RESUMEN
The polypeptide Pep19-2.5 (Aspidasept®) has been described to act efficiently against infection-inducing bacteria by binding and neutralizing their most potent toxins, i.e., lipopolysaccharides (LPS) and lipoproteins/peptides (LP), independent of the resistance status of the bacteria. The mode of action was described to consist of a primary Coulomb/polar interaction of the N-terminal region of Pep19-2.5 with the polar region of the toxins followed by a hydrophobic interaction of the C-terminal region of the peptide with the apolar moiety of the toxins. However, clinical development of Aspidasept as an anti-sepsis drug requires an in-depth characterization of the interaction of the peptide with the constituents of the human immune system and with other therapeutically relevant compounds such as antibiotics and non-steroidal anti-inflammatory drugs (NSAIDs). In this contribution, relevant details of primary and secondary pharmacodynamics, off-site targets, and immunogenicity are presented, proving that Pep19-2.5 may be readily applied therapeutically against the deleterious effects of a severe bacterial infection.
Asunto(s)
Antiinfecciosos/farmacología , Antiinflamatorios/farmacología , Endotoxemia/tratamiento farmacológico , Inflamación , Péptidos/farmacología , Animales , Antiinfecciosos/uso terapéutico , Antiinflamatorios/uso terapéutico , Modelos Animales de Enfermedad , Endotoxemia/inmunología , Humanos , Lipopolisacáridos , Ratones , Péptidos/uso terapéuticoRESUMEN
The activity of antimicrobial peptides (AMPs) has been investigated extensively using model membranes composed of phospholipids or lipopolysaccharides in aqueous environments. However, from a biophysical perspective, there is a large scientific interest regarding the direct interaction of membrane-active peptides with whole bacteria. Working with living bacteria limits the usability of experimental setups and the interpretation of the resulting data because of safety risks and the overlap of active and passive effects induced by AMPs. We killed or inactivated metabolic-active bacteria using γ-irradiation or sodium azide, respectively. Microscopy, flow cytometry, and SYTOX green assays showed that the cell envelope remained intact to a high degree at the minimal bactericidal dose. Furthermore, the tumor-necrosis-factor-α-inducing activity of the lipopolysaccharides and the chemical lipid composition was unchanged. Determining the binding capacity of AMPs to the bacterial cell envelope by calorimetry is difficult because of an overlapping of the binding heat and metabolic activities of the bacteria-induced by the AMPs. The inactivation of all active processes helps to decipher the complex thermodynamic information. From the isothermal titration calorimetry (ITC) results, we propose that the bacterial membrane potential (Δψ) is possibly an underestimated modulator of the AMP activity. The negative surface charge of the outer leaflet of the outer membrane of Gram-negative bacteria is already neutralized by peptide concentrations below the minimal inhibitory concentration. This proves that peptide aggregation on the bacterial membrane surface plays a decisive role in the degree of antimicrobial activity. This will not only enable many biophysical approaches for the investigation between bacteria and membrane-active peptides in the future but will also make it possible to compare biophysical parameters of active and inactive bacteria. This opens up new possibilities to better understand the active and passive interaction processes between AMPs and bacteria.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Bacterias/efectos de los fármacos , Bacterias/efectos de la radiación , Rayos gamma , Viabilidad Microbiana/efectos de los fármacos , Viabilidad Microbiana/efectos de la radiación , Adsorción , Bacterias/ultraestructura , Fenómenos Biofísicos , Membrana Celular/efectos de los fármacos , Membrana Celular/efectos de la radiación , Membrana Celular/ultraestructura , Potenciales de la Membrana/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Fosfolípidos/metabolismo , Unión Proteica/efectos de los fármacos , TermodinámicaRESUMEN
With this study, we investigated the effect of synthetic antimicrobial peptides Pep19-2.5 and Pep194LF alone or in combination with antibiotics on S. mutans growth and biofilm formation/disruption. We also examined the cytotoxic effect of each peptide on monocytes. S. mutans was cultured in the presence of different concentrations of each peptide. We showed that Pep19-2.5 and Pep19-4LF were able to significantly (pâ¯≤â¯0.01) inhibit the growth of S. mutans. The synthetic peptides also decreased biofilm formation by S. mutans. Furthermore, both peptides reduced the viability of S. mutans in already formed biofilms. The combination of each peptide with antibiotics (penicillin/streptomycin, P/S) produced additive interactions which inhibited S. mutans growth and biofilm formation. Pep19-2.5 and Pep19-4LF were nontoxic, as they did not decrease monocyte viability and did not increase the lactate dehydrogenase activity of the exposed cells. In conclusion, synthetic peptides Pep19-2.5 and Pep19-4LF did inhibit S. mutans growth and its capacity to form biofilm. Both peptides were found to be nontoxic to monocytes. These data provide new insight into the efficacy of synthetic peptides Pep19-2.5 and Pep19-4LF against S. mutans. These peptides may thus be useful in controlling the adverse effects of this cariogenic bacterium in human.
