RESUMEN
Diverse computational approaches have been widely used to assist in designing antimicrobial peptides with enhanced activities. This tactic has also been used to address the need for new treatment alternatives to combat resistant bacterial infections. Herein, we have designed eight variants from a natural peptide, pro-adrenomedullin N-terminal 20 peptide (PAMP), using an in silico pattern insertion approach, the Joker algorithm. All the variants show an α-helical conformation, but with differences in the helix percentages according to circular dichroism (CD) results. We found that the C-terminal portion of PAMP may be relevant for its antimicrobial activities, as revealed by the molecular dynamics, CD, and antibacterial results. The analogs showed variable antibacterial potential, but most were not cytotoxic. Nevertheless, PAMP2 exhibited the most potent activities against human and animal-isolated bacteria, showing cytotoxicity only at a substantially higher concentration than its minimal inhibitory concentration (MIC). Our results suggest that the enhanced activity in the profile of PAMP2 may be related to their particular physicochemical properties, along with the adoption of an amphipathic α-helical arrangement with the conserved C-terminus portion. Finally, the peptides designed in this study can constitute scaffolds for the design of improved sequences.
Asunto(s)
Adrenomedulina , Dicroismo Circular , Pruebas de Sensibilidad Microbiana , Simulación de Dinámica Molecular , Humanos , Adrenomedulina/química , Adrenomedulina/farmacología , Secuencia de Aminoácidos , Antibacterianos/farmacología , Antibacterianos/química , Antibacterianos/síntesis química , Animales , Simulación por Computador , Precursores de Proteínas/química , Precursores de Proteínas/farmacología , Precursores de Proteínas/metabolismo , Péptidos Antimicrobianos/química , Péptidos Antimicrobianos/farmacología , Estructura Secundaria de ProteínaRESUMEN
BACKGROUND: The synthetic antimicrobial peptide, PaDBS1R1, has been reported as a powerful anti-Klebsiella pneumoniae antimicrobial. However, there is only scarce knowledge about whether K. pneumoniae could develop resistance against PaDBS1R1 and which resistance mechanisms could be involved. OBJECTIVES: Identify via label-free shotgun proteomics the K. pneumoniae resistance mechanisms developed against PaDBS1R1. METHODS: An adaptive laboratory evolution experiment was performed to obtain a PaDBS1R1-resistant K. pneumoniae lineage. Antimicrobial susceptibility was determined through microdilution assay. Modifications in protein abundances between the resistant and sensitive lineages were measured via label-free quantitative shotgun proteomics. Enriched Gene Ontology terms and KEGG pathways were identified through over-representation analysis. Data are available via ProteomeXchange with identifier PXD033020. RESULTS: K. pneumoniae ATCC 13883 parental strain challenged with increased subinhibitory PaDBS1R1 concentrations allowed the PaDBS1R1-resistant K. pneumoniae lineage to emerge. Proteome comparisons between PaDBS1R1-resistant K. pneumoniae and PaDBS1R1-sensitive K. pneumoniae under PaDBS1R1-induced stress conditions enabled the identification and quantification of 1702 proteins, out of which 201 were differentially abundant proteins (DAPs). The profiled DAPs comprised 103 up-regulated proteins (adjusted P value < 0.05, fold change ≥ 2) and 98 down-regulated proteins (adjusted P value < 0.05, fold change ≤ 0.5). The enrichment analysis suggests that PhoPQ-guided LPS modifications and CpxRA-dependent folding machinery could be relevant resistance mechanisms against PaDBS1R1. CONCLUSIONS: Based on experimental evolution and a label-free quantitative shotgun proteomic approach, we showed that K. pneumoniae developed resistance against PaDBS1R1, whereas PhoPQ-guided LPS modifications and CpxRA-dependent folding machinery appear to be relevant resistance mechanisms against PaDBS1R1.
