Role of the complement system in kidney cell death induced by Loxosceles venom Sphingomyelinases D.

Envenomation by Loxosceles spiders can result in local and systemic pathologies. Systemic loxoscelism, which can lead to death, is characterized by intravascular hemolysis, platelet aggregation, and acute kidney injury. Sphingomyelinase D (SMase D) in Loxosceles spider venom is responsible for both local and systemic pathologies, and has been shown to induce metalloprotease activity. As the complement system is involved in many renal pathologies and is involved in hemolysis in systemic loxoscelism, the aim of this study was to investigate its role and the role of complement regulators and metalloproteases in an in vitro model of Loxosceles venom induced renal pathology. We investigated the effects of the venom/SMase D and the complement system on the HK-2 kidney cell line. Using cell viability assays, western blotting, and flow cytometry, we show that human serum, as a source of complement, enhanced the venom/SMase D induced cell death and the deposition of complement components and properdin. Inhibitors for ADAM-10 and ADAM-17 prevented the venom induced release of the of the complement regulator MCP/CD46 and reduced the venom/SMase D induced cell death. Our results show that the complement system can contribute to Loxosceles venom induced renal pathology. We therefore suggest that patients experiencing systemic loxoscelism may benefit from treatment with metalloproteinase inhibitors and complement inhibitors, but this proposition should be further analyzed in future pre-clinical and clinical assays.

Mechanism of neutrophil dysfunction: neutrophil serine proteases cleave and inactivate the C5a receptor.

Neutrophil dysfunction, resulting in inefficient bacterial clearance, is a feature of several serious medical conditions, including cystic fibrosis (CF) and sepsis. Poorly controlled neutrophil serine protease (NSP) activity and complement activation have been implicated in this phenomenon. The capacity for excess NSP secretion and complement activation to influence the expression and function of the important neutrophil-activating receptor C5aR was investigated. Purified NSPs cathepsin G (CG), neutrophil elastase (NE), and proteinase 3 cleaved C5aR to a 26- to 27-kDa membrane-bound fragment, thereby inactivating its C5a-induced signaling ability. In a supernatant transfer assay, NSPs released from neutrophils in response to C5a induced the cleavage of the C5aR on unstimulated cells. Stimulation of myeolomonocytic U937 cells and purified neutrophils with C5a resulted in downregulation of the C5aR on these cells, which, in the case of U937 cells, was largely caused by NSP-mediated cleavage of C5aR, but in the case of neutrophils, intracellular degradation was likely the main mediator in addition to a small role for NSPs. CG and NE in bronchoalveolar lavage fluid from CF patients both contributed to C5aR cleavage. We propose two converging models for C5a- and NSP-mediated neutrophil dysfunction whereby C5aR cleavage is induced by NSPs, secreted in response to: 1) excess C5a generation or other stimuli; or 2) necrosis. The consequent impairment of C5aR activity contributes to suboptimal local neutrophil priming and bacterial clearance. NSP inhibitors with specificity for both CG and NE may aid the treatment of pathologies associated with neutrophil dysfunction including sepsis and CF.

Sphingomyelinases D From Loxosceles Spider Venoms and Cell Membranes: Action on Lipid Rafts and Activation of Endogenous Metalloproteinases.

