RESUMEN
Saponins are plant and marine animal specific metabolites that are commonly considered as molecular vectors for chemical defenses against unicellular and pluricellular organisms. Their toxicity is attributed to their membranolytic properties. Modifying the molecular structures of saponins by quantitative and selective chemical reactions is increasingly considered to tune the biological properties of these molecules (i) to prepare congeners with specific activities for biomedical applications and (ii) to afford experimental data related to their structure-activity relationship. In the present study, we focused on the sulfated saponins contained in the viscera of Holothuria scabra, a sea cucumber present in the Indian Ocean and abundantly consumed on the Asian food market. Using mass spectrometry, we first qualitatively and quantitatively assessed the saponin content within the viscera of H. scabra. We detected 26 sulfated saponins presenting 5 different elemental compositions. Microwave activation under alkaline conditions in aqueous solutions was developed and optimized to quantitatively and specifically induce the desulfation of the natural saponins, by a specific loss of H2SO4. By comparing the hemolytic activities of the natural and desulfated extracts, we clearly identified the sulfate function as highly responsible for the saponin toxicity.
Asunto(s)
Holothuria/química , Saponinas/química , Saponinas/farmacología , Sulfatos/química , Sulfatos/farmacología , Vísceras/química , Álcalis/química , Animales , Bovinos , Cromatografía Liquida , Hemólisis/efectos de los fármacos , Hemolíticos/análisis , Hemolíticos/química , Hemolíticos/aislamiento & purificación , Hemolíticos/farmacología , Hidrólisis , Océano Índico , Microondas , Saponinas/análisis , Saponinas/aislamiento & purificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Relación Estructura-Actividad , Sulfatos/análisis , Sulfatos/aislamiento & purificación , Espectrometría de Masas en TándemRESUMEN
The snake genus Daboia (Viperidae: Viperinae; Oppel, 1811) contains five species: D. deserti, D. mauritanica, and D. palaestinae, found in Afro-Arabia, and the Russell's vipers D. russelii and D. siamensis, found in Asia. Russell's vipers are responsible for a major proportion of the medically important snakebites that occur in the regions they inhabit, and their venoms are notorious for their coagulopathic effects. While widely documented, the extent of venom variation within the Russell's vipers is poorly characterised, as is the venom activity of other species within the genus. In this study we investigated variation in the haemotoxic activity of Daboia using twelve venoms from all five species, including multiple variants of D. russelii, D. siamensis, and D. palaestinae. We tested the venoms on human plasma using thromboelastography, dose-response coagulometry analyses, and calibrated automated thrombography, and on human fibrinogen by thromboelastography and fibrinogen gels. We assessed activation of blood factors X and prothrombin by the venoms using fluorometry. Variation in venom activity was evident in all experiments. The Asian species D. russelii and D. siamensis and the African species D. mauritanica possessed procoagulant venom, while D. deserti and D. palaestinae were net-anticoagulant. Of the Russell's vipers, the venom of D. siamensis from Myanmar was most toxic and D. russelli of Sri Lanka the least. Activation of both factor X and prothrombin was evident by all venoms, though at differential levels. Fibrinogenolytic activity varied extensively throughout the genus and followed no phylogenetic trends. This venom variability underpins one of the many challenges facing treatment of Daboia snakebite envenoming. Comprehensive analyses of available antivenoms in neutralising these variable venom activities are therefore of utmost importance.
Asunto(s)
Hemolíticos/química , Venenos de Víboras/química , Venenos de Víboras/toxicidad , Animales , Antivenenos , Asia , Factor X/análisis , Hemolíticos/análisis , Humanos , Plasma/efectos de los fármacos , Protrombina/análisis , Daboia , Mordeduras de Serpientes , Venenos de Víboras/análisis , ViperidaeRESUMEN
The worrisome emergence of pathogens resistant to conventional drugs has stimulated the search for new classes of antimicrobial and antiparasitic agents from natural sources. Antimicrobial peptides (AMPs), acting through mechanisms that do not rely on the interaction with a specific receptor, provide new possibilities for the development of drugs against resistant organisms. This study sought to purify and proteomically characterize the antimicrobial and antiparasitic peptidomes of B. atrox and B. jararacussu snake venoms against Gram-positive (Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus-MRSA), Gram-negative (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae) bacteria, and the protozoan parasites Leishmania amazonensis and Plasmodium falciparum (clone W2, resistant to chloroquine). To this end, B. atrox and B. jararacussu venom peptides were purified by combination of 3 kDa cut-off Amicon® ultracentrifugal filters and reverse-phase high-performance liquid chromatography, and then identified by electrospray-ionization Ion-Trap/Time-of-Flight mass spectrometry. Fourteen distinct peptides, with masses ranging from 443.17 to 1383.73 Da and primary structure between 3 and 13 amino acid residues, were sequenced. Among them, 13 contained unique sequences, including 4 novel bradykinin-potentiating-like peptides (BPPs), and a snake venom metalloproteinase tripeptide inhibitor (SVMPi). Although commonly found in Viperidae venoms, except for Bax-12, the BPPs and SVMPi here reported had not been described in B. atrox and B. jararacussu venoms. Among the novel peptides, some exhibited bactericidal activity towards P. aeruginosa and S. aureus, had low hemolytic effect, and were devoid of antiparasitic activity. The identified novel antimicrobial peptides may be relevant in the development of new drugs for the management of multidrug-resistant Gram-negative and Gram-positive bacteria.