Asunto(s)
Antiinfecciosos , Infecciones por Klebsiella , Humanos , Antibacterianos/farmacología , Klebsiella pneumoniae/genética , Péptidos Antimicrobianos , Proteómica , Lipopolisacáridos , Antiinfecciosos/farmacología , Pruebas de Sensibilidad MicrobianaRESUMEN
Klebsiella pneumoniae has been implicated in wide-ranging nosocomial outbreaks, causing severe infections without effective treatments due to antibiotic resistance. Here, we performed genome sequencing of 70 extensively drug resistant clinical isolates, collected from Brasília's hospitals (Brazil) between 2010 and 2014. The majority of strains (60 out of 70) belonged to a single clonal complex (CC), CC258, which has become distributed worldwide in the last two decades. Of these CC258 strains, 44 strains were classified as sequence type 11 (ST11) and fell into two distinct clades, but no ST258 strains were found. These 70 strains had a pan-genome size of 10â366 genes, with a core-genome size of ~4476 genes found in 95â% of isolates. Analysis of sequences revealed diverse mechanisms of resistance, including production of multidrug efflux pumps, enzymes with the same target function but with reduced or no affinity to the drug, and proteins that protected the drug target or inactivated the drug. ß-Lactamase production provided the most notable mechanism associated with K. pneumoniae. Each strain presented two or three different ß-lactamase enzymes, including class A (SHV, CTX-M and KPC), class B and class C AmpC enzymes, although no class D ß-lactamase was identified. Strains carrying the NDM enzyme involved three different ST types, suggesting that there was no common genetic origin.
Asunto(s)
Farmacorresistencia Bacteriana Múltiple/genética , Genómica , Klebsiella pneumoniae/genética , Factores de Virulencia/genética , Brasil , ADN Bacteriano/genética , Humanos , Infecciones por Klebsiella/epidemiología , Klebsiella pneumoniae/aislamiento & purificación , Filogenia , Virulencia/genética , beta-Lactamasas/genéticaRESUMEN
BACKGROUND: Amylin is a 37-amino-acid peptide hormone co-secreted with insulin, which participates in glucose homeostasis. This hormone is able to aggregate in a ß-sheet conformation and deposit in islet amyloids, a hallmark in type II diabetes. Since amylin is a gene-encoded hormone, this peptide has variants caused by point mutations that can impact its functions. METHODS: Here, we analyzed the structural effects caused by S20G and G33R point mutations which, according to the 1000 Genomes Project, have frequency in East Asian and European populations, respectively. The analyses were performed by means of aggrescan server, SNP functional effect predictors, and molecular dynamics. RESULTS: We found that both mutations have aggregation potential and cause changes in the monomeric forms when compared with wild-type amylin. Furthermore, comparative analyses with pramlintide, an amylin drug analogue, allowed us to infer that second α-helix maintenance may be related to the aggregation potential. CONCLUSIONS: The S20G mutation has been described as pathologically related, which is in agreement with our findings. In addition, our data suggest that the G33R mutation might have a deleterious effect. The data presented here also provide new therapy opportunities, whether for creating more effective drugs for diabetes or implementing specific treatment for patients with these mutations. GENERAL SIGNIFICANCE: Our data could help to better understand the impact of mutations on the wild-type amylin sequence, as a starting point for the evaluation and characterization of other variations. Moreover, these findings could improve the health of patients with type II diabetes.
