RESUMO
Tribolium castaneum, also known as the red flour beetle, is a polyphagous pest that seriously damages agricultural products, including stored and processed grains. Researchers have aimed to discover alternative pest control mechanisms that are less harmful to the ecosystem than those currently used. We conduct the purification and characterization of a protease inhibitor from C. plumieri seeds and an in vitro evaluation of its insecticidal potential against the insect pest T. castaneum. The trypsin inhibitor was isolated from C. plumieri seeds in a single-step DEAE-Sepharose column chromatography and had a molecular mass of 50 kDA. When analyzed for interaction with different proteolytic enzymes, the inhibitor exhibited specificity against trypsin and no activity against other serine proteases such as chymotrypsin and elastase-2. The isolated inhibitor was able to inhibit digestive enzymes of T. castaneum from extracts of the intestine of this insect. Therefore, we conclude that the new protease inhibitor, specific in tryptic inhibition, of protein nature from the seeds of C. plumieri was effective in inhibiting the digestive enzymes of T. castaneum and is a promising candidate in the ecological control of pests.
Assuntos
Tribolium , Inibidores da Tripsina , Animais , Inibidores da Tripsina/farmacologia , Inibidores da Tripsina/química , Inibidores da Tripsina/isolamento & purificação , Tribolium/enzimologia , Tribolium/efeitos dos fármacos , Proteínas de Insetos/química , Proteínas de Insetos/isolamento & purificação , Proteínas de Insetos/antagonistas & inibidores , Sementes/química , Inseticidas/farmacologia , Inseticidas/química , Inseticidas/isolamento & purificação , Proteínas de Plantas/farmacologia , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/químicaRESUMO
The incidence of Candida species resistant to traditional antifungal drugs is increasing globally. This issue significantly impacts patients' lives and increases healthcare expenses, confirming the need to develop novel therapeutic strategies. Recently, a thermostable trypsin inhibitor named ShTI (11.558 kDa), which has antibacterial effects on Staphylococcus aureus, was isolated from Salvia hispanica L. (chia) seeds. This study aimed to assess the antifungal effect of ShTI against Candida species and its synergism with fluconazole and to evaluate its mode of action. Preliminary toxicological studies on mouse fibroblasts were also performed. ShTI exhibited antifungal effects against C. parapsilosis (ATCC® 22,019), C. krusei (ATCC® 6258), and six clinical fluconazole-resistant strains of C. albicans (2), C. parapsilosis (2), and C. tropicalis (2). The minimum inhibitory concentration (MIC) values were 4.1 µM (inhibiting 50% of the isolates) and 8.2 µM (inhibiting 100% of the isolates). Additionally, when combined with fluconazole, ShTI had a synergistic effect on C. albicans, altering the morphological structure of the yeast. The mode of action of ShTI against C. krusei (ATCC® 6258) and C. albicans involves cell membrane permeabilization, the overproduction of reactive oxygen species, the formation of pseudohyphae, pore formation, and consequently, cell death. In addition, ShTI (8.65 and 17.3 µM) had noncytotoxic and nongenotoxic effects on L929 mouse fibroblasts. These findings suggest that ShTI could be a promising antimicrobial candidate, but further research is necessary to advance its application as a novel antifungal agent.