Asunto(s)
Antiinfecciosos/química , Antiinfecciosos/farmacología , Venenos de Crotálidos/química , Péptidos/farmacología , Animales , Antibacterianos/química , Antibacterianos/farmacología , Antimaláricos/química , Antimaláricos/farmacología , Bothrops , Venenos de Crotálidos/aislamiento & purificación , Hemolíticos/química , Hemolíticos/farmacología , Humanos , Leishmania/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Péptidos/química , Péptidos/aislamiento & purificación , Plasmodium falciparum/efectos de los fármacos , Pseudomonas aeruginosa/efectos de los fármacos , Espectrometría de Masa por Ionización de Electrospray , Staphylococcus aureus/efectos de los fármacosRESUMEN
A molecular umbrella composed of two O-sulfated cholic acid residues was applied for the construction of conjugates with cispentacin, containing a "trimethyl lock" (TML) or o-dithiobenzylcarbamoyl moiety as a cleavable linker. Three out of five conjugates demonstrated antifungal in vitro activity against C. albicans and C. glabrata but not against C. krusei, with MIC90 values in the 0.22-0.99 mM range and were not hemolytic. Antifungal activity of the most active conjugate 24c, containing the TML-pimelate linker, was comparable to that of intact cispentacin. A structural analogue of 24c, containing the Nap-NH2 fluorescent probe, was accumulated in Candida cells, and TML-containing conjugates were cleaved in cell-free extract of C. albicans cells. These results suggest that a molecular umbrella can be successfully applied as a nanocarrier for the construction of cleavable antifungal conjugates.
Asunto(s)
Antifúngicos/administración & dosificación , Antifúngicos/química , Cicloleucina/análogos & derivados , Portadores de Fármacos/química , Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Candida glabrata/efectos de los fármacos , Ácido Cólico/química , Cicloleucina/química , Portadores de Fármacos/administración & dosificación , Portadores de Fármacos/farmacología , Eritrocitos/efectos de los fármacos , Hemolíticos/química , Hemolíticos/farmacología , Humanos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Relación Estructura-ActividadRESUMEN
A series of new derivatives of 4-(2-chloroethyl)morpholine hydrochloride (5) were efficiently synthesized. Briefly, different aromatic organic acids (1a-f) were refluxed to acquire respective esters (2a-f) using conc. H2SO4 as catalyst. The esters were subjected to nucleophillic substitution by monohydrated hydrazine to acquire hydrazides (3a-f). The hydrazides were cyclized with CS2 in the presence of KOH to yield corresponding oxadiazoles (4a-f). Finally, the derivatives, 6a-f, were prepared by reacting oxadiazoles (4a-f) with 5 using NaH as activator. Structures of all the derivatives were elucidated through 1D-NMR EI-MS and IR spectral data. All these molecules were subjected to antibacterial and hemolytic activities and showed good antibacterial and hemolytic potential relative to the reference standards.
