RESUMO
Leishmaniasis is considered a neglected disease, which makes it an unattractive market for the pharmaceutical industry; hence, efforts in the search for biologically active substances are hampered by this lack of financial motivation. Thus, in the present study, we report the leishmanicidal activity and the possible mechanisms of action of compounds with promising activity against the species Leishmania (V.) braziliensis, the causative agent of the skin disease leishmaniasis. The natural compound 1a (piplartine) and the analog 2a were the most potent against promastigote forms with growth inhibition values for 50% of the parasite population (IC50) = 8.58 and 11.25 µM, respectively. For amastigote forms, the ICa50 values were 1.46 and 16.7 µM, respectively. In the molecular docking study, piplartine showed favorable binding energy (-7.13 kcal/mol) and with 50% inhibition of trypanothione reductase (IC50) = 91.1 µM. Preliminary investigations of the mechanism of action indicate that piplartine increased ROS levels, induced loss of cell membrane integrity, and caused accumulation of lipid bodies after 24 h of incubation at its lowest effective concentration (IC50), which was not observed for the synthetic analog 2a. The mode of action for the leishmanicidal activity of piplartine (1a) was assigned to involve affinity for the trypanothione reductase of Leishmania (V.) braziliensis TR.
Assuntos
Amidas/farmacologia , Leishmania braziliensis/efeitos dos fármacos , Piperidonas/farmacologia , Tripanossomicidas/farmacologia , Amidas/química , Animais , Linhagem Celular Tumoral , Chlorocebus aethiops , Simulação por Computador , Humanos , Simulação de Acoplamento Molecular , NADH NADPH Oxirredutases/antagonistas & inibidores , Piperidonas/química , Células VeroRESUMO
We report a structural and functional proteomics characterization of venoms of the two subspecies (Bothrops bilineatusbilineatus and B. b. smaragdinus) of the South American palm pit viper from the Brazilian state of Rondônia and B. b. smaragdinus from Perú. These poorly known arboreal and mostly nocturnal generalist predators are widely distributed in lowland rainforests throughout the entire Amazon region, where they represent an important cause of snakebites. The three B. bilineatus spp. venom samples exhibit overall conserved proteomic profiles comprising components belonging to 11 venom protein classes, with PIII (34-40% of the total venom proteins) and PI (8-18%) SVMPs and their endogenous tripeptide inhibitors (SVMPi, 8-10%); bradykinin-potentiating-like peptides (BBPs, 10.7-15%); snake venom serine proteinases (SVSP, 5.5-14%); C-type lectin-like proteins (CTL, 3-10%); phospholipases A2 (PLA2, 2.8-7.6%); cysteine-rich secretory proteins (CRISP, 0.9-2.8%); l-amino acid oxidases (LAO, 0.9-5%) representing the major components of their common venom proteomes. Comparative analysis of the venom proteomes of the two geographic variants of B. b. smaragdinus with that of B. b. bilineatus revealed that the two Brazilian taxa share identical molecules between themselves but not with Peruvian B. b. smaragdinus, suggesting hybridization between the geographically close, possibly sympatric, Porto Velho (RO, BR) B. b. smaragdinus and B. b. bilineatus parental populations. However, limited sampling does not allow determining the frequency of this event. The toxin arsenal of the South American palm pit vipers may account for the in vitro recorded collagenolytic, caseinolytic, PLA2, l-amino acid oxidase, thrombin-like and factor X-activating activities, and the clinical features of South American palm pit viper envenomings, i.e., local and progressively ascending pain, shock and loss of consciousness, spontaneous bleeding, and profound coagulopathy. The remarkable cross-reactivity of the Brazilian pentabothropic SAB antivenom toward the heterologous B. b. bilineatus venom suggests that the paraspecific antigenic determinants should have been already present in the venom of the last common ancestor of the Bothrops â³jararacaâ³ and â³taeniatusâ³ clades, about 8.5 Mya in the mid-late Miocene epoch of the Cenozoic era. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifiers PXD020043, PXD020026, and PXD020013.