Asunto(s)
Antiinfecciosos/farmacología , Biopelículas/efectos de los fármacos , Proteínas del Tejido Nervioso/farmacología , Péptidos/farmacología , Streptococcus mutans/efectos de los fármacos , Péptidos Catiónicos Antimicrobianos , Biopelículas/crecimiento & desarrollo , Supervivencia Celular/efectos de los fármacos , Combinación de Medicamentos , Sinergismo Farmacológico , Humanos , L-Lactato Deshidrogenasa/metabolismo , Pruebas de Sensibilidad Microbiana , Monocitos/efectos de los fármacos , Proteínas del Tejido Nervioso/síntesis química , Penicilinas/farmacología , Péptidos/síntesis química , Streptococcus mutans/crecimiento & desarrolloRESUMEN
Antimicrobial peptides (AMPs) are in the focus of scientific research since the 1990s. In most cases, the main aim was laid on the design of AMP to kill bacteria effectively, with particular emphasis on broadband action and independency on antibiotic resistance. However, so far no approved drug on the basis of AMP has entered the market.Our approach of constructing AMP, called synthetic anti-lipopolysaccharide peptides (SALPs), on the basis of inhibiting the inflammatory action of lipopolysaccharide (LPS, endotoxin) from Gram-negative bacteria was focused on the neutralization of the decisive toxins. These are, beside LPS from Gram-negative bacteria, the lipoproteins (LP) from Gram-positive origin. Although some of the SALPs have an antibacterial action, the most important property is the high-affinity binding to LPS and LP, whether as constituent of the bacteria or in free form which prevents the damaging inflammation, that could otherwise lead to life-threatening septic shock. Most importantly, the SALP may inhibit inflammation independently of the resistance status of the bacteria, and so far the repeated use of the peptides apparently does not cause resistance of the attacking pathogens.In this chapter, an overview is given over the variety of possible applications in the field of fighting against severe bacterial infections, from the use in systemic infection/inflammation up to various topical applications such as anti-biofilm action and severe skin and soft tissue infections.
Asunto(s)
Antibacterianos/química , Moléculas de Patrón Molecular Asociado a Patógenos/antagonistas & inhibidores , Péptidos/química , Infecciones Bacterianas/tratamiento farmacológico , Endotoxinas , Bacterias Gramnegativas , Humanos , LipopolisacáridosRESUMEN
OBJECTIVE: To evaluate (1) levels of the host-defense/antimicrobial peptide LL-37 in patients with trauma and hemorrhagic shock (HS) and (2) the effects of a synthetic host-defense peptide; Pep19-4LF on multiple organ failure (MOF) associated with HS. BACKGROUND: HS is a common cause of death in severely injured patients. There is no specific therapy that reduces HS-associated MOF. METHODS: (1) LL-37 was measured in 47 trauma/HS patients admitted to an urban major trauma center. (2) Male Wistar rats were submitted to HS (90âmin, target mean arterial pressure: 27-32âmm Hg) or sham operation. Rats were treated with Pep19-4LF [66 (n = 8) or 333âµg/kgâ·âh (n = 8)] or vehicle (n = 12) for 4 hours following resuscitation. RESULTS: Plasma LL-37 was 12-fold higher in patients with trauma/HS compared to healthy volunteers. HS rats treated with Pep19-4LF (high dose) had a higher mean arterial pressure at the end of the 4-hour resuscitation period (79â±â4 vs 54â±â5âmm Hg) and less renal dysfunction, liver injury, and lung inflammation than HS rats treated with vehicle. Pep19-4LF enhanced (kidney/liver) the phosphorylation of (1) protein kinase B and (2) endothelial nitric oxide synthase. Pep19-4LF attenuated the HS-induced (1) translocation of p65 from cytosol to nucleus, (2) phosphorylation of IκB kinase on Ser, and (3) phosphorylation of IκBα on Ser resulting in inhibition of nuclear factor kappa B and formation of proinflammatory cytokines. Pep19-4LF prevented the release of tumor necrosis factor alpha caused by heparan sulfate in human mononuclear cells by binding to this damage-associated molecular pattern. CONCLUSIONS: Trauma-associated HS results in release of LL-37. The synthetic host-defense/antimicrobial peptide Pep19-4LF attenuates the organ injury/dysfunction associated with HS.