Asunto(s)
Antibacterianos/química , Hemolíticos/química , Morfolinas/química , Oxadiazoles/química , Antibacterianos/síntesis química , Antibacterianos/farmacología , Bacillus subtilis/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Hemolíticos/síntesis química , Hemolíticos/farmacología , Klebsiella pneumoniae/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Morfolinas/síntesis química , Morfolinas/farmacología , Oxadiazoles/síntesis química , Oxadiazoles/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Salmonella typhi/efectos de los fármacos , Espectrofotometría Infrarroja , Staphylococcus aureus/efectos de los fármacosRESUMEN
Six peptides, belonging to the NDBP-4 family of scorpion antimicrobial peptides were structurally and functionally characterized. The sequence of the mature peptides VpCT1, VpCT2, VpCT3 and VpCT4 was inferred by transcriptomic analysis of the venom gland of the scorpion Mesomexovis variegatus. Analysis of their amino acid sequences revealed patterns that are also present in previously reported peptides that show differences in their hemolytic and antimicrobial activities in vitro. Two other variants, VpCT3W and VpCTConsensus were designed to evaluate the effect of sequence changes of interest on their structure and activity. The synthesized peptides were evaluated by circular dichroism to confirm their α-helical conformation in a folding promoting medium. The peptides were assayed on two Gram-positive and three Gram-negative bacterial strains, and on two yeast strains. They preferentially inhibited the growth of Staphylococcus aureus, were mostly ineffective on Pseudomonas aeruginosa, and moderately inhibited the growth of Candida yeasts. All six peptides exhibited hemolytic activity on human erythrocytes in the range of 4.8-83.7⯵M. VpCT3W displayed increased hemolytic and anti-yeast activities, but showed no change in antibacterial activity, relative to its parental peptide, suggesting that Trp6 may potentiate the interaction of VpCT3 with eukaryotic cell membranes. VpCTConsensus showed broader and enhanced antimicrobial activity relative to several of the natural peptides. The results presented here contribute new information on the structure and function of NDBP-4 antimicrobial peptides and provides clues for the design of less hemolytic and more effective antimicrobial peptides.
Asunto(s)
Péptidos Antimicrobianos/química , Péptidos Antimicrobianos/farmacología , Escorpiones/química , Animales , Antibacterianos/química , Antibacterianos/farmacología , Antifúngicos/química , Antifúngicos/farmacología , Péptidos Antimicrobianos/síntesis química , Dicroismo Circular , Hemolíticos/química , Hemolíticos/farmacología , Humanos , Pruebas de Sensibilidad Microbiana , Relación Estructura-ActividadRESUMEN
The mosquito protein AEG12 is up-regulated in response to blood meals and flavivirus infection though its function remained elusive. Here, we determine the three-dimensional structure of AEG12 and describe the binding specificity of acyl-chain ligands within its large central hydrophobic cavity. We show that AEG12 displays hemolytic and cytolytic activity by selectively delivering unsaturated fatty acid cargoes into phosphatidylcholine-rich lipid bilayers. This property of AEG12 also enables it to inhibit replication of enveloped viruses such as Dengue and Zika viruses at low micromolar concentrations. Weaker inhibition was observed against more distantly related coronaviruses and lentivirus, while no inhibition was observed against the nonenveloped virus adeno-associated virus. Together, our results uncover the mechanistic understanding of AEG12 function and provide the necessary implications for its use as a broad-spectrum therapeutic against cellular and viral targets.
Asunto(s)
Antivirales/metabolismo , Hemolíticos/metabolismo , Proteínas de Insectos/metabolismo , Lípidos , Animales , Antivirales/química , Antivirales/farmacología , Línea Celular , Membrana Celular/metabolismo , Culicidae , Eritrocitos/efectos de los fármacos , Ácidos Grasos Insaturados/metabolismo , Hemolíticos/química , Hemolíticos/farmacología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas de Insectos/química , Proteínas de Insectos/farmacología , Ligandos , Lípidos/química , Unión Proteica , Estructura Terciaria de Proteína , Envoltura Viral/metabolismo , Virus/efectos de los fármacos , Virus/metabolismoRESUMEN
The peptides from the ranacyclin family share similar active disulphide loop with plant-derived Bowman-Birk type inhibitors, some of which have the dual activities of trypsin inhibition and antimicrobial. Herein, a novel Bowman-Birk type trypsin inhibitor of the ranacyclin family was identified from the skin secretion of broad-folded frog (Sylvirana latouchii) by molecular cloning method and named as SL-BBI. After chemical synthesis, it was proved to be a potent inhibitor of trypsin with a Ki value of 230.5 nM and showed weak antimicrobial activity against tested microorganisms. Modified analogue K-SL maintains the original inhibitory activity with a Ki value of 77.27 nM while enhancing the antimicrobial activity. After the substitution of active P1 site to phenylalanine and P2' site to isoleucine, F-SL regenerated its inhibitory activity on chymotrypsin with a Ki value of 309.3 nM and exhibited antiproliferative effects on PC-3, MCF-7 and a series of non-small cell lung cancer cell lines without cell membrane damage. The affinity of F-SL for the ß subunits in the yeast 20S proteasome showed by molecular docking simulations enriched the understanding of the possible action mode of Bowman-Birk type inhibitors. Further mechanistic studies have shown that F-SL can activate caspase 3/7 in H157 cells and induce apoptosis, which means it has the potential to become an anticancer agent.