Assuntos
Bothrops , Venenos de Crotalídeos , Crotalinae , Animais , Antivenenos , Proteoma/genética , Proteômica , Venenos de VíborasRESUMO
Polyclonal antibodies raised in Balb-c mice against BnSP-7, a Lys-49 phospholipase A2, were used to measure cross reactivity against other snake venoms. Using ELISA, these antibodies were able to recognize PLA2s isoforms present in venoms of botropic snakes at 1:6400, 1:3200 and 1:100 ratios (w/w). These antibodies highly recognized proteins of low molecular weight (â¼14,000) from crude snake venom Bp and Bm by Western Blotting. PLA2 these venoms, by alignment of primary structures demonstrated high identity with BnSP-7 PLA2, especially in the C-terminal region. However, the crude snake venom Bd and Bj, showed low recognition. The PLA2 activity of Bothrops pauloensis, Bothrops moojeni venoms or BpPLA2-TXI was inhibited significantly when anti-BnSP-7 antibodies were incubated at 1:10 and 1:20 ratios (venoms or toxin:anti-BnSP-7, w/w), respectively. The myotoxic effect induced by the same venoms was also reduced significantly at 1:1, 1:10 and 1:20 ratios, by decreased creatine kinase levels. The anti-PLA2 polyclonal antibodies effectively recognized PLA2s from Bothrops pauloensis and Bothrops moojeni venoms, and neutralized specific catalytic and myotoxic activity.
Assuntos
Anticorpos Monoclonais/imunologia , Bothrops/imunologia , Reações Cruzadas/imunologia , Venenos de Crotalídeos/imunologia , Fosfolipases A2/imunologia , Venenos de Serpentes/imunologia , Sequência de Aminoácidos , Animais , Western Blotting , Bothrops/classificação , Bothrops/metabolismo , Venenos de Crotalídeos/metabolismo , Ensaio de Imunoadsorção Enzimática , Masculino , Camundongos Endogâmicos BALB C , Testes de Neutralização , Fosfolipases A2/genética , Fosfolipases A2/metabolismo , Homologia de Sequência de Aminoácidos , Venenos de Serpentes/metabolismo , Especificidade da EspécieRESUMO
Snake venom toxins are related not only in detention, death and the promotion of initial digestion of prey but also due to their different biochemical, structural and pharmacological effects they can result in new drugs. Among these toxins snake venom serine proteases (SVSPs) should be highlighted because they are responsible for inducing changes in physiological functions such as blood coagulation, fibrinolysis, and platelet aggregation. This article presents the first serine protease (SP) isolated from Bothrops brazili: BbrzSP-32. The new SP showed 36 kDa of relative molecular mass and its absolute mass was confirmed by mass spectrometry as 32,520 Da. It presents 79.48% identity when compared to other SVSPs and was able to degrade the α-chain of fibrinogen, in in vitro models, because of this it is considered a SVTLE-A. It showed dose-dependent activity in the process of degradation of fibrin networks demonstrating greater specificity for this activity when compared to its thrombolytic action. BbrzSP-32 demonstrated proteolytic activity on gelatin and chromogenic substrates for serine proteases and thrombin-like enzymes (S-2288 and S-2238 respectively), besides having coagulant activity on human plasma. After pre-incubation with PMSF and benzamidine the coagulant and proteolytic activities on the S-2288 and S-2238 substrates were reduced. BbrzSP-32 shows stability against pH and temperature variations, demonstrating optimum activity between 30 and 40 °C and in the pH range 7.5 to 8.5. A new SP with potential biotechnological application was isolated.
