RESUMEN
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.
Asunto(s)
Bauhinia , Inhibidores de Tripsina , Animales , Bovinos , Inhibidores de Tripsina/farmacología , Inhibidores de Tripsina/química , Bauhinia/metabolismo , Tripsina/análisis , Tripsina/química , Tripsina/metabolismo , Espectrometría de Masas en Tándem , Semillas/química , Anticoagulantes/farmacología , Anticoagulantes/análisis , Anticoagulantes/químicaRESUMEN
Lectins are proteins of non-immunological origin with the ability to bind to carbohydrates reversibly. They emerge as an alternative to conventional antifungals, given the ability to interact with carbohydrates in the fungal cell wall inhibiting fungal growth. The lectin from D. violacea (DVL) already has its activity described as anti-candida in some species. Here, we observed the anti-candida effect of DVL on C. albicans, C. krusei and C. parapsilosis and its multiple mechanisms of action toward the yeasts. Additionally, it was observed that DVL induces membrane and cell wall damage and ROS overproduction. DVL was also able to cause an imbalance in the redox system of the cells, interact with ergosterol, inhibit ergosterol biosynthesis, and induce cytochrome c release from the mitochondrial membrane. These results endorse the potential application of DVL in developing a new antifungal drug to fight back against fungal resistance.
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Dioclea , Lectinas , Lectinas/farmacología , Candida/metabolismo , Dioclea/metabolismo , Lectinas de Plantas/farmacología , Lectinas de Plantas/metabolismo , Antifúngicos/farmacología , Carbohidratos , Semillas/metabolismo , Ergosterol , Candida albicans , Pruebas de Sensibilidad MicrobianaRESUMEN
The emergence of antibiotic resistance (AMR) is a global public health problem. According to estimates, drug-resistant bacteria infect 2 million patients and perish 23,000 annually. To overcome this problem, antimicrobial peptides became a potential solution based on a new mechanism of action against bacteria. This article addresses the phenomenon of antibacterial resistance in most of its nuances, responding to historical, technical-scientific, and economic aspects. Likewise, it explores new therapeutic approaches to combat multi-resistant pathogens, specifically concerning antibacterial peptides, as a potential therapeutic tool to mitigate the current crisis of antibacterial drugs. It is expected that, with technological advances, especially with the advent and adoption of artificial intelligence, there will be an increase in diversified synthetic peptide production, which can face the challenges that we have in terms of antibacterial drugs.
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Antibacterianos , Inteligencia Artificial , Humanos , Antibacterianos/farmacología , Bacterias , Péptidos , Farmacorresistencia BacterianaRESUMEN
DVL is a Man/Glc-binding lectin from Dioclea violacea seeds that has the ability to interact with the antibiotic gentamicin. The present work aimed to evaluate whether the DVL has the ability to interact with neomycin via CRD and to examine the ability of this lectin to modulate the antibiotic effect of neomycin against multidrug-resistant strains (MDR). The hemagglutinating activity test revealed that neomycin inhibited the hemagglutinating activity of DVL with a minimum inhibitory concentration of 50 mM, indicating that the antibiotic interacts with DVL via the carbohydrate recognition domain (CRD). DVL immobilized on cyanogen bromide-activated Sepharose® 4B bound 41 % of the total neomycin applied to the column, indicating that the DVL-neomycin interaction is efficient for purification processes. Furthermore, the minimum inhibitory concentrations (MIC) obtained for DVL against all strains studied were not clinically relevant. However, when DVL was combined with neomycin, a significant increase in antibiotic activity was observed against S. aureus and P. aeruginosa. These results demonstrate the first report of lectin-neomycin interaction, indicating that immobilized DVL has the potential to isolate neomycin by affinity chromatography. Moreover, DVL increased the antibiotic activity of neomycin against MDR, suggesting that it is a potent adjuvant in the treatment of infectious diseases.