Asunto(s)
Diabetes Mellitus Tipo 2/genética , Polipéptido Amiloide de los Islotes Pancreáticos/química , Polipéptido Amiloide de los Islotes Pancreáticos/genética , Simulación de Dinámica Molecular , Mutación Puntual , Diabetes Mellitus Tipo 2/patología , Humanos , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismoRESUMEN
BACKGROUND: Physical exercise is an essential factor in preventing and treating metabolic diseases by promoting systemic benefits throughout the body. The molecular factors involved in this process are poorly understood. Micro RNAs (miRNAs) are small non-coding RNAs that inhibit mRNA transcription. MiRNAs, which can participate in the benefits of exercise to health, circulate in plasma in extracellular particles (EP). Horses that undergo endurance racing are an excellent model to study the impact of long-duration/low intensity exercise in plasma EP miRNAs. OBJECTIVES: To evaluate the effects of 160 km endurance racing on horse plasma extracellular particles and their miRNA population. STUDY DESIGN: Cohort study. METHODS: We collected plasma from five Arabian horses during five time-points of an endurance ride. Extracellular particles were purified from plasma and characterised by electron microscopy, resistive pulse sensing (qNano) and western blotting. Small RNAs were purified from horse plasma EP, and sequencing was performed. RESULTS: Endurance racing increased EP concentration and average diameter compared to before the race. Western blotting showed a high concentration of extracellular vesicles proteins 2 hours after the race, which returned to baseline 15 hours after the race. MicroRNA differential expression analysis revealed increasing levels of eca-miR-486-5p during and after the race, and decreasing levels of eca-miR-9083 after the end. CONCLUSIONS: This study adds new data about the variation in plasma EP concentrations after long-distance exercise and brings new insights about the roles of exercise-derived EP miRNAs during low-intensity endurance exercise.
Asunto(s)
MicroARNs , Condicionamiento Físico Animal , Animales , Estudios de Cohortes , Caballos , MicroARNs/genética , Resistencia Física , PlasmaRESUMEN
Novel antibiotics are urgently needed to combat multidrug-resistant pathogens. Venoms represent previously untapped sources of novel drugs. Here we repurposed mastoparan-L, the toxic active principle derived from the venom of the wasp Vespula lewisii, into synthetic antimicrobials. We engineered within its N terminus a motif conserved among natural peptides with potent immunomodulatory and antimicrobial activities. The resulting peptide, mast-MO, adopted an α-helical structure as determined by NMR, exhibited increased antibacterial properties comparable to standard-of-care antibiotics both in vitro and in vivo, and potentiated the activity of different classes of antibiotics. Mechanism-of-action studies revealed that mast-MO targets bacteria by rapidly permeabilizing their outer membrane. In animal models, the peptide displayed direct antimicrobial activity, led to enhanced ability to attract leukocytes to the infection site, and was able to control inflammation. Permutation studies depleted the remaining toxicity of mast-MO toward human cells, yielding derivatives with antiinfective activity in animals. We demonstrate a rational design strategy for repurposing venoms into promising antimicrobials.
Asunto(s)
Bacteriemia/tratamiento farmacológico , Proteínas Citotóxicas Formadoras de Poros/química , Venenos de Avispas/química , Animales , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Células HEK293 , Humanos , Ratones , Pruebas de Sensibilidad Microbiana , Proteínas Citotóxicas Formadoras de Poros/uso terapéutico , Proteínas Citotóxicas Formadoras de Poros/toxicidad , Venenos de Avispas/uso terapéutico , Venenos de Avispas/toxicidadRESUMEN