Assuntos
Venenos de Crotalídeos/química , Serina Proteases/isolamento & purificação , Sequência de Aminoácidos , Animais , Bothrops , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Serina Proteases/químicaRESUMO
BACKGROUND/AIMS: To evaluate antileishmanial activity of crotamine, a toxin isolated from Crotalus durissus terrificus, in solution form and encapsulated in biodegradable microparticles in vitro. METHODS: Particles were analyzed on-chip by surface plasmon resonance and characterized by testing their diameters, zeta potential and encapsulation rate. The viability of promastigotes as well as murine macrophages was assessed. Furthermore, the phagocytic index was determined for macrophages, and cell supernatants were collected for the determination of TNF-α levels. An infection assay using Leishmania amazonensis-infected macrophages was also conducted. RESULTS: The diameters and zeta potential of control particles (1.35 µm; -12.3 mV) and of those containing crotamine (3.09 µm; -20.9 mV) were adequate for the assays conducted. Crotamine-loaded particles were better captured by macrophages than control particles (increase of 12% in the phagocytic index), leading to increased TNF-α levels (196 pg/ml), and they also induced a significant decrease in the numbers of amastigotes compared to infected macrophages only. CONCLUSION: The approach presented here opens the possibility of working with safe concentrations of encapsulated toxins to reach antileishmanial effects.
Assuntos
Antiprotozoários/farmacologia , Venenos de Crotalídeos/farmacologia , Leishmania/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , Animais , Antiprotozoários/administração & dosagem , Venenos de Crotalídeos/administração & dosagem , Crotalus , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/farmacologia , Ácido Láctico/química , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/microbiologia , Masculino , Camundongos Endogâmicos BALB C , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: The Combretum leprosum Mart. plant, popularly known as mofumbo, is used in folk medicine for inflammation, pain and treatment of wounds. From this species, it is possible to isolate three triterpenes: (3ß, 6ß, 16ß-trihydroxylup-20(29)-ene) called lupane, arjunolic acid and molic acid. In this study, through preclinical tests, the effect of lupane was evaluated on the cytotoxicity and on the ability to activate cellular function by the production of TNF-α, an inflammatory cytokine, and IL-10, an immuno regulatory cytokine was assessed. The effect of lupane on the enzymes topoisomerase I and II was also evaluated. METHODS: For this reason, peripheral blood mononuclear cells (PBMCs) were obtained and cytotoxicity was assessed by the MTT method at three different times (1, 15 and 24 h), and different concentrations of lupane (0.3, 0.7, 1.5, 6, 3 and 12 µg/mL). The cell function was assessed by the production of TNF-α and IL-10 by PBMCs quantified by specific enzyme immunoassay (ELISA). The activity of topoisomerases was assayed by in vitro biological assays and in silico molecular docking. RESULTS: The results obtained showed that lupane at concentrations below 1.5 µg/mL was not toxic to the cells. Moreover, lupane was not able to activate cellular functions and did not alter the production of IL-10 and TNF-α. Furthermore, the data showed that lupane has neither interfered in the action of topoisomerase I nor in the action of topoisomerase II. CONCLUSION: Based on preclinical results obtained in this study, we highlight that the compound studied (lupane) has moderate cytotoxicity, does not induce the production of TNF-α and IL-10, and does not act on human topoisomerases. Based on the results of this study and taking into consideration the reports about the anti-inflammatory and leishmanicidal activity of 3ß, 6ß, 16ß-trihydroxylup-20(29)-ene, we suggest that this compound may serve as a biotechnological tool for the treatment of leishmaniasis in the future.
Assuntos
Anti-Inflamatórios/toxicidade , Combretum , Leucócitos Mononucleares/efeitos dos fármacos , Triterpenos/toxicidade , Anti-Inflamatórios/farmacologia , DNA Topoisomerases/metabolismo , Flores , Humanos , Interleucina-10/biossíntese , Extratos Vegetais/farmacologia , Extratos Vegetais/toxicidade , Triterpenos/farmacologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Bothrops brazili is a snake found in the forests of the Amazonian region whose commercial therapeutic anti-bothropic serum has low efficacy for local myotoxic effects, resulting in an important public health problem in this area. Catalytically inactive phospholipases A2-like (Lys49-PLA2s) are among the main components from Bothrops genus venoms and are capable of causing drastic myonecrosis. Several studies have shown that the C-terminal region of these toxins, which includes a variable combination of positively charged and hydrophobic residues, is responsible for their activity. In this work we describe the crystal structures of two Lys49-PLA2s (BbTX-II and MTX-II) from B. brazili venom and a comprehensive structural comparison with several Lys49-PLA2s. Based on these results, two independent sites of interaction were identified between protein and membrane which leads to the proposition of a new myotoxic mechanism for bothropic Lys49-PLA2s composed of five different steps. This proposition is able to fully explain the action of these toxins and may be useful to develop efficient inhibitors to complement the conventional antivenom administration.