Asunto(s)
Antineoplásicos/aislamiento & purificación , Ranidae/metabolismo , Inhibidores de Tripsina/aislamiento & purificación , Secuencias de Aminoácidos , Animales , Antiinfecciosos/aislamiento & purificación , Antiinfecciosos/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Quimotripsina/antagonistas & inhibidores , Ensayos de Selección de Medicamentos Antitumorales , Hemolíticos/química , Hemolíticos/aislamiento & purificación , Hemolíticos/farmacología , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Estructura Molecular , Inhibidores de Tripsina/síntesis química , Inhibidores de Tripsina/farmacologíaRESUMEN
The growing prevalence of resistance to antibiotics motivates the search for new antibacterial agents. Antimicrobial peptides are a diverse class of well-studied membrane-active peptides which function as part of the innate host defence system, and form a promising avenue in antibiotic drug research. Some antimicrobial peptides exhibit toxicity against eukaryotic membranes, typically characterised by hemolytic activity assays, but currently, the understanding of what differentiates hemolytic and non-hemolytic peptides is limited. This study leverages advances in machine learning research to produce a novel artificial neural network classifier for the prediction of hemolytic activity from a peptide's primary sequence. The classifier achieves best-in-class performance, with cross-validated accuracy of [Formula: see text] and Matthews correlation coefficient of 0.71. This innovative classifier is available as a web server at https://research.timmons.eu/happenn , allowing the research community to utilise it for in silico screening of peptide drug candidates for high therapeutic efficacies.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Hemólisis/efectos de los fármacos , Hemolíticos/farmacología , Aprendizaje Automático , Redes Neurales de la Computación , Programas Informáticos , Péptidos Catiónicos Antimicrobianos/química , Simulación por Computador , Hemolíticos/química , Humanos , Análisis de Secuencia de ProteínaRESUMEN
Antimicrobial peptides (AMPs) are cationic small peptide chains that have good antimicrobial activity against a variety of bacteria, fungi, and viruses. AMP-17 is a recombinant insect AMP obtained by a prokaryotic expression system. However, the full antifungal activity, physicochemical characteristics, and cytotoxicity of AMP-17 were previously unknown. AMP-17 was shown to have good antifungal activity against five pathogenic fungi, with minimum inhibitory concentrations (MIC) of 9.375-18.75 µg/ml, and minimum fungicidal concentrations (MFC) of 18.75-37.5 µg/ml. Notably, the antifungal activity of AMP-17 against Cryptococcus neoformans was superior to that of other Candida spp. In addition, the hemolytic rate of AMP-17 was only 1.47%, even at the high concentration of 16× MIC. AMP-17 was insensitive to temperature and high salt ion concentration, with temperatures of 98°C and -80°C, and NaCl and MgCl2 concentrations of 50-200 mmol/l, having no significant effect on antifungal activity. However, AMP-17 was sensitive to proteases, trypsin, pepsin, and proteinase K. The elucidation of antifungal activity, physicochemical properties and cytotoxicity of AMP-17 provided an experimental basis for its safety evaluation and application, as well as indicated that AMP-17 might be a promising drug.Antimicrobial peptides (AMPs) are cationic small peptide chains that have good antimicrobial activity against a variety of bacteria, fungi, and viruses. AMP-17 is a recombinant insect AMP obtained by a prokaryotic expression system. However, the full antifungal activity, physicochemical characteristics, and cytotoxicity of AMP-17 were previously unknown. AMP-17 was shown to have good antifungal activity against five pathogenic fungi, with minimum inhibitory concentrations (MIC) of 9.37518.75 µg/ml, and minimum fungicidal concentrations (MFC) of 18.7537.5 µg/ml. Notably, the antifungal activity of AMP-17 against Cryptococcus neoformans was superior to that of other Candida spp. In addition, the hemolytic rate of AMP-17 was only 1.47%, even at the high concentration of 16× MIC. AMP-17 was insensitive to temperature and high salt ion concentration, with temperatures of 98°C and 80°C, and NaCl and MgCl2 concentrations of 50200 mmol/l, having no significant effect on antifungal activity. However, AMP-17 was sensitive to proteases, trypsin, pepsin, and proteinase K. The elucidation of antifungal activity, physicochemical properties and cytotoxicity of AMP-17 provided an experimental basis for its safety evaluation and application, as well as indicated that AMP-17 might be a promising drug.
