RESUMO
This study investigated crotamine (CTA), a peptide derived from the venom of the South American rattlesnake Crotalus durissus terrificus, known for its exceptional cell penetration potential. The objective was to explore the antibacterial and antifungal activity of CTA, its ability to inhibit efflux pumps and evaluate the effectiveness of its pharmacological combination with antibiotics and antifungals. In microbiological assays, CTA in combination with antibiotics was tested against strains of S. aureus and the inhibition of NorA, Tet(K) and MepA efflux pumps was also evaluated. CTA alone did not present clinically relevant direct antibacterial action, presenting MIC > 209.7 µM against strains S. aureus 1199B, IS-58, K2068. The standard efflux pump inhibitor CCCP showed significant effects in all negative relationships to assay reproducibility. Against the S. aureus 1199B strain, CTA (20.5 µM) associated with norfloxacin diluted 10 × (320.67 µM) showed a potentiating effect, in relation to the control. Against the S. aureus IS-58 strain, the CTA associated with tetracycline did not show a significant combinatorial effect, either with 2304 or 230.4 µM tetracycline. CTA at a concentration of 2.05 µM associated with ciprofloxacin at a concentration of 309.4 µM showed a significant potentiating effect. In association with EtBr, CTA at concentrations of 2.05 and 20.5 µM potentiated the effect in all strains tested, reducing the prevention of NorA, Tet(K) and MepA efflux pumps. In the C. albicans strain, a potentiating effect of fluconazole (334.3 µM) was observed when combined with CTA (2.05 µM). Against the C. tropicalis strain, a significant effect was also observed in the association of fluconazole 334.3 µM, where CTA 2.05 µM considerably reduced fungal growth and decreased the potentiation of fluconazole. Against the C. krusei strain, no significant potentiating effect of fluconazole was obtained by CTA. Our results indicate that CTA in pharmacological combination potentiates the effects of antibiotics and antifungal. This represents a new and promising antimicrobial strategy for treating a wide variety of infections.
Assuntos
Antibacterianos , Antifúngicos , Venenos de Crotalídeos , Crotalus , Testes de Sensibilidade Microbiana , Antifúngicos/farmacologia , Antifúngicos/química , Antibacterianos/farmacologia , Venenos de Crotalídeos/farmacologia , Animais , Staphylococcus aureus/efeitos dos fármacos , Sinergismo Farmacológico , Candida albicans/efeitos dos fármacos , Serpentes PeçonhentasRESUMO
Snake venom metalloproteases (SVMPs) are hydrolytic enzymes dependent on metal binding, primarily zinc (Zn2+), at their catalytic site. They are classified into three classes (P-I to P-III). BjussuMP-II, a P-I SVMP isolated from Bothrops jararacussu snake venom, has a molecular mass of 24 kDa. It exhibits inhibitory activity on platelet aggregation and hydrolyzes fibrinogen. TNF-α upregulates the expression of adhesion molecules on endothelial cell surfaces, promoting leukocyte adhesion and migration during inflammation. Literature indicates that SVMPs may cleave the TNF-α precursor, possibly due to significant homology between metalloproteases from mammalian extracellular matrix and SVMPs. This study aimed to investigate BjussuMP-II's effects on human umbilical vein endothelial cells (HUVEC), focusing on viability, detachment, adhesion, release, and cleavage of TNF-α, IL-1ß, IL-6, IL-8, and IL-10. HUVEC were incubated with BjussuMP-II (1.5-50 µg/mL) for 3-24 h. Viability was determined using LDH release, MTT metabolization, and 7AAD for membrane integrity. Adhesion and detachment were assessed by incubating cells with BjussuMP-II and staining with Giemsa. Cytokines were quantified in HUVEC supernatants using EIA. TNF-α cleavage was evaluated using supernatants from PMA-stimulated cells or recombinant TNF-α. Results demonstrated BjussuMP-II's proteolytic activity on casein. It was not toxic to HUVEC at any concentration or duration studied but interfered with adhesion and promoted detachment. PMA induced TNF-α release by HUVEC, but this effect was not observed with BjussuMP-II, which cleaved TNF-α. Additionally, BjussuMP-II cleaved IL-1ß, IL-6, and IL-10. These findings suggest that the zinc metalloprotease BjussuMP-II could be a valuable biotechnological tool for treating inflammatory disorders involving cytokine deregulation.