Asunto(s)
Antiinfecciosos/uso terapéutico , Péptidos Catiónicos Antimicrobianos/sangre , Insuficiencia Multiorgánica/prevención & control , Péptidos/uso terapéutico , Sustancias Protectoras/uso terapéutico , Choque Hemorrágico/tratamiento farmacológico , Heridas y Lesiones/complicaciones , Animales , Biomarcadores/sangre , Estudios de Casos y Controles , Terapia Combinada , Humanos , Masculino , Insuficiencia Multiorgánica/etiología , Ratas , Ratas Wistar , Resucitación , Choque Hemorrágico/sangre , Choque Hemorrágico/complicaciones , Choque Hemorrágico/diagnóstico , Resultado del Tratamiento , CatelicidinasRESUMEN
Bacterial infections, with the most severe form being sepsis, can often not be treated adequately leading to high morbidity and lethality of infected patients in critical care units. In particular, the increase in resistant bacterial strains and the lack of new antibiotics are main reasons for the worsening of the current situation, As a new approach, the use of antimicrobial peptides (AMPs) seems to be promising, combining the ability of broad-spectrum bactericidal activity and low potential of induction of resistance. Peptides based on natural defense proteins or polypeptides such as lactoferrin, Limulus anti-lipopolysaccharide factor (LALF), cathelicidins, and granulysins are candidates due to their high affinity to bacteria and to their pathogenicity factors, in first line lipopolysaccharide (LPS, endotoxin) of Gram-negative origin. In this review, we discuss literature with the focus on the use of AMPs from natural sources and their variants as antibacterial as well as anti-endotoxin (anti-inflammatory) drugs. Considerable progress has been made by the design of new AMPs for acting efficiently against the LPS-induced inflammation reaction in vitro as well as in vivo (mouse) models of sepsis. Furthermore, the data indicate that efficient antibacterial compounds are not necessarily equally efficient as anti-endotoxin drugs and vice versa. The most important reason for this may be the different molecular geometry of LPS in bacteria and in free form. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.
Asunto(s)
Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Infecciones por Escherichia coli/tratamiento farmacológico , Escherichia coli/efectos de los fármacos , Lipopolisacáridos/antagonistas & inhibidores , Sepsis/tratamiento farmacológico , Secuencia de Aminoácidos , Animales , Antibacterianos/síntesis química , Antibacterianos/química , Antígenos de Diferenciación de Linfocitos T/química , Antígenos de Diferenciación de Linfocitos T/farmacología , Péptidos Catiónicos Antimicrobianos/síntesis química , Péptidos Catiónicos Antimicrobianos/química , Proteínas de Artrópodos/síntesis química , Proteínas de Artrópodos/química , Proteínas de Artrópodos/farmacología , Modelos Animales de Enfermedad , Diseño de Fármacos , Farmacorresistencia Bacteriana Múltiple , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Escherichia coli/patogenicidad , Infecciones por Escherichia coli/metabolismo , Infecciones por Escherichia coli/microbiología , Humanos , Lactoferrina/química , Lipopolisacáridos/química , Lipopolisacáridos/metabolismo , Ratones , Datos de Secuencia Molecular , Sepsis/metabolismo , Sepsis/microbiologíaRESUMEN
Lipopolysaccharides (LPS, endotoxin) are complex and indispensable components of the outer membrane of most Gram-negative bacteria. They represent stimuli for many biological effects with pathophysiological character. Recombinant therapeutic proteins that are manufactured using biotechnological processes are prone to LPS contaminations due to their ubiquitous occurrence. The maximum endotoxin load of recombinant therapeutic proteins must be below the pyrogenic threshold. Certain matrices that are commonly used for recombinant therapeutic proteins show a phenomenon called "Low Endotoxin Recovery (LER)". LER is defined as the loss of detectable endotoxin activity over time using compendial Limulus amebocyte lysate (LAL) assays when undiluted products are spiked with known amount of endotoxin standards. Because LER poses potential risks that endotoxin contaminations in products may be underestimated or undetected by the LAL assay, the United States (U.S.) Food and Drug Administration's (FDA's) Center for Drug Evaluation and Research (CDER) has recently started requesting that companies conduct endotoxin spike/hold recovery studies to determine whether a given biological product causes LER. Here, we have performed an analysis of different LPS preparations with relevant detergents studying their acyl chain phase transition, their aggregate structures, their size distributions, and binding affinity with a particular anti-endotoxin peptide, and correlating it with the respective data in the macrophage activation test. In this way, we have worked out biophysical parameters that are important for an understanding of LER.