BACKGROUND: Bacterial infections represent a major worldwide health problem the antimicrobial peptides (AMPs) have been considered as potential alternative agents for treating these infections. Here we demonstrated the antimicrobial activity of EcDBS1R6, a peptide derived from a signal peptide sequence of Escherichia coli that we previously turned into an AMP by making changes through the Joker algorithm. METHODS: Antimicrobial activity was measured by broth microdilution method. Membrane integrity was measured using fluorescent probes and through scanning electron microscopy imaging. A sliding window of truncated peptides was used to determine the EcDBS1R6 active core. Molecular dynamics in TFE/water environment was used to assess the EcDBS1R6 structure. RESULTS: Signal peptides are known to naturally interact with membranes; however, the modifications introduced by Joker transformed this peptide into a membrane-active agent capable of killing bacteria. The C-terminus was unable to fold into an α-helix whereas its fragments showed poor or no antimicrobial activity, suggesting that the EcDBS1R6 antibacterial core was located at the helical N-terminus, corresponding to the signal peptide portion of the parent peptide. CONCLUSION: The strategy of transforming signal peptides into AMPs appears to be promising and could be used to produce novel antimicrobial agents. GENERAL SIGNIFICANCE: The process of transforming an inactive signal peptide into an antimicrobial peptide could open a new venue for creating new AMPs derived from signal peptides.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Escherichia coli/química , Señales de Clasificación de Proteína , Secuencia de Aminoácidos , Bacterias Gramnegativas/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Simulación de Dinámica Molecular , Conformación ProteicaRESUMEN
Diverse peptides have been evaluated for their activity against pathogenic microorganisms. Here, five mastoparan variants were designed based on mastoparan-L, among which two (R1 and R4) were selected for in-depth analysis. Mastoparan-L (parent/control), R1, and R4 inhibited susceptible/resistant bacteria at concentrations ranging from 2 to 32 µM, whereas only R1 and R4 eradicated Pseudomonas aeruginosa biofilms at 16 µM. Moreover, the toxic effects of mastoparan-L toward mammalian cells were drastically reduced in both variants. In skin infections, R1 at 64 µM was the most effective variant, reducing P. aeruginosa bacterial counts 1000 times on day 4 post-infection. Structurally, all of the peptides showed varying levels of helicity and structural stability in aqueous and membrane-like conditions, which may affect the different bioactivities observed here. By computationally modifying the physicochemical properties of R1 and R4, we reduced the cytotoxicity and optimized the therapeutic potential of these mastoparan-like peptides both in vitro and in vivo.
Asunto(s)
Antibacterianos/farmacología , Diseño Asistido por Computadora , Péptidos y Proteínas de Señalización Intercelular/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Venenos de Avispas/farmacología , Antibacterianos/síntesis química , Antibacterianos/química , Relación Dosis-Respuesta a Droga , Péptidos y Proteínas de Señalización Intercelular/síntesis química , Péptidos y Proteínas de Señalización Intercelular/química , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Relación Estructura-Actividad , Venenos de Avispas/síntesis química , Venenos de Avispas/químicaRESUMEN
Bacterial biofilms and associated infections represent one of the biggest challenges in the clinic, and as an alternative to counter bacterial infections, antimicrobial peptides have attracted great attention in the past decade. Here, ten short cationic antimicrobial peptides were generated through a sliding-window strategy on the basis of the 19-amino acid residue peptide, derived from a Pyrobaculum aerophilum ribosomal protein. PaDBS1R6F10 exhibited anti-infective potential as it decreased the bacterial burden in murine Pseudomonas aeruginosa cutaneous infections by more than 1000-fold. Adverse cytotoxic and hemolytic effects were not detected against mammalian cells. The peptide demonstrated structural plasticity in terms of its secondary structure in the different environments tested. PaDBS1R6F10 represents a promising antimicrobial agent against bacteria infections, without harming human cells.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Infecciones por Pseudomonas/tratamiento farmacológico , Pseudomonas aeruginosa/efectos de los fármacos , Pyrobaculum/metabolismo , Proteínas Ribosómicas/química , Secuencia de Aminoácidos , Animales , Péptidos Catiónicos Antimicrobianos/química , Proteínas Arqueales/química , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Modelos Animales de Enfermedad , Humanos , Ratones , Pruebas de Sensibilidad Microbiana , Estructura Secundaria de Proteína , Pseudomonas aeruginosa/fisiologíaRESUMEN