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Dioclea , Fabaceae , Humanos , Masculino , Lectinas/farmacología , Antibacterianos/farmacología , Dioclea/química , Neomicina/farmacología , Lectinas de Plantas/química , Staphylococcus aureus/metabolismo , Fabaceae/metabolismoRESUMEN
Lectins are carbohydrate-binding proteins belonging to the Leguminosae family. In this family stand out proteins extracted from species belonging to Diocleinae subtribe, which includes, for example, the seed lectin from Dioclea violacea (DVL) and the jack bean lectin Concanavalin A (ConA). Here, we report the photosynthesis of silver/silver chloride nanoparticles (NPs) assisted by ConA and DVL. The syntheses were simple processes using a green-chemistry approach. Under electron microscopy, NPs heterogeneous in size, nearly spherical and covered by a thin lectin corona, were observed. Both NPs assisted by lectins were capable to cause strong rabbit erythrocytes agglutination with the same titers of hemagglutinating activities. These results indicate that both lectins maintained their biological activities even after association with the NPs and therefore are able to interact with biological membrane carbohydrates. However, for rabbit erythrocytes treated with proteolytic enzymes were observed different titers of hemagglutinating activities, suggesting differences in the spatial arrangement of the lectins on the surface of the NPs. This study provides evidences that these hybrid lectin-coated silver/silver chloride NPs can be used for selective recognition and interaction with membrane carbohydrates and others biotechnological applications.
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Lectinas , Lectinas de Plantas , Animales , Conejos , Lectinas/química , Lectinas de Plantas/farmacología , Lectinas de Plantas/química , Lectinas de Plantas/metabolismo , Plata/farmacología , Carbohidratos/química , FotosíntesisRESUMEN
The use of schiff base complex against microbial agentes a has recently received more attention as a strategy to combat infections caused by multidrug-resistant bacteria and leishmania. This study aimed to evaluate the toxicity, antibacterial and leishmanicidal activities of the nickel (II) chloride schiff base complex ([Ni(L2)] against Leishmania amazonensis promastigote, multi-resistant bacterial strains and evaluate to modulate antibiotic activity against multi-resistant bacterial. The schiff base complex was characterized by the techniques of elemental analysis, Fourier transform infrared spectroscopy (FTIR), UV-vis absorption spectroscopy and thermal analysis (TGA/DTG/DSC). The [Ni(L2)] complex presented moderate toxicity in saline artemia (LC50 = 150.8 µg/mL). In leishmanicidal assay, the NiL2 complex showed values of IC50 of (6.079 µg/mL ± 0.05656 at the 24 h), (0.854 µg/mL ± 0.02474, 48 h) and (1.076 µg/mL ± 0.04039, 72 h). In antibacterial assay, the [Ni(L2)] complex presented significant inhibited the bacterial growth of P. aeruginosa (MIC = 256 µg/mL). However, [Ni(L2)] complex did not present clinically relevant minimum inhibitory concentration (MIC ≥1024 µg/mL) against S. aureus and E. coli. The combination of [Ni(L2)] complex and antibacterial drugs resulted in the increased antibiotic activity of gentamicin and amikacin against S. aureus and E.coli multi-resistant strains. Thus, our results showed that [Ni(L2)] complex is a promising molecule for the development of new therapies associated with aminoglycoside antibiotics and in disease control related to resistant bacteria and leishmaniasis.
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Antibacterianos/farmacología , Complejos de Coordinación/farmacología , Bases de Schiff/farmacología , Tripanocidas/farmacología , Amicacina/farmacología , Animales , Antibacterianos/química , Artemia/efectos de los fármacos , Complejos de Coordinación/química , Sinergismo Farmacológico , Escherichia coli/efectos de los fármacos , Gentamicinas/farmacología , Leishmania infantum/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Níquel/química , Pruebas de Sensibilidad Parasitaria , Pseudomonas aeruginosa/efectos de los fármacos , Bases de Schiff/química , Staphylococcus aureus/efectos de los fármacos , Tripanocidas/químicaRESUMEN
Lectins are a group of widely distributed and structurally heterogeneous proteins of nonimmune origin. These proteins have the ability to interact with glycans present on cell surfaces and elicit diverse biological activities. Machaerium acutifolium lectin (MaL) is an N-acetyl-D-glucosamine-binding lectin that exhibits antinociceptive activity via transient receptor potential cation channel subfamily V member 1 (TRPV1). Lectins that have the ability to recognize and interact with N-acetyl-D-glucosamine residues are potential candidates for studies of fungicidal activity. In this work, we show that MaL has antifungal activity against Candida species, and we describe its mode of action towards Candida parapsilosis. MaL inhibited the growth of C. albicans and C. parapsilosis. However, MaL was more potent against C. parapsilosis. The candidacidal mode of action of MaL on C. parapsilosis involves enhanced cell permeabilization, alteration of the plasma membrane proton-pumping ATPase function (H+-ATPase), induction of oxidative stress, and DNA damage. MaL also exhibited antibiofilm activity and noncytotoxicity to Vero cells. These results indicate that MaL is a promising candidate for the future development of a new, natural, and safe drug for the treatment of infections caused by C. parapsilosis.
