RESUMO
Characterization of particulate impurities such as aggregates is necessary to develop safe and efficacious adeno-associated virus (AAV) drug products. Although aggregation of AAVs can reduce the bioavailability of the virus, only a limited number of studies focus on the analysis of aggregates. We explored three technologies for their capability to characterize AAV monomers and aggregates in the submicron (<1 µm) size range: (i) mass photometry (MP), (ii) asymmetric flow field flow fractionation coupled to a UV-detector (AF4-UV/Vis) and (iii) microfluidic resistive pulse sensing (MRPS). Although low counts for aggregates impeded a quantitative analysis, MP was affirmed as an accurate and rapid method for quantifying the genome content of empty/filled/double-filled capsids, consistent with sedimentation velocity analytical ultracentrifugation results. MRPS and AF4-UV/Vis enabled the detection and quantification of aggregate content. The developed AF4-UV/Vis method separated AAV monomers from smaller aggregates, thereby enabling a quantification of aggregates <200 nm. MRPS was experienced as a straightforward method to determine the particle concentration and size distribution between 250-2000 nm, provided that the samples do not block the microfluidic cartridge. Overall, within this study we explored the benefits and limitations of the complementary technologies for assessing aggregate content in AAV samples.
Assuntos
Dependovirus , Fracionamento por Campo e Fluxo , Dependovirus/genética , Fracionamento por Campo e Fluxo/métodos , Vírion/genética , Tamanho da PartículaRESUMO
Aspidasept (Pep19-2.5) and its derivative Pep19-4LF ("Aspidasept II") are anti-infective and anti-inflammatory synthetic polypeptides currently in development for application against a variety of moderate to severe bacterial infections that could lead to systemic inflammation, as in the case of severe sepsis and septic shock, as well as application to non-systemic diseases in the case of skin and soft tissue infections (SSTI). In the present study, Aspidasept and Aspidasept II and their part structures were analysed with respect to their toxic behavior in different established models against a variety of relevant cells, and in electrophysiological experiments targeting the hERG channel according to ICH S7B. Furthermore, the effects in mouse models of neurobiological behavior and the local lymph node according to OECD test guideline 429 were investigated, as well as a rat model of repeated dose toxicology according to ICH M3. The data provide conclusive information about potential toxic effects, thus specifying a therapeutic window for the application of the peptides. Therefore, these data allow us to define Aspidasept concentrations for their use in clinical studies as parenteral application.
RESUMO
Zein is an FDA-approved maize protein featured by its manipulative surface and the possibility of fabrication into nanomaterials. Although extensive research has been carried out in zein-based technology, limited work is available for the application of zein in the field of cancer photodynamic therapy (PDT). In this work, we report zein as a carrier for the natural photosensitizer hypericin in the PDT of hepatocellular carcinoma in vitro. Zein was modified through chemical PEGylation to form PEGylated zein micelles that were compared with two zein nanoparticle formulations physically stabilized by either the lecithin/pluronic mixture or sodium caseinate. FT-IR, 1HNMR and HP-SEC MALS approaches were employed to confirm the chemical PEGylation of zein. Our developed zein nanoparticles and micelles were further characterized by photon correlation spectroscopy (PCS) and atomic force microscopy (AFM). The obtained results showed relatively smaller sizes and higher encapsulation of hypericin in the micellar zein than the nanoparticle-based formulations. Phototoxicity on hepatocellular carcinoma (HepG2 cells) manifested a dose-dependent toxicity pattern of all designed zein formulations. However, superior cytotoxicity was prominent for the hypericin-based micelles, which was influenced by the higher cellular uptake profile. Consequently, the treated HepG2 cells manifested a higher level of intracellular generated ROS and disruption of mitochondrial membrane potential, which induced apoptotic cell death. Comparatively, the designed hypericin formulations indicated lower phototoxicity profile in murine fibroblast L929 cells reflecting their safety on normal cells. Our investigations suggested that the surface-modified zein could be employed to enhance the delivery of the hydrophobic hypericin in PDT and pave the way for future in vivo and clinical applications in cancer treatment.
Assuntos
Neoplasias Hepáticas , Perileno , Fotoquimioterapia , Zeína , Animais , Antracenos , Camundongos , Perileno/análogos & derivados , Perileno/farmacologia , Fármacos Fotossensibilizantes/farmacologia , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Flow imaging microscopy (FIM) is widely used to analyze subvisible particles starting from 2 µm in biopharmaceuticals. Recently, an oil-immersion FIM system emerged, the FlowCam Nano, designed to enable the characterization of particle sizes even below 2 µm. The aim of our study was to evaluate oil-immersion FIM (by using FlowCam Nano) in comparison to microfluidic resistive pulse sensing and resonant mass measurement for sizing and counting of particles in the submicron range. Polystyrene beads, a heat-stressed monoclonal antibody formulation and a silicone oil emulsion, were measured to assess the performance on biopharmaceutical relevant samples, as well as the ability to distinguish particle types based on instrument-derived morphological parameters. The determination of particle sizes and morphologies suffers from inaccuracies due to a low image contrast of small particles and light-scattering effects. The ill-defined measured volume impairs an accurate concentration determination. Nevertheless, FlowCam Nano in its current design complements the limited toolbox of submicron particle analysis of biopharmaceuticals by providing particle images in a size range that was previously not accessible with commercial FIM instruments.