Antimicrobial peptides (AMPs) are small molecules present in all living beings. Despite their huge sequence variability, AMPs present great structural conservation, mainly in cysteine-stabilized families. Moreover, in non-model plants, it is possible to detect cysteine-stabilized AMPs (cs-AMPs) with different sequences not covered by conventional searches. Here, we described a threading application for cs-AMP identification in the non-model arum lily (Zantedeschia aethiopica) plant, exploring the spathe transcriptome. By using the predicted proteins from the Z. aethiopica transcriptome as our primary source of sequences, we have filtered by using structural alignments of 12 putative cs-AMP sequences. The two unreported sequences were submitted to PCR validation, and ZaLTP7 gene was confirmed. By using the structure alignments, we classified ZaLTP7 as an LTP type 2-like. The successful threading application for cs-AMP identification is an important advance in transcriptomic and proteomic data mining. Besides, the same approach could be applied to the use of NGS public data to discover molecules to combat multidrug-resistant bacteria.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Proteínas de Plantas/química , Transcriptoma , Zantedeschia/genética , Secuencia de Aminoácidos , Antibacterianos/química , Secuenciación de Nucleótidos de Alto Rendimiento , Simulación de Dinámica Molecular , Reproducibilidad de los ResultadosRESUMEN
Infections caused by Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa foremost among them, constitute a major worldwide health problem. Bioinformatics methodologies are being used to rationally design new antimicrobial peptides, a potential alternative for treating these infections. One of the algorithms used to develop antimicrobial peptides is the Joker, which was used to design the peptide PaDBS1R6. This study evaluates the antibacterial activities of PaDBS1R6 in vitro and in vivo, characterizes the peptide interaction to target membranes, and investigates the PaDBS1R6 structure in contact with mimetic vesicles. Moreover, we demonstrate that PaDBS1R6 exhibits selective antimicrobial activity against Gram-negative bacteria. In the presence of negatively charged and zwitterionic lipids the structural arrangement of PaDBS1R6 transits from random coil to α-helix, as characterized by circular dichroism. The tertiary structure of PaDBS1R6 was determined by NMR in zwitterionic dodecylphosphocholine (DPC) micelles. In conclusion, PaDBS1R6 is a candidate for the treatment of nosocomial infections caused by Gram-negative bacteria, as template for producing other antimicrobial agents.
Asunto(s)
Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Animales , Ratones , Ratones Endogámicos C57BL , Pruebas de Sensibilidad MicrobianaRESUMEN
Antimicrobial peptides (AMPs) are promising candidates for the development of future antibiotics. In an attempt to increase the efficacy of therapeutic AMPs, computer-based design methods appear as a reliable strategy. In this study, we evaluated the antimicrobial efficiency and mechanism of action of a novel designed AMP named PaDBS1R1, previously designed by means of the Joker algorithm, using a fragment of the ribosomal protein L39E from the archaeon Pyrobaculum aerophilum as a template. PaDBS1R1 displayed low micromolar broad-spectrum antimicrobial activity against Gram-negative (MIC of 1.5⯵M) and Gram-positive (MIC of 3⯵M) bacteria, including carbapenem-resistant Klebsiella pneumoniae (MIC of 6.25⯵M) and methicillin-resistant Staphylococcus aureus (MIC of 12.5⯵M), without cytotoxicity towards HEK-293 cells. In addition, membrane permeabilization and depolarization assays, combined with time-kill studies and FEG-SEM imaging, indicated a fast membrane permeation and further leakage of intracellular content. Biophysical studies with lipid vesicles show a preference of PaDBS1R1 for Gram-negative bacteria-like membranes. We investigated the three-dimensional structure of PaDBS1R1 by CD and NMR analyses. Our results suggest that PaDBS1R1 adopts an amphipathic α-helix upon interacting with hydrophobic environments, after an initial electrostatic interaction with negative charges, suggesting a membrane lytic effect. This study reveals that PaDBS1R1 has potential application in antibiotic therapy.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Membrana Celular/efectos de los fármacos , Antibacterianos/farmacología , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Dicroismo Circular , Bacterias Gramnegativas , Células HEK293 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Luz , Lípidos/química , Espectroscopía de Resonancia Magnética , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Micelas , Pruebas de Sensibilidad Microbiana , Microscopía Electrónica de Rastreo , Conformación Proteica en Hélice alfa , Dispersión de RadiaciónRESUMEN