Assuntos
Bothrops , Venenos de Crotalídeos/química , Fosfolipases A2/química , Animais , Venenos de Crotalídeos/genética , Cristalografia por Raios X , Fosfolipases A2/genética , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
The prominent local myotoxic effects induced by Bothrops snake venom are due, in part, to myotoxins. This effect is not neutralized by antivenom, which is the main therapy for victims of snakebite. Two basic myotoxins named MjTX-I and MjTX-II were isolated from Bothrops moojeni venom. Both myotoxins have a Lys-49 phospholipase A2 structure devoid of enzymatic activity, but are highly myonecrotic and edema-inducing. In this study, we analyzed the effect of a low-level laser (LLL) at 685 nm, an energy density of 2.2 J cm(-2), and the irradiation time of 15 s, and a light emitting diode (LED) at 635 or 945 nm at energy densities of 4 and 3.8 J cm(-2), and irradiation times of 41 and 38 s, respectively, applied 30 min and 3 h after edema formation in mice caused by MjTX-I or MjTX-II. MjTX-I or MjTX-II caused a significant edema formation in envenomed paws. LLL and LED irradiation significantly reduced the edema formation by both myotoxins from 1 up to 6 hours after the injection. Both LLL and LEDs were similar in reducing the edema formation induced by myotoxins. The combined photobiostimulation with antivenom had the same effect in reducing edema as treatment with the LLL or LEDs alone. In conclusion, the results of this study indicate that photobiostimulation could be used in association with antivenom therapy for treatment of local effects of Bothrops species venom.
Assuntos
Bothrops/metabolismo , Edema/induzido quimicamente , Fosfolipases A/toxicidade , Peçonhas/metabolismo , Animais , Edema/radioterapia , Terapia com Luz de Baixa Intensidade , Masculino , Camundongos , Fosfolipases A/isolamento & purificação , Fosfolipases A/metabolismoRESUMO
In accidents involving Crotalus snakes, the crotoxin complex (CTX) plays lethal action due to its neurotoxic activity. On the other hand, CTX have potential biotechnological application due to its anti-tumoral, anti-inflammatory, antimicrobial, analgesic and immunomodulatory properties. CTX is a heterodimer composed of Crotoxin A (CA or crotapotin), the acidic nontoxic and non-enzymatic component and; Crotoxin B (CB), a basic, toxic and catalytic PLA2. Currently, there are two classes of CTX isoforms, whose differences in their biological activities have been attributed to features presented in CB isoforms. Here, we present the crystal structure of CB isolated from the Crotalus durissus collilineatus venom. It amino acid sequence was assigned using the SEQUENCE SLIDER software, which revealed that the crystal structure is a heterodimer composed of two new CB isoforms (colCB-A and colCB-B). Bioinformatic and biophysical analyses showed that the toxin forms a tetrameric assembly in solution similar to CB from Crotalus durissus terrificus venom, despite some differences observed at the dimeric interface. By the previously proposed classification, the colCB-B presents features of the class I isoforms while colCB-A cannot be classified into classes I and II based on its amino acid sequence. Due to similar features observed for other CB isoforms found in the NCBI database and the results obtained for colCB-A, we suggest that there are more than two classes of CTX and CB isoforms in crotalic venoms.