Loxosceles spider venom contains Sphingomyelinase D (SMase D), the key toxin causing pathology. SMase D hydrolyzes the main component of lipid rafts, sphingomyelin, which changes the membrane microenvironment resulting in the activation of endogenous metalloproteinase from the ADAMs family. Alterations in membrane microenvironment of lipid rafts contribute to the activation of several cell surface molecules. Serine proteinases convertases acting on the pro-domain of membrane metalloproteinases, such as ADAMs, increase the cleavage and the release of proteins ectodomains and receptors located at the cell surface areas containing lipid rafts. We, therefore, investigated the interaction of SMases D with these membrane microdomains (lipid rafts) in human keratinocytes, to better understand the molecular mechanism of SMases D action, and identify the ADAM(s) responsible for the cleavage of cell surface molecules. Using specific inhibitors, we observed that ADAMs 10 and 17 are activated in the cell membrane after SMase D action. Furthermore, proproteins convertases, such as furin, are involved in the SMase D induced ADAMs activation. One of the signaling pathways that may be involved in the activation of these proteases is the MAPK pathway, since phosphorylation of ERK1/2 was observed in cells treated with SMase D. Confocal analysis showed a strong colocalization between SMase D and GM1 ganglioside present in rafts. Analysis of structural components of rafts, such as caveolin-1 and flotillin-1, showed that the action of SMase D on cell membranes leads to a reduction in caveolin-1, which is possibly degraded by toxin-induced superoxide production in cells. The action of the toxin also results in flotilin-1 increased detection in the cell membrane. These results indicate that SMases D from Loxosceles venoms alter membrane rafts structure, leading to the activation of membrane bound proteases, which may explain why the lipase action of this toxin can result in proteolytic cleavage of cell surface proteins, ultimately leading to pathology.

Characterisation of the complement susceptibility of the rat aortic smooth muscle cell line A7r5.

Complement (C) activation is thought to contribute to the initiation and progression of atherosclerosis. Proliferation of smooth muscle cells plays an important role in atherosclerotic plaque formation. Our aim was to investigate the suitability of the rat aortic smooth muscle cell line A7r5 as an in vitro model to study C-induced events in smooth muscle cells. A7r5 cells abundantly expressed membrane bound C-regulators (CReg) Crry and CD59 as assessed by flow-cytometry, but no DAF or MCP was detected. Using RT-PCR in addition to Crry and CD59, also mRNA for rat DAF but not for MCP was detected. Flow-cytometry of cells removed by EDTA instead of trypsin demonstrated that A7r5 did express cell surface DAF. Upon prolonged culturing under either logarithmic growing conditions or under conditions where cells were kept over-confluent, two different sub cell lines were obtained, one which had lost the expression of CD59, while the other showed increased expression of DAF and Crry. The change in expression of these CReg resulted in a change in C-susceptibility. Incubation of the A7r5 cells with human serum induced membrane attack complex dependent proliferation. Transfection with human CD59 efficiently protected the cells from C-mediated killing and C-induced cell proliferation. Our results show that A7r5 cells can be used as an in vitro model for C-induced events, but care has to be taken to use the cells at an early stage of passaging as they readily change their phenotype.

Loxosceles venom Sphingomyelinase D activates human blood leukocytes: Role of the complement system.

Envenomation by Loxosceles spiders can result in severe systemic and local reactions, which are mainly triggered by Sphingomyelinase D (SMase D), a toxic component of Loxosceles venom. SMase D induces a systemic inflammatory condition similar to the reaction observed during an endotoxic shock. Considering the potent pro-inflammatory potential of Loxosceles venom and the SMase D, in this study we have used the whole human blood model to study the endotoxic-like shock triggered by SMase D. Recombinant purified SMase D from L. intermedia venom, similarly to LPS, induced activation of blood leukocytes, as observed by the increase in the expression of CD11b and TLR4, production of reactive oxygen and nitrogen species (superoxide anion and peroxynitrite) and release of TNF-α. Complement consumption in the plasma was also detected, and complement inhibition by compstatin decreased the SMase D and LPS-induced leukocyte activation, as demonstrated by a reduction in the expression of CD11b and TLR4 and superoxide anion production. Similar results were found for the L. intermedia venom, except for the production of TNF-α. These findings indicate that SMase D present in Loxosceles venom is able to activate leukocytes in a partially complement-dependent manner, which can contribute to the systemic inflammation that follows envenomation by this spider. Thus, future therapeutic management of systemic Loxosceles envenomation could include the use of complement inhibitors as adjunct therapy.

Venom from Bothrops lanceolatus, a Snake Species Native to Martinique, Potently Activates the Complement System.