Asunto(s)
Antifúngicos/farmacología , Moscas Domésticas/química , Péptidos/farmacología , Animales , Antifúngicos/química , Antifúngicos/aislamiento & purificación , Eritrocitos/citología , Eritrocitos/efectos de los fármacos , Hongos/efectos de los fármacos , Hemolíticos/química , Hemolíticos/aislamiento & purificación , Hemolíticos/farmacología , Humanos , Pruebas de Sensibilidad Microbiana , Péptidos/química , Péptidos/aislamiento & purificaciónRESUMEN
Lysobacter are ubiquitous in the environment but remain largely underexplored, although the bacteria are considered "peptide specialists". Here, we identified a new cyclic lipodepsipeptide, WBP-29479A1 (1), through genome mining of L. antibioticus ATCC 29479. 1 is biosynthesized by a large NRPS gene cluster, and its structure, including the six nonproteinogenic residues and 3-hydroxy fatty acid, was determined by extensive spectroscopic analyses and chemical derivatization. 1 exhibits potent anti-MRSA activity in a menaquinone-dependent manner.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Lysobacter/genética , Antibacterianos/metabolismo , Depsipéptidos/química , Depsipéptidos/farmacología , Genoma Bacteriano , Hemolíticos/química , Hemolíticos/farmacología , Humanos , Lysobacter/metabolismo , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Familia de Multigenes , Mutación , Péptido Sintasas/genética , Péptido Sintasas/metabolismo , ValinaRESUMEN
To overcome the disadvantages of chemical and physical methods, phyto-fabricated nanoparticles attained great attention due to their multifarious applications. Here we successfully demonstrated Papaver somniferum L. mediated green synthesis of lead oxide (PbO) and iron oxide (Fe2O3) nanoparticles. Characterization of nanoparticles involved techniques including X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR) and energy dispersive X-ray (EDX) associated with scanning electron microscopy (SEM). XRD analysis confirmed the phase identification and crystalline nature. FTIR analysis confirmed the capping of nanoparticles by plants' phytochemicals. SEM revealed morphological features of PbO and Fe2O3 with size of nanoparticles being 23⯱â¯11â¯nm and 38⯱â¯13â¯nm, respectively. The elemental composition of the nanoparticles was confirmed by EDX. Both bacterial and fungal isolates showed susceptibility towards PbO and Fe2O3 NPs. Both the NPs also showed considerable total antioxidant potential, free radical scavenging potential and reducing power. Insignificant level of α-amylase for both NPs was observed. Fe2O3 NPs showed superior biocompatibility with human RBCs as compared to PbO whereas PbO showed more potent anti-cancer activity as compared to Fe2O3 NPs. Overall our study concluded that both NPs played vital role in multiple biological assays however, extensive research focused on cytotoxic evaluation of NPs in-vivo is required.
Asunto(s)
Compuestos Férricos/química , Plomo/química , Nanopartículas del Metal/química , Óxidos/química , Papaver/metabolismo , Antibacterianos/química , Antibacterianos/farmacología , Antifúngicos/química , Antifúngicos/farmacología , Antioxidantes/química , Antioxidantes/farmacología , Evaluación Preclínica de Medicamentos , Estabilidad de Medicamentos , Eritrocitos/efectos de los fármacos , Hemolíticos/química , Hemolíticos/farmacología , Células Hep G2 , Humanos , Hipoglucemiantes/química , Hipoglucemiantes/farmacología , Nanopartículas del Metal/uso terapéutico , Papaver/química , Espectroscopía Infrarroja por Transformada de Fourier , Difracción de Rayos X , alfa-Amilasas/antagonistas & inhibidores , alfa-Amilasas/metabolismoRESUMEN
The present investigation was undertaken to evaluate the antibacterial, antifungal and hemolytic activities of organic and aqueous fractions of Fumaria indica, Dicliptera bupleuroides and Curcuma zedoaria. The methanolic extracts of the plants were dissolved in the water (distilled) separately and then partitioned with the n-hexane, CHCl3, EtOAc and n-BuOH sequentially. Antibacterial activity was checked against Escherichia coli, Pasturella multocida, Bacillus subtilis and Staphylococcus aureus by the disc diffusion method using streptomycin sulphate, a standard antibiotic, as positive control. Antifungal activity was studied against four fungi i.e. Aspergillus niger, Aspergillus flavus, Ganoderma lucidum and Alternaria alternata by the disc diffusion method using fluconazole, a standard antifungal drug, as positive control. It was revealed that aqueous fraction of F. indica showed very good antibacterial activity against P. multocida with zone of inhibition 26mm and MIC of 98µg/mL. Its CHCl3 and n-BuOH fractions also displayed good results. Its CHCl3 fraction showed good antifungal activity against G. lucidum with zone of inhibition 24mm and MIC of 115µg/mL. Other polar fractions of F. indica showed good activity against somefungal strains. The CHCl3 and EtOAc fractions of D. bupleuroides displayed good antibacterial activity against some bacterial strains. Its EtOAc fraction showed good antifungal activity only against G. lucidum. The CHCl3 fraction of C. zedoaria showed good activity against all studied bacterial strains, while its EtOAc and n-BuOH fractions displayed good results against some bacterial strains. None of the fractions of C. zedoaria displayed antifungal activity against the under test strains. All the studied fractions of three plants showed very less toxicity except n-hexane fraction of D. bupleuroides which showed 79% toxicity.