Asunto(s)
Bioensayo/métodos , Lipopolisacáridos/química , Animales , Endotoxinas/química , Bacterias Gramnegativas/química , Cangrejos Herradura/química , Proteínas de la Membrana/químicaRESUMEN
Lipopolysaccharides (LPS) belong to the strongest immune-modulating compounds known in nature, and are often described as pathogen-associated molecular patterns (PAMPs). In particular, at higher concentrations they are responsible for sepsis and the septic shock syndrome associated with high lethality. Since most data are indicative that LPS aggregates are the bioactive units, their supramolecular structures are considered to be of outmost relevance for deciphering the molecular mechanisms of its bioactivity. So far, however, most of the data available addressing this issue, were published only for the lipid part (lipid A) and the core-oligosaccharide containing rough LPS, representing the bioactive unit. By contrast, it is well known that most of the LPS specimen identified in natural habitats contain the smooth-form (S-form) LPS, which carry additionally a high-molecular polysaccharide (O-chain). To fill this lacuna and going into a more natural system, here various wild-type (smooth form) LPS including also some LPS fractions were investigated by small-angle X-ray scattering with synchrotron radiation to analyze their aggregate structure. Furthermore, the influence of a recently designed synthetic anti-LPS peptide (SALP) Pep19-2.5 on the aggregate structure, on the binding thermodynamics, and on the cytokine-inducing activity of LPS were characterized, showing defined aggregate changes, high affinity binding and inhibition of cytokine secretion. The data obtained are suitable to refine our view on the preferences of LPS for non-lamellar structures, representing the highest bioactive forms which can be significantly influenced by the binding with neutralizing peptides such as Pep19-2.5.
Asunto(s)
Anticuerpos Neutralizantes/química , Enterobacteriaceae/química , Lipopolisacáridos/química , Péptidos/química , Secuencia de Aminoácidos , Anticuerpos Neutralizantes/inmunología , Calorimetría/métodos , Células Cultivadas , Enterobacteriaceae/genética , Enterobacteriaceae/inmunología , Humanos , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Monocitos/citología , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Péptidos/inmunología , Péptidos/farmacología , Unión Proteica , Dispersión del Ángulo Pequeño , Espectroscopía Infrarroja por Transformada de Fourier , Termodinámica , Factor de Necrosis Tumoral alfa/metabolismo , Difracción de Rayos XRESUMEN
Antimicrobial peptides (AMPs) are important components of the innate immune system of animals, plants, fungi and bacteria and are recently under discussion as promising alternatives to conventional antibiotics. We have investigated two cecropin-like synthetic peptides, Gm1, which corresponds to the natural overall uncharged Galleria mellonella native peptide and ΔGm1, a modified overall positively charged Gm1 variant. We have analysed these peptides for their potential to inhibit the endotoxin-induced secretion of tumour necrosis factor-α (TNF-α) from human mononuclear cells. Furthermore, in a conventional microbiological assay, the ability of these peptides to inhibit the growth of the rough mutant bacteria Salmonella enterica Minnesota R60 and the polymyxin B-resistant Proteus mirabilis R45 was investigated and atomic force microscopy (AFM) measurements were performed to characterize the morphology of the bacteria treated by the two peptides. We have also studied their cytotoxic properties in a haemolysis assay to clarify potential toxic effects. Our data revealed for both peptides minor anti-inflammatory (anti-endotoxin) activity, but demonstrated antimicrobial activity with differences depending on the endotoxin composition of the respective bacteria. In accordance with the antimicrobial assay, AFM data revealed a stronger morphology change of the R45 bacteria than for the R60. Furthermore, Gm1 had a stronger effect on the bacteria than ΔGm1, leading to a different morphology regarding indentations and coalescing of bacterial structures. The findings verify the biophysical measurements with the peptides on model systems. Both peptides lack any haemolytic activity up to an amount of 100µg/ml, making them suitable as new anti-infective agents.