Bothrops lanceolatus snake venom causes systemic thrombotic syndrome but also local inflammation involving extensive oedema, pain, and haemorrhage. Systemic thrombotic syndrome may lead to fatal pulmonary embolism and myocardial and cerebral infarction. Here, we investigated the ability of B. lanceolatus venom to activate the Complement system (C) in order to improve the understanding of venom-induced local inflammation. Data presented show that B. lanceolatus venom is able to activate all C-pathways. In human serum, the venom strongly induced the generation of anaphylatoxins, such as C5a and C4a, and the Terminal Complement complex. The venom also induced cleavage of purified human components C3, C4, and C5, with the production of biologically active C5a. Furthermore, the venom enzymatically inactivated the soluble C-regulator and the C1-inhibitor (C1-INH), and significantly increased the expression of bound C-regulators, such as MCP and CD59, on the endothelial cell membrane. Our observations that B. lanceolatus venom activates the three Complement activation pathways, resulting in anaphylatoxins generation, may suggest that this could play an important role in local inflammatory reaction and systemic thrombosis caused by the venom. Inactivation of C1-INH, which is also an important inhibitor of several coagulation proteins, may also contribute to inflammation and thrombosis. Thus, further in vivo studies may support the idea that therapeutic management of systemic B. lanceolatus envenomation could include the use of Complement inhibitors as adjunct therapy.

Tetracycline Reduces Kidney Damage Induced by Loxosceles Spider Venom.

Envenomation by Loxosceles spider can result in two clinical manifestations: cutaneous and systemic loxoscelism, the latter of which includes renal failure. Although incidence of renal failure is low, it is the main cause of death, occurring mainly in children. The sphingomyelinase D (SMase D) is the main component in Loxosceles spider venom responsible for local and systemic manifestations. This study aimed to investigate the toxicity of L. intermedia venom and SMase D on kidney cells, using both In vitro and in vivo models, and the possible involvement of endogenous metalloproteinases (MMP). Results demonstrated that venom and SMase D are able to cause death of human kidney cells by apoptosis, concomitant with activation and secretion of extracellular matrix metalloproteases, MMP-2 and MMP-9. Furthermore, cell death and MMP synthesis and secretion can be prevented by tetracycline. In a mouse model of systemic loxoscelism, Loxosceles venom-induced kidney failure was observed, which was abrogated by administration of tetracycline. These results indicate that MMPs may play an important role in Loxosceles venom-induced kidney injury and that tetracycline administration may be useful in the treatment of human systemic loxoscelism.

Role of matrix metalloproteinases in HaCaT keratinocytes apoptosis induced by loxosceles venom sphingomyelinase D.

Envenomation by the spider Loxosceles (brown spider) can result in dermonecrosis and severe ulceration. We have previously shown that Loxosceles sphingomyelinase D (SMaseD), the enzyme responsible for these pathological effects, induced expression of matrix metalloproteinase-9 (MMP-9), which is possibly one of the main factors involved in the pathogenesis of the cutaneous loxoscelism. The aim of this study was to further investigate the molecular mechanisms triggered by Loxosceles SMaseD involved in the initiation of the dermonecrotic lesion, using HaCaT cultures, a human keratinocyte cell line, as an in vitro model for cutaneous loxoscelism. We show here that SMaseD from Loxosceles spider venom induces apoptosis in human keratinocytes, which is associated with an increased expression of metalloproteinase-2 and -9, and that the use of metalloproteinase inhibitors, such as tetracycline, may prevent cell death and potentially may prevent tissue destruction after envenomation.

C-reactive protein-induced in vitro endothelial cell activation is an artefact caused by azide and lipopolysaccharide.