Asunto(s)
Antibacterianos/farmacología , Antifúngicos/farmacología , Hemolíticos/farmacología , Extractos Vegetales/farmacología , Plantas Medicinales/química , Acanthaceae/química , Antibacterianos/química , Antifúngicos/química , Curcuma/química , Evaluación Preclínica de Medicamentos , Fumaria/química , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Hemólisis/efectos de los fármacos , Hemolíticos/química , Humanos , Pruebas de Sensibilidad Microbiana , Extractos Vegetales/químicaRESUMEN
In the present communication, synthesis of bis-pyrazolones containing aryl motifs (4-14) and their α-glucosidase inhibitory activity, hemolytic and antihemolytic activities were reported. The newly synthesized compounds were characterized by analytical techniques such 1H-NMR, 13C-NMR, IR, mass spectrometry and compound No 4 additionally by X-ray crystallography. Compounds 4, 12, 14 were obtained in more than 85% yield. In comparison to typical acarbose (IC50 = 37.38±0.12µM), all synthesized compounds showed potent activity with IC50 values between 31.26±0.11 to 396.25±0.18µM. The most potent compounds 6, 8 and 11 showed IC50 values within the range of 31.26±0.11 to 37.48±0.12µM. Compounds 7, 10, 12 and 13 showed IC50 values within the range of 65.23±0.12 to 154.87±0.16µM, while compounds 4, 5 and 9 showed moderate inhibition with IC50 values 286.56±0.16 to 396.25±0.18µM. Structure-activity relationship (SAR) studies, suggests that electron withdrawing groups played a crucial role in enhancing α-glucosidase inhibitory effects of title compounds. In addition, results of the hemolytic and antihemolytic activity studies indicated that compound 13 possessed moderate levels of hemolytic and highest anti- hemolytic activity while 8 showed low anti- hemolytic and high hemolytic activity.
Asunto(s)
Inhibidores de Glicósido Hidrolasas/química , Inhibidores de Glicósido Hidrolasas/farmacología , Hemolíticos/química , Hemolíticos/farmacología , Pirazoles/síntesis química , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Hemólisis/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Pirazoles/química , Pirazoles/farmacología , Espectrofotometría Infrarroja , Relación Estructura-ActividadRESUMEN
Vibrio parahaemolyticus is a major seafood-borne pathogen that causes life-threatening gastroenteric diseases in humans through the consumption of contaminated seafoods. V. parahaemolyticus produces different kinds of toxins, including thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH), and some effector proteins belonging to the Type 3 Secretion System, out of which TDH and TRH are considered to be the major factors for virulence. Although TRH is one of the major virulent proteins, there is a dearth of understanding about the structural and functional properties of this protein. This study therefore aimed to amplify the full length trh gene from V. parahaemolyticus and perform sequence-based analyses, followed by structural and functional analyses of the TRH protein using different bioinformatics tools. The TRH protein shares significant conservedness with the TDH protein. A multiple sequence alignment of TRH proteins from Vibrio and non-Vibrio species revealed that the TRH protein is highly conserved throughout evolution. The three dimensional (3D) structure of the TRH protein was constructed by comparative modelling and the quality of the predicted model was verified. Molecular dynamics simulations were performed to understand the dynamics, residual fluctuations, and the compactness of the protein. The structure of TRH was found to contain 19 pockets, of which one (pocket ID: 2) was predicted to be important from the view of drug design. Eleven residues (E138, Y140, C151, F158, C161, K162, S163, and Q164), which are reported to actively participate in the formation of the tetrameric structure, were present in this pocket. This study extends our understanding of the structural and functional dynamics of the TRH protein and as well as provides new insights for the treatment and prevention of V. parahaemolyticus infections.