Asunto(s)
Antibacterianos , Péptidos Catiónicos Antimicrobianos , Endotoxemia/tratamiento farmacológico , Proteínas de Insectos , Leucocitos Mononucleares/metabolismo , Mariposas Nocturnas/química , Animales , Antibacterianos/química , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Células Cultivadas , Endotoxemia/inducido químicamente , Endotoxemia/metabolismo , Endotoxemia/patología , Femenino , Humanos , Proteínas de Insectos/química , Proteínas de Insectos/farmacología , Leucocitos Mononucleares/patología , Lipopolisacáridos/toxicidad , Masculino , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Natural occurring antimicrobial peptides (AMPs) are important components of the innate immune system of animals and plants. They are considered to be promising alternatives to conventional antibiotics. Here we present a comparative study of two synthetic peptides: Gm1, corresponding to the natural overall uncharged peptide from Galleria mellonella (Gm) and ΔGm1, a modified overall positively charged Gm1 variant. We have studied the interaction of the peptides with lipid membranes composed of different kinds of lipopolysaccharides (LPS) and dimyristoylphosphatidylglycerol (DMPG), in some cases also dimyristoylphosphatidylethanolamine (DMPE) as representative lipid components of Gram-negative bacterial membranes, by applying Fourier-transform infrared spectroscopy (FTIR), Förster resonance energy transfer spectroscopy (FRET), differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC). Gm1 generates a destabilizing effect on the gel to liquid crystalline phase transition of the acyl chains of the lipids, as deduced from a decrease in the phase transition temperature and enthalpy, suggesting a fluidization, whereas ΔGm1 led to the opposite behavior. Further, FTIR analysis of the functional groups of the lipids participating in the interaction with the peptides indicated a shift in the band position and intensity of the asymmetric PO2(-) stretching vibration originating from the lipid phosphate groups, a consequence of the sterical changes in the head group region. Interestingly, FRET spectroscopy showed a similar intercalation of both peptides into the DMPG and LPS, but much less into the DMPE membrane systems. These results are discussed in the light of a possible use of the peptides as antimicrobial and anti-endotoxin drugs.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Bacterias/química , Membrana Celular/química , Dimiristoilfosfatidilcolina/química , Proteínas de Insectos/química , Membranas Artificiales , Modelos Químicos , Animales , Antiinfecciosos/química , Mariposas NocturnasRESUMEN
Lipopolysaccharide (LPS, endotoxin) is ubiquitous and represents a harmful contaminant of pharmaceutical compounds, recombinant biologicals and drug products. The pyrogen can induce severe immune responses and pathology in vitro and in vivo. Health authorities require strict control of endotoxin in parenteral drugs. However, for research and pre-clinical compound analysis, endotoxin testing is not a required quality control, which may cause potential drawbacks in the translational pipeline. Endotoxin testing is usually performed by the Limulus amebocyte lysate (LAL) assay, which is hampered by the so-called low endotoxin recovery (LER) effect when certain drug formulations are tested. A comprehensive study including structural, biophysical, and biological analyses was conducted to identify LER root cause for phosphate- and polysorbate-containing parenteral drug products. LPS in water showed extended ribbon-like aggregate structures. In placebo (formulation buffer without drug) and in drug product (drug in formulation buffer), a reaggregation of LPS into a network of interlinked micelles with hidden head group charges, and a strong reduction of the negative surface potential was observed. The non-accessibility of the LPS backbone has a direct impact leading (i) to a loss of activation of the LAL-cascade, (ii) reduced activation of the TLR4/MD-2 receptor system, and (iii) increased survival in a mouse model of endotoxemia. These data provide a structure-based explanation of the LER-underlying mechanisms. A human whole blood assay is shown to resolve LER and detect the pyrogenic activity of endotoxin with high sensitivity. This may open new test options to improve quality control in drug development and drug safety.
Asunto(s)
Endotoxinas , Lipopolisacáridos , Animales , Ratones , Humanos , Micelas , Prueba de Limulus , Composición de MedicamentosRESUMEN
Due to the increase in bacterial resistance to common antibiotics and the lack of newly approved drugs, antimicrobial peptides (AMP) have been shown to be an alternative to combat infections caused by drug-resistant organisms. In particular, synthetic anti-lipopolysaccharide peptides (SALP) with the lead structure Aspidasept (Pep19-2.5) display a high anti-inflammatory activity in vitro and in vivo systems of endotoxemia and bacteremia. This was found not only when SALP were applied systemically (i.e. against sepsis), but also in topical therapies aimed at treating wound infections. A further important application involves combating common pathologies of the gastrointestinal tract, such as chronic infections of the small intestine and the colon (e.g., Crohn's disease). For the necessary oral application, the active pharmaceutical ingredient (API), Aspidasept®, must be encapsulated to ensure its protection against the low pH and the hydrolytic enzymes of the gastrointestinal tract. Here, the encapsulation of Aspidasept in polysaccharide matrices, essentially alginate and pectin, was systematically investigated with a variety of physico-chemical techniques. Specifically, we characterized key features of the nanoparticles such as their sizes and size distributions, as well as their stability in different environments mimicking digestive fluids. Finally, we studied the release of the drug from the polysaccharide matrices and the ability of nanoparticles to neutralize endotoxemia in vitro. We showed that our lead formulations exert an optimum inhibitory activity on immune cells stimulated by lipopolysaccharide.
Asunto(s)
Péptidos , Sepsis , Antibacterianos , Antiinflamatorios , Humanos , LipopolisacáridosRESUMEN
INTRODUCTION: Gram-negative bacterial infections represent still a severe problem of human health care, regarding the increase in multi-resistance against classical antibiotics and the lack of newly developed antimicrobials. For the fight against these germs, anti-infective agents must overcome and/or bind to the Gram-negative outer membrane consisting of a lipopolysaccharide (LPS, endotoxin) outer leaflet and an inner leaflet from phospholipids, with additional peripheral or integral membrane proteins (OMP's). AREAS COVERED: The current article reviews data of existing therapeutic options and summarizes newer approaches for targeting and neutralizing endotoxins, ranging from in vitro over in vivo animal data to clinical applications by using databases such as Medline. EXPERT OPINION: Conventional antibiotic treatment of the bacteria leads to their killing, but not necessary LPS neutralization, which may be a severe problem in particular for the systemic pathway. This is the reason why there is an increasing number of therapeutic approaches, which - besides combating whole bacteria - at the same time try to neutralize endotoxin within or outside the bacterial cells mainly responsible for the high inflammation induction in Gram-negative species.