OBJECTIVE: C-reactive protein (CRP) has been proposed to be an independent risk factor for cardiovascular disease. In vitro studies investigating the mechanism behind this have used purified commercial CRP (cCRP) and endothelial cells. We investigated the role of contaminants in cCRP preparations. METHODS AND RESULTS: Human umbilical vein endothelial cells and the human endothelial cell line EA.hy926 were incubated with Escherichia coli-derived cCRP, in-house-generated azide-free recombinant, and ascites-purified CRP, azide, or lipopolysaccharide (LPS) equivalent to the concentration present in cCRP preparations. Cells were investigated for change in cell proliferation, morphology, apoptosis, and expression of endothelial NO synthase and intercellular adhesion molecule-1. Cell supernatants were assessed for monocyte chemoattractant protein-1 (MCP-1), interleukin-8, von Willebrand factor secretion, and pH change. Only cCRP was able to induce all activation events analyzed; however, this ability was lost on extensive dialysis, suggesting that low molecular weight contaminants were responsible for these events. Indeed, the effects of cCRP were mirrored by azide or LPS. CONCLUSIONS: We investigated a wide range of effects on endothelial cells ascribed to CRP; however, azide and LPS, but never CRP itself, were responsible for the cell activation events. We conclude that CRP, per se, does not activate endothelial cells.

Sphingomyelinase D from Loxosceles laeta Venom Induces the Expression of MMP7 in Human Keratinocytes: Contribution to Dermonecrosis.

Envenomation by Loxosceles spider is characterized by the development of dermonecrosis. In previous studies, we have demonstrated that increased expression/secretion of matrix metalloproteinases 2 and 9, induced by Loxosceles intermedia venom Class 2 SMases D (the main toxin in the spider venom), contribute to the development of cutaneous loxoscelism. In the present study we show that the more potent venom containing the Class 1 SMase D from Loxosceles laeta, in addition to increasing the expression/secretion of MMP2 and MMP9, also stimulates the expression of MMP7 (Matrilysin-1), which was associated with keratinocyte cell death. Tetracycline, a matrix metalloproteinase inhibitor, prevented cell death and reduced MMPs expression. Considering that L. laeta venom is more potent at inducing dermonecrosis than L. intermedia venom, our results suggest that MMP7 may play an important role in the severity of dermonecrosis induced by L. laeta spider venom SMase D. In addition, the inhibition of MMPs by e.g. tetracyclines may be considered for the treatment of the cutaneous loxoscelism.

Microcirculation abnormalities provoked by Loxosceles spiders' envenomation.

Loxoscelism is caused by envenomation by spiders from Loxosceles genus. Clinical symptoms only appear a few hours after envenomation and can evolve in local reactions, such as dermonecrosis, and systemic reactions, including intravascular haemolysis, intravascular coagulation and renal failure. Considering that alterations in the microcirculatory network are involved in the pathogenesis of different diseases, including the inflammatory process, the aim of this study was to investigate the action of venoms of males and females of Loxosceles intermedia and Loxosceles laeta on the microcirculatory network and examine the systemic production of inflammatory mediators in a murine model of loxoscelism. We observed that during systemic envenomation, the alterations in the microcirculation include increase in the number of rolling cells, which was more intense in animals injected with female Loxosceles spider venoms. This positively correlated with increase in TNF-α and NO serum levels, induction of which was higher by female venoms when compared with male venoms. The increase of leukocytes rolling was not accompanied by increase of cell adhesion. The absence of leukocyte extravasation may explain why in mice, in contrast to humans, no cutaneous loxoscelism occurs. Thus, targeting the neutrophil adhesion and extravasation in Loxosceles envenomed patients may prevent cutaneous pathology.

Characterization of a Gene Coding for the Complement System Component FB from Loxosceles laeta Spider Venom Glands.