Asunto(s)
Proteínas Hemolisinas/química , Simulación de Dinámica Molecular , Vibrio parahaemolyticus/química , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Proteínas Hemolisinas/genética , Hemolíticos/química , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homología de Secuencia de Aminoácido , Vibrio parahaemolyticus/genéticaRESUMEN
INTRODUCTION: Being important representatives of various proteomes, membrane-active cationic peptides (CPs) are attractive objects as lead compounds in the design of new antibacterial, anticancer, antifungal, and antiviral molecules. Numerous CPs are found in insect and snake venoms, where many of them reveal cytolytic properties. Due to advances in omics technologies, the number of such peptides is growing dramatically. Areas covered: To understand structure-function relationships for CPs in a living cell, detailed analysis of their hydrophobic/hydrophilic properties is indispensable. We consider two structural classes of membrane-active CPs: latarcins (Ltc) from spider and cardiotoxins (CTXs) from snake venoms. While the former are void off disulfide bonds and conformationally flexible, the latter are structurally rigid and cross-linked with disulfide bonds. In order to elucidate structure-activity relationships behind their antibacterial, anticancer, and hemolytic effects, the properties of these polypeptides are considered on a side-by-side basis. Expert commentary: An ever-increasing number of venom-derived membrane-active polypeptides require new methods for identification of their functional propensities and sequence-based design of novel pharmacological substances. We address these issues considering a number of the designed peptides, based either on Ltc or CTX sequences. Experimental and computer modeling techniques required for these purposes are delineated.
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Péptidos Catiónicos Antimicrobianos/farmacología , Cardiotoxinas/farmacología , Péptidos/química , Péptidos/farmacología , Animales , Antibacterianos/química , Antibacterianos/farmacología , Antifúngicos/química , Antifúngicos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Antineoplásicos/química , Antineoplásicos/farmacología , Cardiotoxinas/química , Disulfuros/química , Diseño de Fármacos , Hemolíticos/química , Hemolíticos/farmacología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Péptidos/metabolismo , Venenos de Araña/química , Relación Estructura-ActividadRESUMEN
The current study found that an extract from the fruiting bodies of the edible mushroom Hypsizygus marmoreus exhibited hemolytic activity against sheep red blood cells when its pH was lowered. Although hemolytic activity was not detected when an extract had a neutral pH, an extract with a low pH exhibited potent hemolytic activity. The maximal hemolytic activity was exhibited by an extract with a pH of 5.5. A heat-treated extract did not exhibit hemolytic activity before its pH was lowered, and that activity was inhibited in the presence of PMSF and EDTA. The turbidity of the extract increased during lowering of its pH, and the precipitate fraction exhibited hemolytic activity. Fractionation by a modified Bligh and Dyer method and TLC analyses suggested that a hemolytic compound in the extract might be a type of lipid. These results suggest that a hemolytic lipid-like compound in an extract of H. marmoreus fruiting bodies may be released by a non-active precursor substance(s) through metalloenzyme(s) while the extract has a low pH.
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Agaricales/metabolismo , Cuerpos Fructíferos de los Hongos/metabolismo , Proteínas Hemolisinas/toxicidad , Hemólisis/efectos de los fármacos , Hemolíticos/toxicidad , Animales , Fraccionamiento Químico , Eritrocitos/efectos de los fármacos , Proteínas Hemolisinas/química , Proteínas Hemolisinas/aislamiento & purificación , Hemolíticos/química , Hemolíticos/aislamiento & purificación , Concentración de Iones de Hidrógeno , Lipoproteínas , OvinosRESUMEN
Due to the rapid emergence of resistance to classical antibiotics, novel antimicrobial compounds are needed. It is desirable to selectively kill pathogenic bacteria without targeting other beneficial bacteria in order to prevent the negative clinical consequences caused by many broad-spectrum antibiotics as well as reducing the development of antibiotic resistance. Antimicrobial peptides (AMPs) represent an alternative to classical antibiotics and it has been previously demonstrated that Cap18 has high antimicrobial activity against a broad range of bacterial species. In this study we report the design of a positional scanning library consisting of 696 Cap18 derivatives and the subsequent screening for antimicrobial activity against Y. ruckeri, A. salmonicida, S. Typhimurium and L. lactis as well as for hemolytic activity measuring the hemoglobin release of horse erythrocytes. We show that the hydrophobic face of Cap18, in particular I13, L17 and I24, is essential for its antimicrobial activity against S. Typhimurium, Y. ruckeri, A. salmonicida, E. coli, P. aeruginosa, L. lactis, L. monocytogenes and E. faecalis. In particular, Cap18 derivatives harboring a I13D, L17D, L17P, I24D or I24N substitution lost their antimicrobial activity against any of the tested bacterial strains. In addition, we were able to generate species-specific Cap18 derivatives by particular amino acid substitutions either in the hydrophobic face at positions L6, L17, I20, and I27, or in the hydrophilic face at positions K16 and K18. Finally, our data showed the proline residue at position 29 to be essential for the inherent low hemolytic activity of Cap18 and that substitution of the residues K16, K23, or G21 by any hydrophobic residues enhances the hemolytic activity. This study demonstrates the potential of generating species-specific AMPs for the selective elimination of bacterial pathogens.