Assuntos
Antifúngicos , Candida , Farmacorresistência Fúngica , Fluconazol , Testes de Sensibilidade Microbiana , Salvia , Sementes , Inibidores da Tripsina , Antifúngicos/farmacologia , Antifúngicos/toxicidade , Fluconazol/farmacologia , Fluconazol/toxicidade , Candida/efeitos dos fármacos , Salvia/química , Sementes/química , Animais , Camundongos , Inibidores da Tripsina/farmacologia , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Fibroblastos/efeitos dos fármacos , Sinergismo Farmacológico , Candidíase/microbiologia , Candidíase/tratamento farmacológicoRESUMO
A novel trypsin inhibitor from Cajanus cajan (TIC) fresh leaves was partially purified by affinity chromatography. SDS-PAGE revealed one band with about 15 kDa with expressive trypsin inhibitor activity by zymography. TIC showed high affinity for trypsin (Ki = 1.617 µM) and was a competitive inhibitor for this serine protease. TIC activity was maintained after 24 h of treatment at 70 °C, after 1 h treatments with different pH values, and ß-mercaptoethanol increasing concentrations, and demonstrated expressive structural stability. However, the activity of TIC was affected in the presence of oxidizing agents. In order to study the effect of TIC on secreted serine proteases, as well as on the cell culture growth curve, SK-MEL-28 metastatic human melanoma cell line and CaCo-2 colon adenocarcinoma was grown in supplemented DMEM, and the extracellular fractions were submitted salting out and affinity chromatography to obtain new secreted serine proteases. TIC inhibited almost completely, 96 to 89%, the activity of these serine proteases and reduced the melanoma and colon adenocarcinoma cells growth of 48 and 77% respectively. Besides, it is the first time that a trypsin inhibitor was isolated and characterized from C. cajan leaves and cancer serine proteases were isolated and partial characterized from SK-MEL-28 and CaCo-2 cancer cell lines. Furthermore, TIC shown to be potent inhibitor of tumor protease affecting cell growth, and can be one potential drug candidate to be employed in chemotherapy of melanoma and colon adenocarcinoma.
Assuntos
Cajanus , Folhas de Planta , Humanos , Cajanus/química , Folhas de Planta/química , Células CACO-2 , Proliferação de Células/efeitos dos fármacos , Linhagem Celular Tumoral , Inibidores da Tripsina/farmacologia , Inibidores da Tripsina/química , Inibidores da Tripsina/isolamento & purificação , Proteínas de Plantas/farmacologia , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Serina Proteases/química , Serina Proteases/isolamento & purificação , Serina Proteases/metabolismoRESUMO
INTRODUCTION: Trypsin inhibitors (TIs) have the ability to competitively or non-competitively bind to trypsin and inhibit its action. These inhibitors are commonly found in plants and are used in protease inhibition studies involved in biochemical pathways of pharmacological interest. OBJECTIVES: This work aimed to purify a trypsin inhibitor from Bauhinia pulchella seeds (BpuTI), describing its kinetic mechanism and anticoagulant effect. METHODS: Affinity chromatography, protein assay, and SDS-PAGE were used to purify the inhibitor. Mass spectrometry, inhibition assays, and enzyme kinetics were used to characterize the inhibitor. In vitro assays were performed to verify its ability to prolong blood clotting time. RESULTS: Affinity chromatography on a Trypsin-Sepharose 4B column gave a yield of 43.1. BpuTI has an apparent molecular mass of 20 kDa with glycosylation (1.15%). Protein identification was determined by MS/MS, and BpuTI showed similarity to several Kunitz-type trypsin inhibitors. BpuTI inhibited bovine trypsin as an uncompetitive inhibitor with IC50 (3 x 10-6 M) and Ki (1.05 x 10-6 M). Additionally, BpuTI showed high stability to temperature and pH variations, maintaining its activity up to 100ºC and in extreme pH ranges. However, the inhibitor was susceptible to reducing agents, such as DTT, which completely abolished its activity. BpuTI showed an anticoagulant effect in vitro at a concentration of 33 µM, prolonging clotting time by 2.6 times. CONCLUSION: Our results suggest that BpuTI can be a biological tool to be used in blood clotting studies.
Assuntos
Bauhinia , Inibidores da Tripsina , Animais , Bovinos , Inibidores da Tripsina/farmacologia , Inibidores da Tripsina/química , Bauhinia/metabolismo , Tripsina/análise , Tripsina/química , Tripsina/metabolismo , Espectrometria de Massas em Tandem , Sementes/química , Anticoagulantes/farmacologia , Anticoagulantes/análise , Anticoagulantes/químicaRESUMO
Lepidopteran pests have been successfully managed by the adoption of insect resistant transgenic plants expressing Cry and/or Vip insecticidal proteins derived from Bacillus thuringiensis (Bt plants). Among such pests, Spodoptera frugiperda (Smith, 1797) (Lepidoptera: Noctuidae) is highlighted for its destructive potential in maize crops and for cases of field-evolved resistance to Bt plants. Cry insecticidal proteins expressed in Bt plants are known for their interaction with insect midgut receptors and subsequent midgut cell disruption that leads to target pest death. In the midgut of lepidopteran larval pests such as S. frugiperda, serine proteases are important in dietary protein digestion and activation or degradation of insecticidal proteins. This work was conducted to evaluate if the use of a soybean trypsin inhibitor (SBTI) could disrupt the development of a Bt-susceptible and a Bt-resistant population of S. frugiperda ingesting Bt (expressing Cry1F, Cry1A.105, and Cry2Ab2 Cry proteins) and non-Bt maize plants. The SBTI was produced and purified using recombinant expression in E. coli followed by purification in Ni-Sepharose. Bioassays using non-Bt maize leaves indicated that the development of susceptible and resistant populations of S. frugiperda was not influenced by the ingestion of SBTI. However, when the resistant population consumed Bt maize plants amended with SBTI, high mortality along with a reduction in larval weight and reduced activity of digestive trypsins were observed. Although the mode of action was not elucidated, it is possible that the consumption of SBTI increased susceptibility to Bt maize in the resistant population of S. frugiperda.