Asunto(s)
Antibacterianos/administración & dosificación , Bacterias Gramnegativas/efectos de los fármacos , Infecciones por Bacterias Gramnegativas/tratamiento farmacológico , Animales , Antibacterianos/farmacología , Desarrollo de Medicamentos , Endotoxinas/antagonistas & inhibidores , Bacterias Gramnegativas/aislamiento & purificación , Infecciones por Bacterias Gramnegativas/microbiología , Humanos , Inflamación/tratamiento farmacológico , Inflamación/microbiología , Lipopolisacáridos/antagonistas & inhibidoresRESUMEN
Increasing failure of conventional antibiotics to combat bacterial infections requires the urgent development of new antibacterial drugs; a promising class of new drugs based on antimicrobial peptides. Here, we studied the molecular interaction of polycationic synthetic antilipopolysaccharide peptides (SALPs) with various gram-negative and gram-positive bacteria, including resistant strains. The analysis of antimicrobial activity by conventional techniques and atomic force microscopy showed a strict dependence on amino acid (aa) sequences, with the type of amino acid, its position within the primary structure, and the sequence length being critical parameters. By monitoring lipopolysaccharide (LPS)- or bacteria-induced cytokine production in human mononuclear cells and whole blood, we found a direct link between the binding of the lead compound Pep19-2.5 to Salmonella enterica and the anti-inflammatory activity of the peptide. Thermodynamic analysis of Pep19-2.5 binding to the bacterial cell envelope showed an exothermic reaction with saturation characteristics, whereas small-angle X-ray scattering data indicated a direct attachment of Pep19-2.5 to the bacterial cell envelope. This binding preferentially takes place to the LPS outer monolayer, as evidenced by the change in the LPS acyl chain and phosphate vibrational bands seen by Fourier-transform infrared spectroscopy. We report here that the anti-inflammatory activity of Pep19-2.5 is not only connected with neutralization of cell-free bacterial toxins but also with a direct binding of the peptide to the outer leaflet of the bacterial outer membrane.
Asunto(s)
Antibacterianos/farmacología , Toxinas Bacterianas/metabolismo , Péptidos/metabolismo , Péptidos/farmacología , Antibacterianos/química , Antibacterianos/metabolismo , Calorimetría , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Membrana Celular/microbiología , Radioisótopos de Cesio/toxicidad , Citocinas/metabolismo , Citometría de Flujo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/microbiología , Lipopolisacáridos/farmacología , Pruebas de Sensibilidad Microbiana , Microscopía de Fuerza Atómica , Péptidos/síntesis química , Salmonella enterica/efectos de los fármacos , Salmonella enterica/metabolismo , Salmonella enterica/efectos de la radiación , Dispersión del Ángulo Pequeño , Espectroscopía Infrarroja por Transformada de Fourier , Difracción de Rayos XRESUMEN
Dysregulation of the disintegrin-metalloproteinase ADAM10 may contribute to the development of diseases including tumorigenesis and Alzheimer's disease. The mechanisms underlying ADAM10 sheddase activation are incompletely understood. Here, we show that transient exposure of the negatively charged phospholipid phosphatidylserine (PS) is necessarily required. The soluble PS headgroup was found to act as competitive inhibitor of substrate cleavage. Overexpression of the Ca2+-dependent phospholipid scramblase Anoctamin-6 (ANO6) led to increased PS externalization and substrate release. Transfection with a constitutively active form of ANO6 resulted in maximum sheddase activity in the absence of any stimulus. Calcium-dependent ADAM10 activation could not be induced in lymphocytes of patients with Scott syndrome harbouring a missense mutation in ANO6. A putative PS-binding motif was identified in the conserved stalk region. Replacement of this motif resulted in strong reduction of sheddase activity. In conjunction with the recently described 3D structure of the ADAM10 extracellular domain, a model is advanced to explain how surface-exposed PS triggers ADAM10 sheddase function.