Assuntos
Anticorpos Monoclonais/química , Produtos Biológicos/química , Química Farmacêutica/métodos , Microscopia/métodos , Química Farmacêutica/instrumentação , Técnicas Analíticas Microfluídicas , Microscopia/instrumentação , Tamanho da Partícula , Agregados Proteicos , Óleos de Silicone/químicaRESUMO
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.
Assuntos
Antibacterianos/química , Moléculas com Motivos Associados a Patógenos/antagonistas & inibidores , Peptídeos/química , Infecções Bacterianas/tratamento farmacológico , Endotoxinas , Bactérias Gram-Negativas , Humanos , LipopolissacarídeosRESUMO
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.
Assuntos
Anti-Inflamatórios/uso terapêutico , Inflamação/tratamento farmacológico , Inflamação/etiologia , Lipopolissacarídeos/efeitos adversos , Lipoproteínas/efeitos adversos , Peptídeos/uso terapêutico , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Anti-Inflamatórios/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Endotoxinas/efeitos adversos , Endotoxinas/antagonistas & inibidores , Endotoxinas/química , Humanos , Inflamação/metabolismo , Peptídeos/farmacologiaRESUMO
Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder worldwide and characterized by the loss of dopaminergic neurons in the patients' midbrains. Both the presence of the protein α-synuclein in intracellular protein aggregates in surviving neurons and the genetic linking of the α-synuclein encoding gene point towards a major role of α-synuclein in PD etiology. The exact pathogenic mechanisms of PD development are not entirely described to date, neither is the specific role of α-synuclein in this context. Previous studies indicate that one aspect of α-synuclein-related cellular toxicity might be direct proteasome impairment. The 20/26S proteasomal machinery is an important instrument of intracellular protein degradation. Thus, direct proteasome impairment by α-synuclein might explain or at least contribute to the formation of intracellular protein aggregates. Therefore this study investigates direct proteasomal impairment by α-synuclein both in vitro using recombinant α-synuclein and isolated proteasomes as well as in living cells. Our experiments demonstrate that the impairment of proteasome activity by α-synuclein is highly dependent upon the cellular background and origin. We show that recombinant α-synuclein oligomers and fibrils scarcely affect 20S proteasome function in vitro, neither does transient α-synuclein expression in U2OS ps 2042 (Ubi(G76V)-GFP) cells. However, stable expression of both wild-type and mutant α-synuclein in dopaminergic SH-SY5Y and PC12 cells results in a prominent impairment of the chymotrypsin-like 20S/26S proteasomal protein cleavage. Thus, our results support the idea that α-synuclein in a specific cellular environment, potentially present in dopaminergic cells, cannot be processed by the proteasome and thus contributes to a selective vulnerability of dopaminergic cells to α-synuclein pathology.
Assuntos
Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , alfa-Sinucleína/farmacologia , Animais , Western Blotting , Neurônios Dopaminérgicos/efeitos dos fármacos , Imunofluorescência , Humanos , Microscopia de Força Atômica , Células PC12 , Doença de Parkinson/etiologia , Complexo de Endopeptidases do Proteassoma/ultraestrutura , Ratos , Proteínas RecombinantesRESUMO
Measurement and characterization of subvisible particles (including proteinaceous and non-proteinaceous particulate matter) is an important aspect of the pharmaceutical development process for biotherapeutics. Health authorities have increased expectations for subvisible particle data beyond criteria specified in the pharmacopeia and covering a wider size range. In addition, subvisible particle data is being requested for samples exposed to various stress conditions and to support process/product changes. Consequently, subvisible particle analysis has expanded beyond routine testing of finished dosage forms using traditional compendial methods. Over the past decade, advances have been made in the detection and understanding of subvisible particle formation. This article presents industry case studies to illustrate the implementation of strategies for subvisible particle analysis as a characterization tool to assess the nature of the particulate matter and applications in drug product development, stability studies and post-marketing changes.
Assuntos
Nefelometria e Turbidimetria/métodos , Material Particulado/análise , Preparações Farmacêuticas/análise , Ar , Anticorpos Monoclonais/análise , Terapia Biológica , Composição de Medicamentos , Contaminação de Medicamentos , Embalagem de Medicamentos , Liofilização , Microbolhas , Técnicas Analíticas Microfluídicas , Tamanho da Partícula , Proteínas Recombinantes/análise , Espalhamento de Radiação , Óleos de Silicone , Espectrometria por Raios X , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
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.