Assuntos
Bacillus thuringiensis , Inseticidas , Animais , Spodoptera , Zea mays , Inibidores da Tripsina/farmacologia , Glycine max/genética , Endotoxinas/farmacologia , Escherichia coli/metabolismo , Toxinas de Bacillus thuringiensis , Resistência a Inseticidas , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Proteínas Hemolisinas/farmacologia , Proteínas Hemolisinas/genética , Inseticidas/farmacologia , Bacillus thuringiensis/genética , Larva/fisiologia , Plantas Geneticamente Modificadas/genéticaRESUMO
Serine proteases play crucial biological roles and have their activity controlled by inhibitors, such as the EcTI, a serine protease inhibitor purified from Enterolobium contortisiliquum seeds, which has anticancer activity. This study aimed to conjugate EcTI with quantum dots (QDs), fluorophores with outstanding optical properties, and investigate the interaction of QDs-EcTI nanoprobe with cancer cells. The conjugation was evaluated by fluorescence correlation spectroscopy (FCS) and fluorescence microplate assay (FMA). EcTI inhibitory activity after interaction with QDs was also analyzed. From FCS, the conjugate presented a hydrodynamic diameter about 4× greater than bare QDs, suggesting a successful conjugation. This was supported by FMA, which showed a relative fluorescence intensity of ca. 3815% for the nanosystem, concerning bare QDs or EcTI alone. The EcTI inhibitory activity remained intact after its interaction with QDs. From flow cytometry analyses, approximately 62% of MDA-MB-231 and 90% of HeLa cells were labeled with the QD-EcTI conjugate, suggesting that their membranes have different protease levels to which EcTI exhibits an affinity. Concluding, the QD-EcTI represents a valuable nanotool to study the interaction of this inhibitor with cancer cells using fluorescence-based techniques with the potential to unravel the intricate dynamics of interplays between proteases and inhibitors in cancer biology.
Assuntos
Fabaceae , Neoplasias , Pontos Quânticos , Humanos , Inibidores da Tripsina/farmacologia , Células HeLa , Fabaceae/química , Serina Proteases , CorantesRESUMO
Spiders are important predators of insects and their venoms play an essential role in prey capture. Spider venoms have several potential applications as pharmaceutical compounds and insecticides. However, transcriptomic and proteomic analyses of the digestive system (DS) of spiders show that DS is also a rich source of new peptidase inhibitor molecules. Biochemical, transcriptomic and proteomic data of crude DS extracts show the presence of molecules with peptidase inhibitor potential in the spider Nephilingis cruentata. Therefore, the aims of this work were to isolate and characterize molecules with trypsin inhibitory activity. The DS of fasting adult females was homogenized under acidic conditions and subjected to heat treatment. After that, samples were submitted to ion exchange batch and high-performance reverse-phase chromatography. The fractions with trypsin inhibitory activity were confirmed by mass spectrometry, identifying six molecules with inhibitory potential. The inhibitor NcTI (Nephilingis cruentata trypsin inhibitor) was kinetically characterized, showing a KD value of 30.25 nM ± 8.13. Analysis of the tertiary structure by molecular modeling using Alpha-Fold2 indicates that the inhibitor NcTI structurally belongs to the MIT1-like atracotoxin family. This is the first time that a serine peptidase inhibitory function is attributed to this structural family and the inhibitor reactive site residue is identified. Sequence analysis indicates that these molecules may be present in the DS of other spiders and could be associated to the inactivation of prey trypsin (serine peptidase) ingested by the spiders.