Computer-aided screening of antimicrobial peptides (AMPs) is a promising approach for discovering novel therapies against multidrug-resistant bacterial infections. Here, we functionally and structurally characterized an Escherichia coli-derived AMP (EcDBS1R5) previously designed through pattern identification [α-helical set (KK[ILV](3)[AILV])], followed by sequence optimization. EcDBS1R5 inhibited the growth of Gram-negative and Gram-positive, susceptible and resistant bacterial strains at low doses (2-32 µM), with no cytotoxicity observed against non-cancerous and cancerous cell lines in the concentration range analyzed (<100 µM). Furthermore, EcDBS1R5 (16 µM) acted on Pseudomonas aeruginosa pre-formed biofilms by compromising the viability of biofilm-constituting cells. The in vivo antibacterial potential of EcDBS1R5 was confirmed as the peptide reduced bacterial counts by two-logs 2 days post-infection using a skin scarification mouse model. Structurally, circular dichroism analysis revealed that EcDBS1R5 is unstructured in hydrophilic environments, but has strong helicity in 2,2,2-trifluoroethanol (TFE)/water mixtures (v/v) and sodium dodecyl sulfate (SDS) micelles. The TFE-induced nuclear magnetic resonance structure of EcDBS1R5 was determined and showed an amphipathic helical segment with flexible termini. Moreover, we observed that the amide protons for residues Met2-Ala8, Arg10, Ala13-Ala16, and Trp19 in EcDBS1R5 are protected from the solvent, as their temperature coefficients values are more positive than -4.6 ppb·K-1. In summary, this study reports a novel dual-antibacterial/antibiofilm α-helical peptide with therapeutic potential in vitro and in vivo against clinically relevant bacterial strains.
Asunto(s)
Antibacterianos/administración & dosificación , Antibacterianos/química , Péptidos Catiónicos Antimicrobianos/administración & dosificación , Péptidos Catiónicos Antimicrobianos/química , Biopelículas/efectos de los fármacos , Escherichia coli/química , Infecciones por Pseudomonas/tratamiento farmacológico , Animales , Dicroismo Circular , Diseño Asistido por Computadora , Diseño de Fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Femenino , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ratones , Pruebas de Sensibilidad Microbiana , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/fisiologíaRESUMEN
Physical exercise stimulates organs, mainly the skeletal muscle, to release a broad range of molecules, recently dubbed exerkines. Among them, RNAs, such as miRNAs, piRNAs, and tRNAs loaded in extracellular vesicles (EVs) have the potential to play a significant role in the way muscle and other organs communicate to translate exercise into health. Low, moderate and high intensity treadmill protocols were applied to rat groups, aiming to investigate the impact of exercise on serum EVs and their associated small RNA molecules. Transmission electron microscopy, resistive pulse sensing, and western blotting were used to investigate EVs morphology, size distribution, concentration and EVs marker proteins. Small RNA libraries from EVs RNA were sequenced. Exercise did not change EVs size, while increased EVs concentration. Twelve miRNAs were found differentially expressed after exercise: rno-miR-128-3p, 103-3p, 330-5p, 148a-3p, 191a-5p, 10b-5p, 93-5p, 25-3p, 142-5p, 3068-3p, 142-3p, and 410-3p. No piRNA was found differentially expressed, and one tRNA, trna8336, was found down-regulated after exercise. The differentially expressed miRNAs were predicted to target genes involved in the MAPK pathway. A single bout of exercise impacts EVs and their small RNA load, reinforcing the need for a more detailed investigation into EVs and their load as mediators of health-promoting exercise.