Assuntos
Venenos de Crotalídeos , Crotoxina , Serpentes Peçonhentas , Animais , Crotoxina/química , Fosfolipases A2/química , Crotalus/metabolismo , Venenos de Crotalídeos/química , Isoformas de Proteínas/metabolismoRESUMO
INTRODUCTION: Hantavirus, a zoonotic pathogen, causes severe syndromes like hemorrhagic fever with renal syndrome (HFRS), sometimes fatal in humans. Considering the importance of detecting the hantavirus antigen, the construction of an immunosensor is essential. The structural and functional characteristics of camelid nanobodies (VHHs) encourage their application in the areas of nanobiotechnology, therapeutics, diagnostics, and basic research. Therefore, this study aimed to standardize stable bioconjugates using gold nanoparticles (AuNPs) and VHHs, in order to develop immunobiosensors for the diagnosis of hantavirus infection. METHODS: Immobilized metal affinity chromatography (IMAC) was performed to obtain purified recombinant anti-hantavirus nucleocapsid nanobodies (anti-prNΔ85 VHH), while AuNPs were synthesized for bioconjugation. UV-visible spectrophotometry and transmission electron microscopy (TEM) analysis were employed to characterize AuNPs. RESULTS: The bioconjugation stability parameters (VHH-AuNPs), analyzed by spectrophotometry, showed that the ideal pH value and VHH concentration were obtained at 7.4 and 50 µg/mL, respectively, after addition of 1 M NaCl, which induces AuNP aggregation. TEM performed before and after bioconjugation showed uniform, homogeneous, well-dispersed, and spherical AuNPs with an average diameter of ~ 14 ± 0.57 nm. Furthermore, high-resolution images revealed a thin white halo on the surface of the AuNPs, indicating the coating of the AuNPs with protein. A biosensor simulation test (dot blot-like [DB-like]) was performed in stationary phase to verify the binding and detection limits of the recombinant nucleocapsid protein from the Araucária hantavirus strain (prN∆85). DISCUSSION: Using AuNPs/VHH bioconjugates, a specific interaction was detected between 5 and 10 min of reaction in a dose-dependent manner. It was observed that this test was sensitive enough to detect prNΔ85 at concentrations up to 25 ng/µL. Considering that nanostructured biological systems such as antibodies conjugated with AuNPs are useful tools for the development of chemical and biological sensors, the stability of the bioconjugate indicates proficiency in detecting antigens. The experimental results obtained will be used in a future immunospot assay or lateral flow immunochromatography analysis for hantavirus detection.
Assuntos
Técnicas Biossensoriais , Ouro , Nanopartículas Metálicas , Orthohantavírus , Anticorpos de Domínio Único , Ouro/química , Nanopartículas Metálicas/química , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/química , Orthohantavírus/imunologia , Humanos , Técnicas Biossensoriais/métodos , Anticorpos Antivirais/imunologia , Animais , Infecções por Hantavirus/diagnósticoRESUMO
The pursuit of novel treatment alternatives to address the accumulated resistance to antimicrobials over the years has prompted the scientific community to explore biodiversity, particularly animal venom, as a potential source of new antimicrobial drugs. Snake venoms, with their complex mixtures of components, are particularly promising targets for investigation in this regard. The search for novel molecules exhibiting antimicrobial activity against multidrug-resistant strains is of paramount importance for public health and numerous research groups worldwide. High expectations within the healthcare field are supported by the scientific literature, which highlights the potential development of innovative drugs through in vivo and in vitro application, depending on dose titration. Snake venoms and their molecules and peptides offer exponential possibilities for biotechnological applications as antimicrobial agents. However, many uncertainties and unexplored avenues remain, presenting opportunities for discoveries and research.
RESUMO
Snake venom-secreted phospholipase A2 (svPLA2) enzymes, both catalytically active and inactive, are a central component in envenoming. These are responsible for disrupting the cell membrane's integrity, inducing a wide range of pharmacological effects, such as the necrosis of the bitten limb, cardiorespiratory arrest, edema, and anticoagulation. Although extensively characterized, the reaction mechanisms of enzymatic svPLA2 are still to be thoroughly understood. This review presents and analyses the most plausible reaction mechanisms for svPLA2, such as the "single-water mechanism" or the "assisted-water mechanism" initially proposed for the homologous human PLA2. All of the mechanistic possibilities are characterized by a highly conserved Asp/His/water triad and a Ca2+ cofactor. The extraordinary increase in activity induced by binding to a lipid-water interface, known as "interfacial activation," critical for the PLA2s activity, is also discussed. Finally, a potential catalytic mechanism for the postulated noncatalytic PLA2-like proteins is anticipated.