Assuntos
Venenos de Aranha , Aranhas , Feminino , Animais , Inibidores da Tripsina/farmacologia , Tripsina , Proteômica , Venenos de Aranha/farmacologia , Venenos de Aranha/química , Sistema Digestório , SerinaRESUMO
The Leucaena leucocephala trypsin inhibitor (LTI) + Bacillus thuringiensis (Bt) protoxins mix has been proposed as a novel larvicide agent in order to control the vector mosquito of dengue virus, Aedes aegypti, in their aquatic breeding sites. However, use of this insecticide formulation has raised concerns about its impacts on aquatic biota. In this context, this work aimed to assess the effects of LTI and Bt protoxins, separately or in combination, in zebrafish, in regard to the evaluation of toxicity at early life stages and to the presence of LTI inhibitory effects on intestinal proteases of this fish. Results showed that LTI and Bt concentrations (250 mg/L, and 0.13 mg/L, respectively), and LTI + Bt mix (250 mg/L + 0.13 mg/L) - 10 times superior to those with insecticidal action - did not cause death nor did it induce morphological changes during embryonic and larval development (3 to 144 h post-fertilization) of zebrafish. Molecular docking analyses highlighted a possible interaction between LTI and zebrafish trypsin, especially through hydrophobic interactions. In concentrations near to those with larvicidal action, LTI (0.1 mg/mL) was able to inhibit in vitro intestinal extracts of trypsin in female and male fish by 83 % and 85 %, respectively, while LTI + Bt mix promoted trypsin inhibition of 69 % in female and 65 % in male ones. These data show that the larvicidal mix can potentially promote deleterious effects to nutrition and survival in non-target aquatic organisms, especially those with trypsin-like dependent protein digestion.
Assuntos
Inseticidas , Animais , Inseticidas/toxicidade , Peixe-Zebra , Inibidores de Proteases/farmacologia , Tripsina , Larva , Simulação de Acoplamento Molecular , Mosquitos Vetores , Inibidores da Tripsina/farmacologia , Antivirais/farmacologia , Proteínas de Bactérias/toxicidadeRESUMO
Bacterial infections have become a global concern, stimulating the growing demand for natural and biologically safe therapeutic agents with antibacterial action. This study was evaluated the genotoxicity of the trypsin inhibitor isolated from tamarind seeds (TTI) and the antibacterial effect of TTI theoric model, number 56, and conformation number 287 (TTIp 56/287) and derived peptides in silico. TTI (0.3 and 0.6 mg.mL-1) did not cause genotoxicity in cells (p > 0.05). In silico, a greater interaction of TTIp 56/287 with the Gram-positive membrane (GP) was observed, with an interaction potential energy (IPE) of -1094.97 kcal.mol-1. In the TTIp 56/287-GP interaction, the Arginine, Threonine (Thr), and Lysine residues presented lower IPE. In molecular dynamics (MD), Peptidotrychyme59 (TVSQTPIDIPIGLPVR) showed an IPE of -518.08 kcal.mol-1 with the membrane of GP bacteria, and the Thr and Arginine residues showed the greater IPE. The results highlight new perspectives on TTI and its derived peptides antibacterial activity.
Assuntos
Tamarindus , Inibidores da Tripsina , Inibidores da Tripsina/farmacologia , Tamarindus/química , Peptídeos/química , Sementes/química , Antibacterianos/farmacologia , Antibacterianos/análise , Arginina/análise , Arginina/químicaRESUMO
The emergence of antibiotic resistance poses a serious and challenging threat to healthcare systems, making it imperative to discover novel therapeutic options. This work reports the isolation and characterization of a thermostable trypsin inhibitor from chia (Salvia hispanica L.) seeds, with antibacterial activity against Staphylococcus aureus sensitive and resistant to methicillin. The trypsin inhibitor ShTI was purified from chia seeds through crude extract heat treatment, followed by affinity and reversed-phase chromatography. Tricine-SDS-PAGE revealed a single glycoprotein band of ~ 11 kDa under nonreducing conditions, confirmed by mass spectrometry analysis (11.558 kDa). ShTI was remarkably stable under high temperatures (100 °C; 120 min) and a broad pH range (2-10; 30 min). Upon exposure to DTT (0.1 M; 120 min), ShTI antitrypsin activity was partially lost (~ 38%), indicating the participation of disulfide bridges in its structure. ShTI is a competitive inhibitor (Ki = 1.79 × 10-8 M; IC50 = 1.74 × 10-8 M) that forms a 1:1 stoichiometry ratio for the ShTI:trypsin complex. ShTI displayed antibacterial activity alone (MICs range from 15.83 to 19.03 µM) and in combination with oxacillin (FICI range from 0.20 to 0.33) against strains of S. aureus, including methicillin-resistant strains. Overproduction of reactive oxygen species and plasma membrane pore formation are involved in the antibacterial action mode of ShTI. Overall, ShTI represents a novel candidate for use as a therapeutic agent for the bacterial management of S. aureus infections.