Assuntos
Adesão Celular , Citocinas , Células Endoteliais da Veia Umbilical Humana , Metaloproteases , Humanos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Citocinas/metabolismo , Metaloproteases/metabolismo , Adesão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Bothrops/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Venenos de Crotalídeos/metabolismo , Venenos de Crotalídeos/toxicidade , Proteólise/efeitos dos fármacosRESUMO
Lectins are a large group of proteins found in many snake venoms. BjcuL is a C-type lectin from Bothrops jararacussu snake venom that does not present cytotoxicity action on human peripheral blood mononuclear cells (PBMCs) at concentrations of 5 and 10 µg/mL. BjcuL demonstrates an immunomodulatory role in PBMCs with the production of pro- and anti-inflammatory cytokines (IL-2, IL-10, IFN-γ, IL-6, TNF-α, and IL-17) in addition to stimulate T cells to produce reactive oxygen species (ROS) that could play a role in the acute inflammatory reaction observed in the victims. Inflammasomes are an essential arm in cells of innate immunity to detect and sense a range of endogenous or exogenous, sterile, or infectious stimuli to elicit cellular responses and effector mechanisms. NLRP3 inflammasome is a significant target for this study, because the lectin is responsible for leukocyte activation stimulating the release of inflammatory mediators, which results in dynamic cellular responses to remove the detrimental process to the body in snakebites. Thus, this study aimed to investigate how isolated BjcuL from B. jararacussu venom affects NLRP3 inflammasome activation on PBMCs. For this, the cells were isolated by density gradient and incubated with BjcuL at different periods and concentrations for the evaluation of the activation of the NLRP3 inflammasome through gene and protein expressions of ASC, CASPASE-1, and NLRP3 by RT-qPCR, Western blot, and immunofluorescence, as well as the participation of Toll-like receptor 4 (TLR4) and ROS in the IL-1ß production, a product resultant of the NLRP3 inflammasome activation. Herein, BjcuL interacts with TLR4 as demonstrated by in vitro and in silico studies and induces cytokines release via NF-κB signaling. By genic and protein expression assays, BjcuL activates NLRP3 inflammasome, and the pharmacological modulation with LPS-RS, an antagonist of TLR4; LPS-SM, an agonist of TLR4; MCC950, a specific NLRP3 inhibitor, and rotenone, an inhibitor of mitochondrial ROS, confirmed the participation of TLR4 and ROS in the NLRP3 inflammasome activation and IL-1ß liberation. The effects of BjcuL on the regulation and activation of the NLRP3 inflammasome complex via TLR4 activation with ROS participation may be determinant for the development of the inflammatory local effects seen in snakebite victims. In addition, in silico together with in vitro studies provide information that may be useful in the rational design of TLR agonists as well as new adjuvants for immunomodulatory therapy.
Assuntos
Inflamassomos , Leucócitos Mononucleares , Humanos , Inflamassomos/metabolismo , Leucócitos Mononucleares/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Receptor 4 Toll-Like/metabolismo , Lectinas Tipo C/metabolismo , Lipopolissacarídeos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Citocinas/metabolismo , Interleucina-1beta/metabolismoRESUMO
BACKGROUND: The venom of Crotalus durissus terrificus, as well as its fractions, has intrigued research groups worldwide who are working to isolate, characterize, and find possible biotechnological applications. A number of studies have elucidated that these fractions and their derivatives possess pharmacological properties, which can enable the development of new drug prototypes with anti-inflammatory, antinociceptive, antitumor, antiviral, and antiparasitic applications. OBJECTIVE: This review presents a systematic study on Crotalus durissus terrificus, the most notable crotalid subspecies in South America, focusing on the composition, toxicological mechanisms, structural aspects, and applications of the main venom toxins (convulxin, gyroxin, crotamine, crotoxin, and their subunits). CONCLUSION: The authors have found that research on this snake and its toxins is still an area of focus, despite that almost a century has passed since the isolation of crotoxin. Several applications of these proteins in the development of novel drugs and bioactive substances have also been demonstrated.
Assuntos
Venenos de Crotalídeos , Crotoxina , Animais , Crotoxina/farmacologia , Crotoxina/uso terapêutico , Crotoxina/química , Crotalus , Venenos de Crotalídeos/química , América do Sul , BiologiaRESUMO
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 â 23â¯kDa. BmooMPα-I was able to inhibit in vitro growth of W2 P. falciparum, with an IC50 value of 16.14⯵g/mL. Virtual screening with Pep1 BM demonstrated two PfPNP target binding regions, with ΔG values at the interaction interface of -10.75â¯kcal/mol and -11.74â¯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.