Assuntos
Venenos de Crotalídeos , Venenos de Serpentes , Humanos , Fosfolipases A2/química , Fosfolipases A2/metabolismo , ÁguaRESUMO
Animal venoms and their chemical compounds have aroused both empirical and scientific attention for ages. However, there has been a significant increase in scientific investigations in recent decades, allowing the production of various formulations that are helping in the development of many important tools for biotechnological, diagnostic, or therapeutic use, both in human and animal health, as well as in plants. Venoms are composed of biomolecules and inorganic compounds that may have physiological and pharmacological activities that are not related to their principal actions (prey immobilization, digestion, and defense). Snake venom toxins, mainly enzymatic and non-enzymatic proteins, and peptides have been identified as potential prototypes for new drugs and/or models for the development of pharmacologically active structural domains for the treatment of cancer, cardiovascular diseases, neurodegenerative and autoimmune diseases, pain, and infectious-parasitic diseases. This minireview aims to provide an overview of the biotechnological potential of animal venoms, with a focus on snakes, and to introduce the reader to the fascinating world of Applied Toxinology, where animal biodiversity can be used to develop therapeutic and diagnostic applications for humans.
Assuntos
Neoplasias , Venenos de Serpentes , Animais , Humanos , Venenos de Serpentes/química , Serpentes/metabolismo , Proteínas/química , Peptídeos/farmacologia , Neoplasias/tratamento farmacológicoRESUMO
Snake envenomation is an ongoing global health problem and tropical neglected disease that afflicts millions of people each year. The only specific treatment, antivenom, has several limitations that affects its proper distribution to the victims and its efficacy against local effects, such as myonecrosis. The main responsible for this consequence are the phospholipases A2 (PLA2) and PLA2-like proteins, such as BthTX-I from Bothrops jararacussu. Folk medicine resorts to plants such as Tabernaemontana catharinensis to palliate these and other snakebite effects. Here, we evaluated the effect of its root bark extract and one of its isolated compounds, 12-methoxy-4-methyl-voachalotine (MMV), against the in vitro paralysis and muscle damage induced by BthTX-I. Secondary and quaternary structures of BthTX-I were not modified by the interaction with MMV. Instead, this compound interacted in an unprecedented way with the region inside the toxin hydrophobic channel and promoted a structural change in Val31, loop 58-71 and Membrane Disruption Site. Thus, we hypothesize that MMV inhibits PLA2-like proteins by preventing entrance of fatty acid into the hydrophobic channel. These data may explain the traditional use of T. catharinensis extract and confirm MMV as a promising candidate to complement antivenom or a structural guide to develop more effective inhibitors.
Assuntos
Bothrops , Venenos de Crotalídeos , Tabernaemontana , Animais , Antivenenos/farmacologia , Antivenenos/química , Tabernaemontana/metabolismo , Fosfolipases A2/química , Venenos de Serpentes , Venenos de Crotalídeos/química , Bothrops/metabolismoRESUMO
Varespladib (LY315920) is a potent inhibitor of human group IIA phospholipase A2 (PLA2) originally developed to control inflammatory cascades of diseases associated with high or dysregulated levels of endogenous PLA2. Recently, varespladib was also found to inhibit snake venom PLA2 and PLA2-like toxins. Herein, ex vivo neuromuscular blocking activity assays were used to test the inhibitory activity of varespladib. The binding affinity between varespladib and a PLA2-like toxin was quantified and compared with other potential inhibitors for this class of proteins. Crystallographic and bioinformatic studies showed that varespladib binds to PrTX-I and BthTX-I into their hydrophobic channels, similarly to other previously characterized PLA2-like myotoxins. However, a new finding is that an additional varespladib binds to the MDiS region, a particular site that is related to muscle cell disruption by these toxins. The present results further advance the characterization of the molecular interactions of varespladib with PLA2-like myotoxins and provide additional evidence for this compound as a promising inhibitor candidate for different PLA2 and PLA2-like toxins.