Assuntos
Oxacilina , Staphylococcus aureus , Oxacilina/farmacologia , Oxacilina/análise , Inibidores da Tripsina/farmacologia , Inibidores da Tripsina/análise , Salvia hispanica , Antibacterianos/farmacologia , Sementes/química , Combinação de MedicamentosRESUMO
Pest management is challenged with resistant herbivores and problems regarding human health and environmental issues. Indeed, the greatest challenge to modern agriculture is to protect crops from pests and still maintain environmental quality. This study aimed to analyze by in silico, in vitro, and in vivo approaches to the feasibility of using the inhibitory protein extracted from mammals - Bovine Pancreatic Trypsin Inhibitor (BPTI) as a potential inhibitor of digestive trypsins from the pest Anticarsia gemmatalis and comparing the results with the host-plant inhibitor - Soybean Kunitz Trypsin Inhibitor (SKTI). BPTI and SKTI interacts with A. gemmatalis trypsin-like enzyme competitively, through hydrogen and hydrophobic bonds. A. gemmatalis larvae exposed to BPTI did not show two common adaptative mechanisms i.e., proteolytic degradation and overproduction of proteases, presenting highly reduced trypsin-like activity. On the other hand, SKTI-fed larvae did not show reduced trypsin-like activity, presenting overproduction of proteases and SKTI digestion. In addition, the larval survival was reduced by BPTI similarly to SKTI, and additionally caused a decrease in pupal weight. The non-plant protease inhibitor BPTI presents intriguing element to compose biopesticide formulations to help decrease the use of conventional refractory pesticides into integrated pest management programs.
Assuntos
Agentes de Controle Biológico , Glycine max , Mariposas , Praguicidas , Animais , Aprotinina/farmacologia , Agentes de Controle Biológico/farmacologia , Bovinos , Hidrogênio/farmacologia , Larva , Peptídeo Hidrolases/metabolismo , Praguicidas/farmacologia , Inibidores de Proteases/farmacologia , Tripsina , Inibidores da Tripsina/farmacologiaRESUMO
Cell adhesion and migration are crucial for cancer progression and malignancy. Drugs available for the treatment of metastatic melanoma are expensive and unfit for certain patients. Therefore, there is still a need to identify new drugs that block tumor cell development. We investigated the effects of Enterolobium contortisiliquum trypsin inhibitor (EcTI), a protease inhibitor, on cell viability, cell migration, invasion, cell adhesion, and cell death (hallmarks of cancer) in vitro using human melanoma cells (SK-MEL-28 and CHL-1). Although EcTI did not affect non-tumor cells, it significantly inhibited the proliferation, migration, invasion, and adhesion of melanoma cells. Investigation of the underlying mechanisms revealed that EcTI triggered apoptosis and nuclear shrinkage, increased PI uptake, activated effector caspases-3/7, and produced reactive oxygen species (ROS). Furthermore, EcTI disrupted the mitochondrial membrane potential, altered calcium homeostasis, and modified proteins associated with survival and apoptosis/autophagy regulation. Acridine orange staining indicated acidic vesicular organelle formation upon EcTI treatment, demonstrating a cell death display. Electronic microscopy corroborated the apoptotic pattern by allowing the visualization of apoptotic bodies, mitochondrial cristae disorganization, and autophagic vesicles. Taken together, these results provide new insights into the anti-cancer properties of the natural EcTI protein, establishing it as a promising new therapeutic drug for use in melanoma treatment.
