiments. In studies with independent-injection, in which Batx-I was injected and then, at the same site, a concentration of 600 µM of each compound were administered at either 0, 5 or 10 min, BA showed a significant reduction of hemorrhage at 0 and 5 min. In addition, these compounds inhibited myotoxicity and edema-forming activity of Batx-I at 600 µM concentration. Molecular docking studies suggested that these compounds could occupy part of the substrate binding cleft of the enzyme affecting its catalytic cycle. In this manner, triterpenic acids are candidates for the development of inhibitors for the prevention of local tissue damage in snakebite envenomation.

New findings from the first transcriptome of the Bothrops moojeni snake venom gland.

Snakebites are a serious health problem in tropical countries. In Brazil, the genus Bothrops (Viperidae family) causes most of the ophidic accidents, characterized by proteolysis and haemorrhage. Snake venoms are rich sources of toxins with great therapeutic and biotechnological potential and omics approaches is a valuable tool for identification of new bioactive components in the venom. In this study, we described the first transcriptome of the venom gland of Bothrops moojeni snake, using the next-generation sequencing with the Illumina platform. We identified: (i) 20 venom components classes, among which metalloproteases were the most expressed ones, followed by serine proteases and phospholipases; and (ii) the 33 full-length amino acid sequences of toxins that have never been reported before in B. moojeni venom, such as one cysteine-rich secretory protein (Moojin), two hyaluronidases (BmooHyal-1 and BmooHyal-2), and one three-finger toxin (Bmoo-3FTx). Altogether, the transcripts identified herein represent a starting point for the analysis of structure-function relationships of toxins, which shall help develop novel biological tools and therapeutic drugs.

In vitro cytotoxicity of L-amino acid oxidase from the venom of Crotalus mitchellii pyrrhus.

L-amino acid oxidase isolated from Crotalus mitchellii pyrrhus (Cmp-LAAO) exhibited cytotoxicity against LNCaP prostate adenocarcinoma cells. The viability of LNCaP cells decreased in a concentration- and time-dependent manner upon administration of Cmp-LAAO. Cmp-LAAO induced apoptosis as evidenced by AnnexinV/PI staining using flow cytometry. An increase in caspase-9 and caspase-3 activity were also observed. The damaging effect of LAAO appears to be due to its enzymatic activity, that produces hydrogen peroxide which can then induce oxidative stress within the cells. As expected, the level of oxidative stress in LNCaP cells increased with Cmp-LAAO treatment as confirmed by 2', 7'-dichlorofluorescin diacetate (DCFDA) fluorescence assay. Co-treatment with catalase significantly reduced the cytotoxic effect of Cmp-LAAO, thereby affirming that hydrogen peroxide is probably the main mediator of Cmp-LAAO cytotoxicity. Hence, Cmp-LAAO may be a potential cancer therapeutic for prostate cancer.

In vitro cleavage of bioactive peptides by peptidases from Bothrops jararaca venom and its neutralization by bothropic antivenom produced by Butantan Institute: Major contribution of serine peptidases.

In Brazil, envenomation by Bothrops pitvipers is responsible for over 73% of snakebites, and their venom is a rich source of proteolytic enzymes. Most studies have demonstrated that Bothrops jararaca venom acts on macromolecular substrates, causing an imbalance in the victim's hemostatic system. In contrast, fewer studies have examined the proteolytic activity on small molecules such as peptides. In this study, we used a set of bioactive peptides (insulin B chain, Met-enkephalin, Leu-enkephalin, neuropeptide Y, peptide YY, pancreatic polypeptide, substance P and somatostatin) to identify new peptide substrates for the metallopeptidases and serine peptidases from the B. jararaca venom. The majority of these peptides were substrates for the venom, but neuropeptide Y and pancreatic polypeptide presented higher hydrolyses rates. Although most of the peptides were simultaneously substrates for both classes of proteases, serine peptidases were the most active. Substance P was an exclusive substrate for metallopeptidases, while somatostatin was a selective substrate for serine peptidases. The neutralizing efficacy of the bothropic antivenom produced by the Butantan Institute was also assessed and found to totally prevent substance P hydrolysis, whereas somatostatin cleavage was not inhibited. Thus, the antivenom effectively inhibited metallopeptidase activity, but did not neutralize some of the serine peptidases. These results indicate that, in addition to cleaving proteins, the proteolytic enzymes from this venom also hydrolyze bioactive peptides, and this peptidase activity could effectively contribute to some of the many dire manifestations of envenomation.