Assuntos
Bothrops , Venenos de Crotalídeos , Toxinas Biológicas , Animais , Humanos , Bothrops/metabolismo , Neurotoxinas , Cetoácidos , Venenos de Crotalídeos/toxicidade , Venenos de Crotalídeos/química , Fosfolipases A2/químicaRESUMO
Bothrops venom contains a high amount of secreted phospholipase A2 (sPLA2s) enzymes responsible for the inflammatory reaction and activation of leukocytes in cases of envenoming. PLA2s are proteins that have enzymatic activity and can hydrolyze phospholipids at the sn-2 position, thereby releasing fatty acids and lysophospholipids precursors of eicosanoids, which are significant mediators of inflammatory conditions. Whether these enzymes have a role in the activation and function of peripheral blood mononuclear cells (PBMCs) is not known. Here we show for the first time how two secreted PLA2s (BthTX-I and BthTX-II) isolated from the venom of Bothrops jararacussu affect the function and polarization of PBMCs. Neither BthTX-I nor BthTX-II exhibited significant cytotoxicity to isolated PBMCs compared with the control at any of the time points studied. RT-qPCR and enzyme-linked immunosorbent assays were used to determine changes in gene expression and the release of pro-inflammatory (TNF-α, IL-6, and IL-12) and anti-inflammatory (TGF-ß and IL-10) cytokines, respectively, during the cell differentiation process. Lipid droplets formation and phagocytosis were also investigated. Monocytes/macrophages were labeled with anti-CD14, -CD163, and -CD206 antibodies to assay cell polarization. Both toxins caused a heterogeneous morphology (M1 and M2) on days 1 and 7 based on immunofluorescence analysis, revealing the considerable flexibility of these cells even in the presence of typical polarization stimuli. Thus, these findings indicate that the two sPLA2s trigger both immune response profiles in PBMCs indicating a significant degree of cell plasticity, which may be crucial for understanding the consequences of snake envenoming.
Assuntos
Bothrops , Venenos de Crotalídeos , Fosfolipases A2 Secretórias , Mordeduras de Serpentes , Humanos , Animais , Antivenenos , Leucócitos Mononucleares , Venenos de Serpentes , Poliésteres , Venenos de Crotalídeos/toxicidadeRESUMO
L-Amino acid oxidase (LAAO) is an enzyme found in snake venom that has multifaceted effects, including the generation of hydrogen peroxide (H2O2) during oxidative reactions, leading to various biological and pharmacological outcomes such as apoptosis, cytotoxicity, modulation of platelet aggregation, hemorrhage, and neutrophil activation. Human neutrophils respond to LAAO by enhancing chemotaxis, and phagocytosis, and releasing reactive oxygen species (ROS) and pro-inflammatory mediators. Exosomes cellular nanovesicles play vital roles in intercellular communication, including immune responses. This study investigates the impact of Calloselasma rhodostoma snake venom-derived LAAO (Cr-LAAO) on human neutrophil exosome release, including activation patterns, exosome formation, and content. Neutrophils isolated from healthy donors were stimulated with Cr-LAAO (100 µg/mL) for 3 h, followed by exosome isolation and analysis. Results show that Cr-LAAO induces the release of exosomes with distinct protein content compared to the negative control. Proteomic analysis reveals proteins related to the regulation of immune responses and blood coagulation. This study uncovers Cr-LAAO's ability to activate human neutrophils, leading to exosome release and facilitating intercellular communication, offering insights into potential therapeutic approaches for inflammatory and immunological disorders.
Assuntos
Exossomos , L-Aminoácido Oxidase , Humanos , L-Aminoácido Oxidase/farmacologia , L-Aminoácido Oxidase/metabolismo , Neutrófilos , Exossomos/metabolismo , Peróxido de Hidrogênio/farmacologia , Proteômica , Venenos de SerpentesRESUMO
Two myotoxic and noncatalytic Lys49-phospholipases A(2) (braziliantoxin-II and MT-II) and a myotoxic and catalytic phospholipase A(2) (braziliantoxin-III) from the venom of the Amazonian snake Bothrops brazili were crystallized. The crystals diffracted to resolutions in the range 2.56-2.05â Å and belonged to space groups P3(1)21 (braziliantoxin-II), P6(5)22 (braziliantoxin-III) and P2(1) (MT-II). The structures were solved by molecular-replacement techniques. Both of the Lys49-phospholipases A(2) (braziliantoxin-II and MT-II) contained a dimer in the asymmetric unit, while the Asp49-phospholipase A(2) braziliantoxin-III contained a monomer in its asymmetric unit. Analysis of the quaternary assemblies of the braziliantoxin-II and MT-II structures using the PISA program indicated that both models have a dimeric conformation in solution. The same analysis of the braziliantoxin-III structure indicated that this protein does not dimerize in solution and probably acts as a monomer in vivo, similar to other snake-venom Asp49-phospholipases A(2).