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Antifúngicos/farmacología , Candida parapsilosis/metabolismo , Estructuras de la Membrana Celular/química , Fabaceae/química , Lectinas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Animales , Antifúngicos/administración & dosificación , Antifúngicos/aislamiento & purificación , Apoptosis/efectos de los fármacos , Biopelículas/efectos de los fármacos , Candida albicans/efectos de los fármacos , Candida albicans/metabolismo , Candida parapsilosis/citología , Candida parapsilosis/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Estructuras de la Membrana Celular/metabolismo , Chlorocebus aethiops , Medios de Cultivo/análisis , Medios de Cultivo/química , Daño del ADN , Lectinas/administración & dosificación , Lectinas/aislamiento & purificación , Microscopía Electrónica de Rastreo , Propidio/metabolismo , Semillas/química , Células VeroRESUMEN
Lectins have been studied in the past few years as an alternative to inhibit the development of pathogenic bacteria and gastrointestinal nematodes of small ruminants. The development of new antibacterial and anthelmintic compounds is necessary owing to the increase in drug resistance among important pathogens. Therefore, this study aimed to evaluate the capacity of a glucose/mannose-binding lectin from Parkia platycephala seeds (PPL) to inhibit the development of Haemonchus contortus and to modulate antibiotic activity against multi-resistant bacterial strains, thereby confirming its efficacy when used in combination with gentamicin. PPL at the concentration of 1.2â¯mg/mL did not show inhibitory activity on H. contortus in the egg hatch test or the exsheathment assay. However, it did show significant inhibition of H. contortus larval development with an IC50 of 0.31â¯mg/mL. The minimum inhibitory concentration (MIC) obtained for PPL against all tested bacterial strains was not clinically relevant (MICâ¯≥â¯1024⯵g/mL). However, when PPL was combined with gentamicin, a significant increase in antibiotic activity was observed against S. aureus and E.coli multi-resistant strains. The inhibition of hemagglutinating activity by gentamicin (MICâ¯=â¯50â¯mM) revealed that it may be interacting with the carbohydrate-binding site of PPL. It is this interaction between the antibiotic and lectin carbohydrate-binding site that may be responsible for the enhanced activity of gentamicin against multi-resistant strains. It can be concluded that PPL showed selective anthelmintic effect, inhibiting the development of H. contortus larvae and that it increased the effect of the antibiotic gentamicin against multi-resistant bacterial strains, thus constituting a potential therapeutic resource against resistant bacterial strains and H. contortus.
Asunto(s)
Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Fabaceae/química , Haemonchus/efectos de los fármacos , Haemonchus/crecimiento & desarrollo , Lectinas/farmacología , Extractos Vegetales/farmacología , Animales , Antihelmínticos/farmacología , Gentamicinas/farmacología , Haemonchus/microbiología , Larva/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Semillas/química , Staphylococcus aureus/efectos de los fármacosRESUMEN
A new mannose/N-acetyl-dglucosamine-specific lectin, named MaL, was purified from seeds of Machaerium acutifolium by precipitation with ammonium sulfate, followed by affinity and ion-exchange chromatography. MaL haemagglutinates either native rabbit erythrocytes or those treated with proteolytic enzymes. MaL is highly stable by the ability to maintain its haemagglutinating activity after exposure to temperatures up to 50⯰C. The lectin haemagglutinating activity was optimum between pH 6.0 and 7.0 and inhibited after incubation with d-mannose and N-acetyl-d-glucosamine and α-methyl-d-mannopyranoside. MaL is a glycoprotein with relative molecular mass of 29â¯kDa (α-chain), 13â¯kDa (ß-chain) and 8â¯kDa (γ-chain) with secondary structure composed of 3% α-helix, 44% ß-sheet, 21% ß-turn, and 32% coil. The orofacial antinociceptive activity of the lectin was also evaluated. MaL (0.03â¯mgâ¯mL-1) reduced orofacial nociception induced by capsaicin, an effect that occurred via carbohydrate recognition domain interaction, suggesting an interaction of MaL with the transient receptor potential cation channel subfamily V member 1 (TRPV1) receptor. Our results confirm the potential pharmacological relevance of MaL as an inhibitor of acute orofacial mediated by TRPV1.