Assuntos
Fabaceae , Melanoma , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Humanos , Melanoma/metabolismo , Processos Neoplásicos , Inibidores de Proteases/farmacologia , Inibidores de Proteases/uso terapêutico , Inibidores da Tripsina/farmacologiaRESUMO
This systematic review (SR) aimed to gather studies describing the antibacterial action mechanisms and mode of trypsin inhibitors. The review protocol was registered (PROSPERO: CRD42020189069). Original articles resulting from studies in animal models, in bacterial culture, and using cells that describe antibacterial action of trypsin inhibitor-type peptides or proteins were selected in PubMed, Science Direct, Scopus, Web of Science, BVS, and EMBASE. The methodological quality assessment was performed using the PRISMA and OHAT tool. 2382 articles were retrieved, 17 of which were eligible. Four studies demonstrated the action mechanism directly on the bacterial membrane, and the fifth study on endogenous proteases extracted from the bacteria themselves. The antibacterial action mode was presented in the other studies, which can generate bacteriostatic or bactericidal effects without describing the mechanisms. This study generated information to enable new preclinical or clinical studies with molecules contributing to public health.
Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Inibidores da Tripsina/farmacologia , Tripsina/metabolismo , Antibacterianos/química , Bactérias/metabolismo , Testes de Sensibilidade Microbiana , Inibidores da Tripsina/químicaRESUMO
Protease inhibitors (PIs) are important biotechnological tools of interest in agriculture. Usually they are the first proteins to be activated in plant-induced resistance against pathogens. Therefore, the aim of this study was to characterize a Theobroma cacao trypsin inhibitor called TcTI. The ORF has 740 bp encoding a protein with 219 amino acids, molecular weight of approximately 23 kDa. rTcTI was expressed in the soluble fraction of Escherichia coli strain Rosetta [DE3]. The purified His-Tag rTcTI showed inhibitory activity against commercial porcine trypsin. The kinetic model demonstrated that rTcTI is a competitive inhibitor, with a Ki value of 4.08 × 10-7 mol L-1. The thermostability analysis of rTcTI showed that 100% inhibitory activity was retained up to 60 °C and that at 70-80 °C, inhibitory activity remained above 50%. Circular dichroism analysis indicated that the protein is rich in loop structures and ß-conformations. Furthermore, in vivo assays against Helicoverpa armigera larvae were also performed with rTcTI in 0.1 mg mL-1 spray solutions on leaf surfaces, which reduced larval growth by 70% compared to the control treatment. Trials with cocoa plants infected with Mp showed a greater accumulation of TcTI in resistant varieties of T. cacao, so this regulation may be associated with different isoforms of TcTI. This inhibitor has biochemical characteristics suitable for biotechnological applications as well as in resistance studies of T. cacao and other crops.
Assuntos
Cacau/química , Cacau/parasitologia , Inibidores da Tripsina/isolamento & purificação , Inibidores da Tripsina/farmacologia , Agaricales/efeitos dos fármacos , Agaricales/crescimento & desenvolvimento , Animais , Cacau/metabolismo , Estabilidade de Medicamentos , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Isoformas de Proteínas , Temperatura , Inibidores da Tripsina/química , Inibidores da Tripsina/metabolismoRESUMO
Several studies have sought new therapies for obesity and liver diseases. This study investigated the effect of the trypsin inhibitor isolated from tamarind seeds (TTI), nanoencapsulated in chitosan and whey protein isolate (ECW), on the liver health status of the Wistar rats fed with a high glycemic index (HGLI) diet. The nanoformulations without TTI (CW) and ECW were obtained by nanoprecipitation technique, physically and chemically characterized, and then administered to the animals. The adult male Wistar rats (n = 20) were allocated to four groups: HGLI diet + water; standard diet + water; HGLI diet + ECW (12.5 mg/kg); and HGLI diet + CW (10.0 mg/kg), 1 mL per gagave, for ten days. They were evaluated using biochemical and hematological parameters, Fibrosis-4 Index for Liver Fibrosis (FIB-4), AST to Platelet Ratio Index (APRI) scores, and liver morphology. Both nanoparticles presented spherical shape, smooth surface, and nanometric size [120.7 nm (ECW) and 136.4 nm (CW)]. In animals, ECW reduced (p < 0.05) blood glucose (17%), glutamic oxalacetic transaminase (39%), and alkaline phosphatase (24%). Besides, ECW reduced (p < 0.05) APRI and FIB-4 scores and presented a better aspect of hepatic morphology. ECW promoted benefits over a liver injury caused by the HGLI diet.