Assuntos
Bothrops , Venenos de Crotalídeos/química , Fosfolipases A2/química , Animais , Cristalização , Cristalografia por Raios X , Isoenzimas/químicaRESUMO
CONTEXT: Sapindus saponaria L. (Sapindaceae) bark, root, and fruits are used as sedatives and to treat gastric ulcer and also demonstrate diuretic and expectorant effects. OBJECTIVE: The anti-snake venom properties of callus of S. saponaria are investigated here for the first time. MATERIALS AND METHODS: In vitro cultivated callus of Sapindus saponaria were lyophilized, and the extracts were prepared with different solvents, before submitting to phytochemical studies and evaluation of the anti-ophidian activity. Crude extracts were fractionated by liquid-liquid partition and the fractions were monitored by thin layer chromatography (TLC). Subsequently, anti-ophidian activities were analyzed toward Bothrops jararacussu Lacerda (Viperidae), B. moojeni Hoge (Viperidae), B. alternates Duméril (Viperidea) and Crotalus durissus terrificus Lineu (Viperidae) venoms and isolated myotoxins and phospholipase A(2) (PLA(2)). RESULTS: Fractions A1, A2 and the extract in MeOH:H(2)O (9:1) significantly inhibited the toxic and pharmacological activities induced by snake venoms and toxins, when compared to other extracts and fractions. The lethal, clotting, phospholipase, edema-inducing, hemorrhagic and myotoxic activities were partially inhibited by the different extracts and fractions. TLC profiles of the crude extracts (B and C) and fractions (A1 and A2) showed ß-sitosterol and stigmasterol as their main compounds. Stigmasterol exhibited inhibitory effects on enzymatic and myotoxic activities of PLA(2). DISCUSSION AND CONCLUSION: Sapindus saponaria extracts and fractions presented anti-ophidian activity and could be used as an adjuvant to serum therapy or for its supplementation, and in addition, as a rich source of potential inhibitors of enzymes involved in several pathophysiological human and animal diseases.
Assuntos
Antivenenos/farmacologia , Extratos Vegetais/farmacologia , Sapindus/química , Venenos de Víboras/antagonistas & inibidores , Animais , Antivenenos/isolamento & purificação , Bothrops , Cromatografia em Camada Fina , Crotalus , Masculino , Camundongos , Fosfolipases A2/metabolismo , Sitosteroides/isolamento & purificação , Sitosteroides/farmacologia , Estigmasterol/isolamento & purificação , Estigmasterol/farmacologia , Venenos de Víboras/toxicidadeRESUMO
The fascination and fear of snakes dates back to time immemorial, with the first scientific treatise on snakebite envenoming, the Brooklyn Medical Papyrus, dating from ancient Egypt. Owing to their lethality, snakes have often been associated with images of perfidy, treachery and death. However, snakes did not always have such negative connotations. The curative capacity of venom has been known since antiquity, also making the snake a symbol of pharmacy and medicine. Today, there is renewed interest in pursuing snake-venom-based therapies. This Review focuses on the chemistry of snake venom and the potential for venom to be exploited for medicinal purposes in the development of drugs. The mixture of toxins that constitute snake venom is examined, focusing on the molecular structure, chemical reactivity and target recognition of the most bioactive toxins, from which bioactive drugs might be developed. The design and working mechanisms of snake-venom-derived drugs are illustrated, and the strategies by which toxins are transformed into therapeutics are analysed. Finally, the challenges in realizing the immense curative potential of snake venom are discussed, and chemical strategies by which a plethora of new drugs could be derived from snake venom are proposed.