RESUMEN
Since the early 19th century, host-defense peptides (HDPs) have been known to play a crucial role in innate host defense. Subsequent work has demonstrated their role in adaptive immunity as well as their involvement in cancer and also a number of inflammatory and/or autoimmune diseases. In addition to these multiple functional activities, several studies have shown that HDP accumulation might be correlated with various human diseases and, therefore, could be used as a biomarkers for such. Thus, research has aimed to validate the clinical use of HDPs for diagnosis, prognosis, and further treatment. In this review, we outline the most recent findings related to the use of HDPs as biomarkers, their clinical and epidemiological value, and the techniques used to determine the levels of HDPs.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/metabolismo , Enfermedades del Sistema Inmune/diagnóstico , Enfermedades del Sistema Inmune/metabolismo , Inmunidad Adaptativa , Péptidos Catiónicos Antimicrobianos/inmunología , Bacterias/inmunología , Bacterias/metabolismo , Biomarcadores/metabolismo , Diagnóstico Precoz , Interacciones Huésped-Patógeno , Humanos , Enfermedades del Sistema Inmune/inmunología , Enfermedades del Sistema Inmune/terapia , Inmunidad Innata , Valor Predictivo de las Pruebas , PronósticoRESUMEN
Plants are extensively used in traditional medicine, and several plant antimicrobial peptides have been described as potential alternatives to conventional antibiotics. However, after more than four decades of research no plant antimicrobial peptide is currently used for treating bacterial infections, due to their length, post-translational modifications or high dose requirement for a therapeutic effect . Here we report the design of antimicrobial peptides derived from a guava glycine-rich peptide using a genetic algorithm. This approach yields guavanin peptides, arginine-rich α-helical peptides that possess an unusual hydrophobic counterpart mainly composed of tyrosine residues. Guavanin 2 is characterized as a prototype peptide in terms of structure and activity. Nuclear magnetic resonance analysis indicates that the peptide adopts an α-helical structure in hydrophobic environments. Guavanin 2 is bactericidal at low concentrations, causing membrane disruption and triggering hyperpolarization. This computational approach for the exploration of natural products could be used to design effective peptide antibiotics.
Asunto(s)
Antibacterianos/química , Péptidos Catiónicos Antimicrobianos/química , Proteínas de Plantas/química , Infecciones por Pseudomonas/tratamiento farmacológico , Pseudomonas aeruginosa/efectos de los fármacos , Psidium/química , Algoritmos , Secuencia de Aminoácidos , Animales , Antibacterianos/biosíntesis , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/biosíntesis , Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/farmacología , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Técnicas Químicas Combinatorias , Diseño de Fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Interacciones Hidrofóbicas e Hidrofílicas , Ratones , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/genética , Proteínas de Plantas/farmacología , Estructura Secundaria de Proteína , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/crecimiento & desarrollo , Psidium/metabolismo , Piel/efectos de los fármacos , Piel/microbiología , Relación Estructura-ActividadRESUMEN
ApoE3 is the major chylomicron apolipoprotein, binding in a specific liver peripheral cell receptor, allowing transport and normal catabolism of triglyceride-rich lipoprotein constituents. Point mutations in ApoE3 have been associated with Alzheimer's disease, type III hyperlipoproteinemia, atherosclerosis, telomere shortening and impaired cognitive function. Here, we evaluate the impact of missense SNPs in APOE retrieved from dbSNP through 16 computational prediction tools, and further evaluate the structural impact of convergent deleterious changes using 100 ns molecular dynamics simulations. We have found structural changes in four analyzed variants (Pro102Arg, Arg132Ser, Arg176Cys and Trp294Cys), two of them (Pro102Arg and Arg176Cys) being previously associated with human diseases. In all cases, except for Trp294Cys, there was a loss in the number of hydrogen bonds between CT and NT domains that could result in their detachment. In conclusion, data presented here could increase the knowledge of ApoE3 activity and be a starting point for the study of the impact of variations on APOE gene.