The human complement system is composed of more than 30 proteins and many of these have conserved domains that allow tracing the phylogenetic evolution. The complement system seems to be initiated with the appearance of C3 and factor B (FB), the only components found in some protostomes and cnidarians, suggesting that the alternative pathway is the most ancient. Here, we present the characterization of an arachnid homologue of the human complement component FB from the spider Loxosceles laeta. This homologue, named Lox-FB, was identified from a total RNA L. laeta spider venom gland library and was amplified using RACE-PCR techniques and specific primers. Analysis of the deduced amino acid sequence and the domain structure showed significant similarity to the vertebrate and invertebrate FB/C2 family proteins. Lox-FB has a classical domain organization composed of a control complement protein domain (CCP), a von Willebrand Factor domain (vWFA), and a serine protease domain (SP). The amino acids involved in Mg2+ metal ion dependent adhesion site (MIDAS) found in the vWFA domain in the vertebrate C2/FB proteins are well conserved; however, the classic catalytic triad present in the serine protease domain is not conserved in Lox-FB. Similarity and phylogenetic analyses indicated that Lox-FB shares a major identity (43%) and has a close evolutionary relationship with the third isoform of FB-like protein (FB-3) from the jumping spider Hasarius adansoni belonging to the Family Salcitidae.

Loxosceles sphingomyelinase induces complement-dependent dermonecrosis, neutrophil infiltration, and endogenous gelatinase expression.

Envenomation by the spider Loxosceles can result in dermonecrosis and severe ulceration. Our aim was to investigate the role of the complement system and of the endogenous metalloproteinases in the initiation of the pathology of dermonecrosis. Histological analysis of skin of rabbits injected with Loxosceles intermedia venom and purified or recombinant sphingomyelinases showed a large influx of neutrophils, concomitant with dissociation of the collagenous fibers in the dermis. Decomplementation, using cobra venom factor, largely prevented the influx of neutrophils, while influx of neutrophils was also reduced in genetically C6-deficient rabbits, suggesting roles for both C5a and the membrane attack complex in the induction of dermonecrosis. However, C-depletion and C6 deficiency did not prevent the haemorrhage and the collagen injury. Zymography analysis of skin extracts showed the induction of expression of the endogenous gelatinase MMP-9 in the skin of envenomated animals. Rabbit neutrophils contained high levels of MMP-9, expression of which was further increased after incubation with venom, suggesting that these cells may be a source of the MMP-9 found in the skin of envenomated animals. Furthermore, skin fibroblasts also secreted MMP-9 and MMP-2 upon incubation with venom, suggesting that locally produced MMPs can also contribute to proteolytic tissue destruction.

C-reactive protein-induced in vitro vasorelaxation is an artefact caused by the presence of sodium azide in commercial preparations.

OBJECTIVE: Although C-reactive protein (CRP) is increasingly recognized as an independent risk factor for acute myocardial events, recent evidence suggests that it can directly induce vasorelaxation. This study aimed to investigate the mechanism of this CRP-induced response. METHODS AND RESULTS: Isometric tension recordings were used to measure endothelium-dependent and endothelium-independent vascular smooth muscle relaxation in isolated rabbit aortic rings. CRP generated in-house by genetic engineering and expressed in Chinese hamster ovary cells, CRP purified from ascites, and CRP obtained from commercial sources were assessed for vasorelaxing properties. Only the commercial CRP preparation induced vasorelaxation; more than half maximal relaxation was observed at 0.025 microg/mL and maximum relaxation attained at 0.25 microg/mL. Commercial CRP contains high levels of sodium azide, a well-known vasorelaxant. Removal of this agent by dialysis abolished the vasodilatory effect of commercial CRP. Sodium azide alone at concentrations equivalent to that present in the commercial CRP produced a near-identical relaxation pattern to the undialyzed commercial product. CONCLUSIONS: CRP has no vasorelaxant properties per se, and the reported vasorelaxant ability of CRP is an artifact caused by sodium azide present in commercial preparations of this agent.

Analysis of the toxic potential of venom from Loxosceles adelaida, a Brazilian brown spider from karstic areas.