Assuntos
Quitosana/química , Dieta , Índice Glicêmico , Fígado/lesões , Nanopartículas/química , Tamarindus/química , Inibidores da Tripsina/farmacologia , Proteínas do Soro do Leite/química , Animais , Glicemia/metabolismo , Jejum/sangue , Homeostase , Insulina/sangue , Resistência à Insulina , Rim/fisiopatologia , Fígado/efeitos dos fármacos , Fígado/patologia , Fígado/fisiopatologia , Masculino , Nanopartículas/ultraestrutura , Ratos Wistar , Valores de ReferênciaRESUMO
Trypsin inhibitors from tamarind seed have been studied in vitro and in preclinical studies for the treatment of obesity, its complications and associated comorbidities. It is still necessary to fully understand the structure and behaviour of these molecules. We purifed this inhibitor, sequenced de novo by MALDI-TOF/TOF, performed its homology modelling, and assessed the interaction with the trypsin enzyme through molecular dynamics (MD) simulation under physiological conditions. We identified additional 75 amino acid residues, reaching approximately 72% of total coverage. The four best conformations of the best homology modelling were submitted to the MD. The conformation n°287 was selected considering the RMSD analysis and interaction energy (-301.0128 kcal.mol-1). Residues Ile (54), Pro (57), Arg (59), Arg (63), and Glu (78) of pTTI presented the highest interactions with trypsin, and arginine residues were mainly involved in its binding mechanism. The results favour bioprospecting of this protein for pharmaceutical health applications.
Assuntos
Simulação de Dinâmica Molecular , Extratos Vegetais/farmacologia , Tamarindus/química , Inibidores da Tripsina/farmacologia , Tripsina/metabolismo , Relação Dose-Resposta a Droga , Estrutura Molecular , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Sementes/química , Relação Estrutura-Atividade , Inibidores da Tripsina/química , Inibidores da Tripsina/isolamento & purificaçãoRESUMO
BACKGROUND: Protease inhibitors have been isolated from plants and present several biological activities, including immunomodulatory action. OBJECTIVE: This work aimed to evaluate a Moringa oleifera flower trypsin inhibitor (MoFTI) for acute toxicity in mice, hemolytic activity on mice erythrocytes and immunomodulatory effects on mice splenocytes. METHODS: The acute toxicity was evaluated using Swiss female mice that received a single dose of the vehicle control or MoFTI (300 mg/kg, i.p.). Behavioral alterations were observed 15-240 min after administration, and survival, weight gain, and water and food consumption were analyzed daily. Organ weights and hematological parameters were analyzed after 14 days. Hemolytic activity of MoFTI was tested using Swiss female mice erythrocytes. Splenocytes obtained from BALB/c mice were cultured in the absence or presence of MoFTI for the evaluation of cell viability and proliferation. Mitochondrial membrane potential (Δψm) and reactive oxygen species (ROS) levels were also determined. Furthermore, the culture supernatants were analyzed for the presence of cytokines and nitric oxide (NO). RESULTS: MoFTI did not cause death or any adverse effects on the mice except for abdominal contortions at 15-30 min after administration. MoFTI did not exhibit a significant hemolytic effect. In addition, MoFTI did not induce apoptosis or necrosis in splenocytes and had no effect on cell proliferation. Increases in cytosolic and mitochondrial ROS release, as well as Δψm reduction, were observed in MoFTI-treated cells. MoFTI was observed to induce TNF-α, IFN-γ, IL-6, IL-10, and NO release. CONCLUSION: These results contribute to the ongoing evaluation of the antitumor potential of MoFTI and its effects on other immunological targets.