Asunto(s)
Proteína ADAM10/metabolismo , Membrana Celular/metabolismo , Activación Enzimática , Proteína ADAM10/química , Secuencia de Aminoácidos , Animales , Anoctaminas/metabolismo , Biomarcadores , Células COS , Línea Celular , Chlorocebus aethiops , Eritrocitos/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Fosfoserina/metabolismo , Conejos , Relación Estructura-ActividadRESUMEN
Outer membrane vesicles (OMVs) are secreted by Gram-negative bacteria and induce a stronger inflammatory response than pure LPS. After endocytosis of OMVs by macrophages, lipopolysaccharide (LPS) is released from early endosomes to activate its intracellular receptors followed by non-canonical inflammasome activation and pyroptosis, which are critically involved in sepsis development. Previously, we could show that the synthetic anti-endotoxin peptide Pep19-2.5 neutralizes inflammatory responses induced by intracellular LPS. Here, we aimed to investigate whether Pep19-2.5 is able to suppress cytoplasmic LPS-induced inflammation under more physiological conditions by using OMVs which naturally transfer LPS to the cytosol. Isothermal titration calorimetry revealed an exothermic reaction between Pep19-2.5 and Escherichia coli OMVs and the Limulus Amebocyte Lysate assay indicated a strong endotoxin blocking activity. In THP-1 macrophages and primary human macrophages Pep19-2.5 and polymyxin B reduced interleukin (IL)-1ß and tumor necrosis factor (TNF) release as well as pyroptosis induced by OMVs, while the Toll-like receptor 4 signaling inhibitor TAK-242 suppressed OMV-induced TNF and IL-1ß secretion, but not pyroptosis. Internalization of Pep19-2.5 was at least partially mediated by the P2X7 receptor in macrophages but not in monocytes. Additionally, a cell-dependent difference in the neutralization efficiency of Pep19-2.5 became evident in macrophages and monocytes, indicating a critical role for peptide-mediated IL-1ß secretion via the P2X7 receptor. In conclusion, we provide evidence that LPS-neutralizing peptides inhibit OMV-induced activation of the inflammasome/IL-1 axis and give new insights into the mechanism of peptide-mediated neutralization of cytoplasmic LPS suggesting an essential and cell-type specific role for the P2X7 receptor.
Asunto(s)
Antiinflamatorios/farmacología , Membrana Externa Bacteriana/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Lipopolisacáridos/antagonistas & inhibidores , Macrófagos/efectos de los fármacos , Péptidos/farmacología , Membrana Externa Bacteriana/inmunología , Línea Celular , Escherichia coli/inmunología , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/microbiología , Humanos , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Inflamación/microbiología , Lipopolisacáridos/inmunología , Macrófagos/inmunología , Piroptosis/efectos de los fármacosRESUMEN
In previous years, we developed anti-infective drugs based on antimicrobial peptides (AMPs), which have been shown to effectively block severe infections and inflammation in vitro as well as in vivo. Besides systemic application, the occurrence of severe local infections necessitates a topical application for example in the case of severe skin and soft tissue infections (SSTI). Recent investigations show that the synthetic anti-lipopolysaccharide peptide (SALP) Pep19-2.5 (Aspidasept® I) and a variant called Pep19-4LF (Aspidasept® II) are able to supress inflammation reactions also in keratinocytes, Langerhans cells, and dendritic cells from the skin. For topical application, a possible formulation represents the drug dispersed into a pharmaceutical cream (DAC base cream). Here, we present investigations on the stability of the peptides using this formulation in dependence on time, which includes the evaluation of the extraction procedure, the quantitative analysis of the peptides after extraction, its sensitivity to protease degradation and its ability to maintain activity against LPS-induced inflammation in vitro. We have developed an extraction procedure for the peptides with an optimum yield and showed that Pep19-2.5 is present as a dimer after extraction from the cream, whereas Pep19-4LF retains its monomeric form. Both peptides show no degradation by chymotrypsin after extraction for at least 1â¯h, which is indicative for an attachment of constituents of the base cream, inhibiting the cutting into peptidic part structures. The extracted peptides and in particular the dimeric Pep19-2.5 are still able to inhibit the LPS-induced inflammation reaction in human mononuclear cells.
Asunto(s)
Antiinflamatorios/química , Pomadas/química , Péptidos/química , Antibacterianos/química , Antiinflamatorios/farmacología , Células Cultivadas , Química Farmacéutica/métodos , Estabilidad de Medicamentos , Humanos , Inflamación/tratamiento farmacológico , QueratinocitosRESUMEN
The most potent cell wall-derived inflammatory toxins ("pathogenicity factors") of Gram-negative and -positive bacteria are lipopolysaccharides (LPS) (endotoxins) and lipoproteins (LP), respectively. Despite the fact that the former signals via toll-like receptor 4 (TLR4) and the latter via TLR2, the physico-chemistry of these compounds exhibits considerable similarity, an amphiphilic molecule with a polar and charged backbone and a lipid moiety. While the exterior portion of the LPS (i.e., the O-chain) represents the serologically relevant structure, the inner part, the lipid A, is responsible for one of the strongest inflammatory activities known. In the last years, we have demonstrated that antimicrobial peptides from the Pep19-2.5 family, which were designed to bind to LPS and LP, act as anti-inflammatory agents against sepsis and endotoxic shock caused by severe bacterial infections. We also showed that this anti-inflammatory activity requires specific interactions of the peptides with LPS and LP leading to exothermic reactions with saturation characteristics in calorimetry assays. Parallel to this, peptide-mediated neutralization of LPS and LP involves changes in various physical parameters, including both the gel to liquid crystalline phase transition of the acyl chains and the three-dimensional aggregate structures of the toxins. Furthermore, the effectivity of neutralization of pathogenicity factors by peptides was demonstrated in several in vivo models together with the finding that a peptide-based therapy sensitizes bacteria (also antimicrobial resistant) to antibiotics. Finally, a significant step in the understanding of the broad anti-inflammatory function of Pep19-2.5 was the demonstration that this compound is able to block the intracellular endotoxin signaling cascade.