Loxosceles adelaida spiders (Araneae, Sicariidae) are found near and inside the caves in the Parque Estadual Turistico do Alto Ribeira (PETAR), Sao Paulo, Brazil, which are visited by thousands of tourists every year. Several Loxosceles species are a public health problem in many regions of the world, by causing severe dermonecrosis and/or complement dependent haemolysis upon envenomation. The aim of this study was to characterize the biochemical and biological properties of L. adelaida venom and evaluate the toxic potential of envenomation by this non-synanthropic Loxosceles species. The biological activities of the L. adelaida venom was compared to that of Loxosceles gaucho, a synanthropic species of medical importance in Brazil. L. adelaida venom showed a similar potential to induce haemolysis, dermonecrosis and lethality as L. gaucho venom. L. adelaida crude venom was purified, yielding a 31 kDa component endowed with haemolytic and dermonecrotic activities. In conclusion, we show here that the troglophile Loxosceles species, L. adelaida, commonly found in the complex of caves from PETAR, is potentially able to cause envenomation with the same gravity of those produced by synanthropic species.

Molecular cloning, expression, function and immunoreactivities of members of a gene family of sphingomyelinases from Loxosceles venom glands.

Loxoscelism is the clinical condition produced by the venom of spiders belonging to the genus Loxosceles, which can be observed as two well-defined clinical variants: cutaneous loxoscelism and systemic or viscerocutaneous loxoscelism. We have recently identified, purified and characterised the toxins (sphingomyelinases) from Loxosceles intermedia venom that are responsible for all the local (dermonecrosis) and systemic effects (complement dependent haemolysis) induced by whole venom. In the present study, we have cloned and expressed the two functional sphingomyelinases isoforms, P1 and P2, and shown that the recombinant proteins display all the functional characteristics of whole L. intermedia venom, e.g., dermonecrotic and complement-dependent hemolytic activities and ability of hydrolyzing sphingomyelin. We have also compared the cross-reactivities of antisera raised against the toxins from different Loxosceles species and show here that the cross-reactivity is high when toxins are from the same species (P1 and P2 from L. intermedia) but low when the toxins are from different species (L. intermedia versus L. laeta). These data suggest that in order to obtain a suitable comprehensive neutralizing antiserum using the recombinant toxin as an immunogen, a mixture of the recombinant toxins from the different species has to be used. The use of anti-recombinant toxin antisera may have clinical benefits to those individuals displaying acute loxoscelic lesions.

A serine protease isolated from the bristles of the Amazonic caterpillar, Premolis semirufa, is a potent complement system activator.

BACKGROUND: The caterpillar of the moth Premolis semirufa, commonly named pararama, is found in the Brazilian Amazon region. Accidental contact with the caterpillar bristles causes an intense itching sensation, followed by symptoms of an acute inflammation, which last for three to seven days after the first incident. After multiple accidents a chronic inflammatory reaction, called "Pararamose", characterized by articular synovial membrane thickening with joint deformities common to chronic synovitis, frequently occurs. Although complement mediated inflammation may aid the host defense, inappropriate or excessive activation of the complement system and generation of anaphylatoxins can lead to inflammatory disorder and pathologies. The aim of the present study was to evaluate, in vitro, whether the Premolis semirufa's bristles extract could interfere with the human complement system. RESULTS: The bristles extract was able to inhibit the haemolytic activity of the alternative pathway, as well as the activation of the lectin pathway, but had no effect on the classical pathway, and this inhibition seemed to be caused by activation and consumption of complement components. The extract induced the production of significant amounts of all three anaphylatoxins, C3a, C4a and C5a, promoted direct cleavage of C3, C4 and C5 and induced a significant generation of terminal complement complexes in normal human serum. By using molecular exclusion chromatography, a serine protease of 82 kDa, which activates complement, was isolated from P. semirufa bristles extract. The protease, named here as Ps82, reduced the haemolytic activity of the alternative and classical pathways and inhibited the lectin pathway. In addition, Ps82 induced the cleavage of C3, C4 and C5 and the generation of C3a and C4a in normal human serum and it was capable to cleave human purified C5 and generate C5a. The use of Phenanthroline, metalloprotease inhibitor, in the reactions did not significantly interfere with the activity of the Ps82, whereas the presence of PMSF, serine protease inhibitor, totally blocked the activity. CONCLUSION: These data show that a serine protease present in the Premolis semirufa's bristles extract has the ability to activate the complement system, which may contribute to the inflammatory process presented in humans after envenomation.