Assuntos
Moringa oleifera/enzimologia , Proteínas de Plantas , Inibidores da Tripsina , Animais , Comportamento Animal/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Feminino , Flores/química , Hemólise/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Proteínas de Plantas/farmacologia , Proteínas de Plantas/toxicidade , Baço/citologia , Testes de Toxicidade Aguda , Inibidores da Tripsina/química , Inibidores da Tripsina/metabolismo , Inibidores da Tripsina/farmacologia , Inibidores da Tripsina/toxicidadeRESUMO
Breast cancer is the most common malignant tumor among women worldwide, and triple-negative breast cancer is the most aggressive type of breast cancer, which does not respond to hormonal therapies. The protease inhibitor, EcTI, extracted from seeds of Enterolobium contortisiliquum, acts on the main signaling pathways of the MDA-MB-231 triple-negative breast cancer cells. This inhibitor, when bound to collagen I of the extracellular matrix, triggers a series of pathways capable of decreasing the viability, adhesion, migration, and invasion of these cells. This inhibitor can interfere in the cell cycle process through the main signaling pathways such as the adhesion, Integrin/FAK/SRC, Akt, ERK, and the cell death pathway BAX and BCL-2. It also acts by reducing the main inflammatory cytokines such as TGF-α, IL-6, IL-8, and MCP-1, besides NFκB, a transcription factor, responsible for the aggressive and metastatic characteristics of this type of tumor. Thus, the inhibitor was able to reduce the main processes of carcinogenesis of this type of cancer.
Assuntos
Citocinas/antagonistas & inibidores , Fabaceae/química , Glicosaminoglicanos/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Inibidores da Tripsina/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Citocinas/biossíntese , Feminino , Humanos , Metaloproteinases da Matriz/metabolismo , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Inibidores da Tripsina/uso terapêuticoRESUMO
BACKGROUND: Disease vector insects are barriers for human development. The use of synthetic chemicals to control these vectors has caused damage to the environment and contributed to the arising of resistant insect populations. This has led to an increased search for plant-derived molecules with insecticidal activity or that show synergistic effects with known insecticidal substances, such as protease inhibitors. Thus, we aimed to evaluate the effect of Enterolobium contortisiliquum trypsin inhibitor (EcTI) on Aedes aegypti development as well as its effect on insecticidal activity of Bacillus thuringiensis toxins. RESULTS: EcTI showed an apparent molecular mass about of 20 kDa in SDS-PAGE and was able to inhibit in vitro the activity of trypsin and proteases from midgut of Ae. aegypti larvae. EcTI was not able to cause acute toxicity on mosquito larvae even at 1000 µg mL-1 , however it promoted a delay in larval development after prolonged exposure. The zymogram results for EcTI-treated larvae (from 50 to 200 µg mL-1 ) showed an increase of midgut proteases activity as a larvae defense mechanism, however no changes in the enzyme profile was observed. These same concentrations were able to enhance up to three fold the insecticidal activity of B. thuringiensis toxins without causing toxicity to Artemia sp. nauplii, a non-target organism. CONCLUSIONS: The results offer a novel approach by combining EcTI and B. thuringiensis toxins for combating Ae. aegypti larvae. © 2020 Society of Chemical Industry.
Assuntos
Aedes , Bacillus thuringiensis , Animais , Larva , Mosquitos Vetores , Sementes , Inibidores da Tripsina/farmacologiaRESUMO
The economic loss in soybean crops caused by the Lepidoptera insects has encouraged the search for new strategies to control this pest, which are currently based on synthetic insecticides. This paper evaluated the ability of ApTI (Adenanthera pavonina trypsin inhibitor) to inhibit trypsin-like proteins from Anticarsia gemmatalis by docking, molecular dynamics, and enzymatic and survival assay. The docking and molecular dynamic simulation between trypsin and ApTI were performed using the program CLUSPRO and NAMD, respectively. The inhibitory constant Ki and the inhibition type were determined through chromogenic assays. The survival assay of neonatal larvae under treatment with artificial diet supplemented with ApTI was also performed. The ApTI binding site was predicted to block substrate access to trypsin due to four interactions with the enzyme, producing a complex with a surface area of 1,183.7 Å2 . The kinetic analysis revealed a noncompetitive tight-binding mechanism. The survival curves obtained using Kaplan-Meier estimators indicated that the highest larvae mortality was 60%, using 1.2 mg of ApTI per 100 ml of artificial diet. The in vitro, in vivo, and in silico studies demonstrated that ApTI is a strong noncompetitive inhibitor of trypsin with biotechnological potential for the control of A. gemmatalis insect.