Asunto(s)
Antiinflamatorios/uso terapéutico , Inflamación/tratamiento farmacológico , Inflamación/etiología , Lipopolisacáridos/efectos adversos , Lipoproteínas/efectos adversos , Péptidos/uso terapéutico , Animales , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Antiinflamatorios/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Catiónicos Antimicrobianos/uso terapéutico , Endotoxinas/efectos adversos , Endotoxinas/antagonistas & inhibidores , Endotoxinas/química , Humanos , Inflamación/metabolismo , Péptidos/farmacologíaRESUMEN
Sepsis, which is induced by severe bacterial infections, is a major cause of death worldwide, and therapies combating the disease are urgently needed. Because many drugs have failed in clinical trials despite their efficacy in mouse models, the development of reliable animal models of sepsis is in great demand. Several studies have suggested that rabbits reflect sepsis-related symptoms more accurately than mice. In this study, we evaluated a rabbit model of acute sepsis caused by the intravenous inoculation of Salmonella enterica. The model reproduces numerous symptoms characteristic of human sepsis including hyperlactatemia, hyperglycemia, leukopenia, hypothermia and the hyperproduction of several pro-inflammatory cytokines. Hence, it was chosen to investigate the proposed ability of Pep19-2.5-an anti-endotoxic peptide with high affinity to lipopolysaccharide and lipoprotein-to attenuate sepsis-associated pathologies in combination with an antibiotic (ceftriaxone). We demonstrate that a combination of Pep19-2.5 and ceftriaxone administered intravenously to the rabbits (1) kills bacteria and eliminates bacteremia 30 min post challenge; (2) inhibits Toll-like receptor 4 agonists in serum 90 min post challenge; (3) reduces serum levels of pro-inflammatory cytokines (interleukin-6 and tumor necrosis factor α); and (4) reverts to hypothermia and gives rise to temperature values indistinguishable from basal levels 330 min post challenge. The two components of the combination displayed synergism in some of these activities, and Pep19-2.5 notably counteracted the endotoxin-inducing potential of ceftriaxone. Thus, the combination therapy of Pep19-2.5 and ceftriaxone holds promise as a candidate for human sepsis therapy.
Asunto(s)
Antibacterianos/uso terapéutico , Bacteriemia/tratamiento farmacológico , Ceftriaxona/uso terapéutico , Péptidos/uso terapéutico , Salmonella enterica/efectos de los fármacos , Animales , Antibacterianos/administración & dosificación , Ceftriaxona/administración & dosificación , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Quimioterapia Combinada , Células HEK293 , Humanos , Hiperlactatemia , Hipotermia , Interleucina-6/sangre , Leucopenia , Lipopolisacáridos/sangre , Masculino , Péptidos/administración & dosificación , Conejos , Receptor Toll-Like 4/antagonistas & inhibidores , Receptor Toll-Like 4/sangre , Factor de Necrosis Tumoral alfa/sangreRESUMEN
Hemoglobin and its structures have been described since the 1990s to enhance a variety of biological activities of endotoxins (LPS) in a dose-dependent manner. To investigate the interaction processes in more detail, the system was extended by studying the interactions of newly designed peptides from the γ-chain of human hemoglobin with the adjuvant monophosphoryl lipid A (MPLA), a partial structure of lipid A lacking its 1-phosphate. It was found that some selected Hbg peptides, in particular two synthetic substructures designated Hbg32 and Hbg35, considerably increased the bioactivity of MPLA, which alone was only a weak activator of immune cells. These findings hold true for human mononuclar cells, monocytes and T lymphocytes. To understand the mechanisms of action in more detail, biophysical techniques were applied. These showed a peptide-induced change of the MPLA aggregate structure from multilamellar into a non-lamellar, probably inverted, cubic structure. Concomitantly, the peptides incorporated into the tightly packed MPLA aggregates into smaller units down to monomers. The fragmentation of the aggregates was an endothermic process, differing from a complex formation but rather typical for a catalytic reaction.