Targeting Loxosceles spider Sphingomyelinase D with small-molecule inhibitors as a potential therapeutic approach for loxoscelism

Loxosceles spiders' venoms consist of a mixture of proteins, including the sphingomyelinases D (SMases D), which are the main toxic components responsible for local and systemic effects in human envenomation. Herein, based on the structural information of SMase D from Loxosceles laeta spider venom and virtual docking-based screening approach, three benzene sulphonate compounds (named 1, 5 and 6) were identified as potential Loxosceles SMase D inhibitors. All compounds inhibited the hydrolysis of the sphingomyelin substrate by both recombinant and native SMases D. Compounds 5 and 6 acted as SMases D uncompetitive inhibitors with Ki values of 0.49 mu M and 0.59 mu M, respectively. Compound 1 is a mixed type inhibitor, and presented a Ki value of 0.54 mu M. In addition, the three compounds inhibited the binding of SMases D to human erythrocytes and the removal of glycophorin C from the cell surface, which are important events in the complement-dependent haemolysis induced by Loxosceles venom. Moreover, compounds 5 and 6 reduced the binding of SMases to human keratinocytes membrane and the venom induced cell death. Importantly, compounds 5 and 6 also controlled the development of the necrotic lesion in an in vivo model of loxoscelism. Together, our findings indicate that the novel SMase D inhibitors presented here are able to suppress both local and systemic reactions induced by Loxosceles venoms. Since the number of Loxosceles envenomation accidents is currently growing worldwide, our results indicate that both inhibitors are promising scaffolds for the rational design of new drugs targeting SMases D from these spiders.

Animal venoms/toxins and the complement system.

Nature is a wealthy source of agents that have been shown to be beneficial to human health, but nature is also a rich source of potential dangerous health damaging compounds. This review will summarise and discuss the agents from the animal kingdom that have been shown to interact with the human complement (C) system. Most of these agents are toxins found in animal venoms and animal secretions. In addition to the mechanism of action of these toxins, their contribution to the field of complement, their role in human pathology and the potential benefit to the venomous animal itself will be discussed. Potential therapeutic applications will also be discussed.

Targeting Loxosceles spider Sphingomyelinase D with small-molecule inhibitors as a potential therapeutic approach for loxoscelism

Loxosceles spiders' venoms consist of a mixture of proteins, including the sphingomyelinases D (SMases D), which are the main toxic components responsible for local and systemic effects in human envenomation. Herein, based on the structural information of SMase D from Loxosceles laeta spider venom and virtual docking-based screening approach, three benzene sulphonate compounds (named 1, 5 and 6) were identified as potential Loxosceles SMase D inhibitors. All compounds inhibited the hydrolysis of the sphingomyelin substrate by both recombinant and native SMases D. Compounds 5 and 6 acted as SMases D uncompetitive inhibitors with Ki values of 0.49 mu M and 0.59 mu M, respectively. Compound 1 is a mixed type inhibitor, and presented a Ki value of 0.54 mu M. In addition, the three compounds inhibited the binding of SMases D to human erythrocytes and the removal of glycophorin C from the cell surface, which are important events in the complement-dependent haemolysis induced by Loxosceles venom. Moreover, compounds 5 and 6 reduced the binding of SMases to human keratinocytes membrane and the venom induced cell death. Importantly, compounds 5 and 6 also controlled the development of the necrotic lesion in an in vivo model of loxoscelism. Together, our findings indicate that the novel SMase D inhibitors presented here are able to suppress both local and systemic reactions induced by Loxosceles venoms. Since the number of Loxosceles envenomation accidents is currently growing worldwide, our results indicate that both inhibitors are promising scaffolds for the rational design of new drugs targeting SMases D from these spiders.