Asunto(s)
Apolipoproteína E3/genética , Mutación Missense/genética , Polimorfismo de Nucleótido Simple/genética , Relación Estructura-Actividad , Apolipoproteína E3/química , Apolipoproteínas E/química , Apolipoproteínas E/genética , Simulación por Computador , Humanos , Simulación de Dinámica Molecular , Unión ProteicaRESUMEN
The antimicrobial activity prediction tools aim to help the novel antimicrobial peptides (AMP) sequences discovery, utilizing machine learning methods. Such approaches have gained increasing importance in the generation of novel synthetic peptides by means of rational design techniques. This study focused on predictive ability of such approaches to determine the antimicrobial sequence activities, which were previously characterized at the protein level by in vitro studies. Using four web servers and one standalone software, we evaluated 78 sequences generated by the so-called linguistic model, being 40 designed and 38 shuffled sequences, with â¼60 and â¼25% of identity to AMPs, respectively. The ab initio molecular modelling of such sequences indicated that the structure does not affect the predictions, as both sets present similar structures. Overall, the systems failed on predicting shuffled versions of designed peptides, as they are identical in AMPs composition, which implies in accuracies below 30%. The prediction accuracy is negatively affected by the low specificity of all systems here evaluated, as they, on the other hand, reached 100% of sensitivity. Our results suggest that complementary approaches with high specificity, not necessarily high accuracy, should be developed to be used together with the current systems, overcoming their limitations.
Asunto(s)
Antiinfecciosos/síntesis química , Benchmarking/métodos , Diseño de Fármacos , Péptidos/síntesis química , Secuencia de Aminoácidos , Antiinfecciosos/química , Péptidos/farmacología , Sensibilidad y Especificidad , Programas Informáticos , Aprendizaje Automático SupervisadoRESUMEN
Guanylin peptides (GPs) are small cysteine-rich peptide hormones involved in salt absorption, regulation of fluids and electrolyte homeostasis. This family presents four members: guanylin (GN), uroguanylin (UGN), lymphoguanylin (LGN) and renoguanylin (RGN). GPs have been used as templates for the development of drugs for the treatment of gastrointestinal disorders. Currently, LGN is the only GP with only one disulfide bridge, making it a remarkable member of this family and a potential drug template; however, there is no structural information about this peptide. In fact, LGN is predicted to be highly disordered and flexible, making it difficult to obtain structural information using in vitro methods. Therefore, this study applied a series of 1µs molecular dynamics simulations in order to understand the structural behavior of LGN, comparing it to the C115Y variant of GN, which shows the same Cys to Tyr modification. LGN showed to be more flexible than GN C115Y. While the negatively charged N-terminal, despite its repellent behavior, seems to be involved mainly in pH-dependent activity, the hydrophobic core showed to be the determinant factor in LGN's flexibility, which could be essential in its activity. These findings may be determinant in the development of new medicines to help in the treatment of gastrointestinal disorders. Moreover, our investigation of LGN structure clarified some issues in the structure-activity relationship of this peptide, providing new knowledge of guanylin peptides and clarifying the differences between GN C115Y and LGN.
Asunto(s)
Enfermedades Gastrointestinales/tratamiento farmacológico , Péptidos/química , Péptidos/farmacología , Conformación Proteica , Secuencia de Aminoácidos , Animales , Simulación por Computador , Hormonas Gastrointestinales/química , Hormonas Gastrointestinales/genética , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Mutación Missense , Péptidos Natriuréticos/química , Péptidos Natriuréticos/genética , Péptidos/genética , Homología de Secuencia de Aminoácido , Electricidad Estática , Relación Estructura-ActividadRESUMEN
The extraction and purification of parigidin-br3, a cyclotide analogue belonging to the "bracelet" subfamily, from Palicourea rigida leaves is discussed. Unlike conventional cyclotides, parigidin-br3 has free N- and C-termini, as identified by MALDI-TOF/TOF analysis and confirmed by gene structure elucidation, and is one of a small number of acyclotides discovered during recent years. Parigidin-br3 showed cytotoxic activity against MCF-7 (breast cancer) and CACO2 (colorectal adenocarcinoma) cells, with IC50 values of â¼2.5 µM and less than 10% hemolytic activity. Overall, parigidin-br3 is a promising new molecule with cytotoxic properties against tumor cell lines and, unlike many synthetic acyclic analogues, demonstrates that cytotoxic activity is not limited to conventional (i.e., cyclic) cyclotides.