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Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Hemolíticos/farmacología , Aeromonas salmonicida/efectos de los fármacos , Sustitución de Aminoácidos , Animales , Antiinfecciosos/química , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/genética , Diseño de Fármacos , Eritrocitos/efectos de los fármacos , Hemolíticos/química , Caballos , Pruebas de Sensibilidad Microbiana , Biblioteca de Péptidos , Salmonella typhimurium/efectos de los fármacos , Yersinia ruckeri/efectos de los fármacos , CatelicidinasRESUMEN
Besides key roles in prey capture and predator defense, scorpion venom also functions as internal immune agents protecting the venom gland from infection and external immune agents cleaning saprophytic microbes from their own body surfaces. However, antimicrobials (typically antimicrobial peptides, AMPs) in the venom often exist in low abundance that might exclude their immune role alone, leaving an open question with regard to their in vivo biological function. Here, we report the bactericidal activity of seven peptides isolated from the scorpion Mesobuthus eupeus venom, including one classical α-helical AMP and five ion channel-targeted neurotoxins. This AMP of 49 amino acids (named Meucin-49) is a multifunctional molecule that displays a wide-spectrum and highly potent activity against Gram-positive and Gram-negative bacteria with strong hemotoxicity on scorpion's predators (i.e., mammals, lizards, and birds) and high insecticidal activity. Although the neurotoxins targeting voltage-gated sodium (Nav) and/or large conductance calcium-activated potassium (BK) channels showed only marginal activity towards several species of bacteria, they were capable of significantly potentiating the bactericidal potency of Meucin-49. This observation highlights, for the first time, the venom's antibacterial immune function mediated by a joint action between neurotoxins and AMPs. The findings that traditionally defined neurotoxins possess (synergistic) bactericidal activity, while the classical AMPs play predatory and defensive roles, provide new evidence in favor of a general and intrinsic multifunctionality of scorpion venom components.
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Antibacterianos/química , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Neurotoxinas/química , Neurotoxinas/farmacología , Venenos de Escorpión/química , Secuencia de Aminoácidos , Animales , Antibacterianos/inmunología , Antibacterianos/aislamiento & purificación , Péptidos Catiónicos Antimicrobianos/inmunología , Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Línea Celular , Permeabilidad de la Membrana Celular/efectos de los fármacos , Columbidae , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Hemolíticos/química , Hemolíticos/aislamiento & purificación , Hemolíticos/farmacología , Moscas Domésticas/efectos de los fármacos , Humanos , Inmunidad Innata , Lagartos , Ratones , Neurotoxinas/inmunología , Neurotoxinas/aislamiento & purificación , Canales de Potasio Calcio-Activados/antagonistas & inhibidores , Conformación Proteica , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacologíaRESUMEN
Recent recognition about snake bite envenomation on June, 2017 as neglected tropical disease under category-A by World Health Organization advocated again its undeniable importance. Present circumstances reasoned to work on a neglected subspecies of Naja naja, i.e., Naja naja karachiensis (N. n. karachensis) has been documented for frequent deaths in Pakistan. In this study median lethal toxic dose (LD50) was determined intraperitoneally in Swiss albino mice and was found to be 2.0µg/g (2.0mg/kg) equal in potency to Naja pallida (red spitting African cobra). Total protein contents (188±0.011µg / 200µg of dry weight) were high enough (94%) to represent an arsenal of proteins. Furthermore, 99mTc was labeled 99.9% with venom and didn't find to alter hemolytic activity of venom in dose dependent manner at 125µg/ml (p>0.5), 250 µg/ml (p>0.1) and 500 µg/ml (p>0.1) when compared with its crude form. Present work will pave the way for proteomics study in effective production of antidote against specific species of snakes as dare demand of it has been felt since long period of time in Pakistan.