Assuntos
Antibacterianos , Peptídeos Catiônicos Antimicrobianos , Endotoxemia/tratamento farmacológico , Proteínas de Insetos , Leucócitos Mononucleares/metabolismo , Mariposas/química , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Células Cultivadas , Endotoxemia/induzido quimicamente , Endotoxemia/metabolismo , Endotoxemia/patologia , Feminino , Humanos , Proteínas de Insetos/química , Proteínas de Insetos/farmacologia , Leucócitos Mononucleares/patologia , Lipopolissacarídeos/toxicidade , Masculino , Fator de Necrose Tumoral alfa/metabolismoRESUMO
This report presents evidence that ibuprofen interacts with red cell membranes as follows: a) in scanning electron microscopy (SEM) studies on human erythrocytes induced shape changes at a concentration as low as 10µM; b) in isolated unsealed human erythrocyte membranes (IUM) induced mild increase in the water content or in their molecular dynamics at the hydrophobic-hydrophilic interphase, while a corresponding ordering decrease at the deep phospholipids acyl chain level; c) at physiological temperature (37°C), 300µM ibuprofen induced a significant increase in the generalized polarization (GP) of dimyristoylphosphatidylcholine (DMPC) large unilamellar vesicles (LUV), an indication that ibuprofen molecules locate in the head polar group region of DMPC; d) X-ray diffraction studies showed that ibuprofen concentrations≥300µM induced increasing structural perturbation to DMPC bilayers; e) differential scanning calorimetry (DSC) data showed that ibuprofen was able to alter the cooperativity of DMPC phase transition in a concentration-dependent manner, to destabilize the gel phase and that ibuprofen did not significantly perturb the organization of the lipid hydrocarbon chains. Additionally, the effect on the viability of both human promyelocytic leukemia HL-60 and human cervical carcinoma HeLa cells was studied.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Eritrócitos/efeitos dos fármacos , Ibuprofeno/farmacologia , Modelos Moleculares , Varredura Diferencial de Calorimetria , Linhagem Celular Tumoral , Dimiristoilfosfatidilcolina/química , Dimiristoilfosfatidilcolina/metabolismo , Eritrócitos/ultraestrutura , Células HeLa , Humanos , Microscopia Eletrônica de Varredura , Temperatura , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismo , Difração de Raios XRESUMO
Bacterial endotoxins (lipopolysaccharides (LPS)) are strong elicitors of the human immune system by interacting with serum and membrane proteins such as lipopolysaccharide-binding protein (LBP) and CD14 with high specificity. At LPS concentrations as low as 0.3 ng/ml, such interactions may lead to severe pathophysiological effects, including sepsis and septic shock. One approach to inhibit an uncontrolled inflammatory reaction is the use of appropriate polycationic and amphiphilic antimicrobial peptides, here called synthetic anti-LPS peptides (SALPs). We designed various SALP structures and investigated their ability to inhibit LPS-induced cytokine secretion in vitro, their protective effect in a mouse model of sepsis, and their cytotoxicity in physiological human cells. Using a variety of biophysical techniques, we investigated selected SALPs with considerable differences in their biological responses to characterize and understand the mechanism of LPS inactivation by SALPs. Our investigations show that neutralization of LPS by peptides is associated with a fluidization of the LPS acyl chains, a strong exothermic Coulomb interaction between the two compounds, and a drastic change of the LPS aggregate type from cubic into multilamellar, with an increase in the aggregate sizes, inhibiting the binding of LBP and other mammalian proteins to the endotoxin. At the same time, peptide binding to phospholipids of human origin (e.g., phosphatidylcholine) does not cause essential structural changes, such as changes in membrane fluidity and bilayer structure. The absence of cytotoxicity is explained by the high specificity of the interaction of the peptides with LPS.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Fenômenos Biofísicos , Interações Hidrofóbicas e Hidrofílicas , Lipopolissacarídeos/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/farmacologia , Materiais Biomiméticos/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Citocinas/metabolismo , Feminino , Caranguejos Ferradura/efeitos dos fármacos , Caranguejos Ferradura/metabolismo , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Bicamadas Lipídicas/metabolismo , Lipopolissacarídeos/química , Lipopolissacarídeos/toxicidade , Camundongos , Fosfolipídeos/metabolismo , Ligação ProteicaRESUMO
Sepsis (blood poisoning) is a severe infectious disease with high mortality, and no effective therapy is actually known. In the case of Gram-negative bacteria, endotoxins (lipopolysaccharides) are known to be responsible for the strong inflammation reaction leading to the systemic infection. Peptides based on endotoxin-binding domains of human or animal proteins represent a promising approach in sepsis research. Although so far no medicament is available, the progress in recent years might lead to a breakthrough in this field. In this review, recent investigations are summarised, which may lead to an understanding of the mechanisms of action of peptides to suppress the inflammation reaction in vitro and in vivo (animal models) and thus may allow the development of effective anti-septic drugs.
Assuntos
Antibacterianos/uso terapêutico , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Peptídeos/uso terapêutico , Sepse/tratamento farmacológico , Animais , Antibacterianos/imunologia , Endotoxinas/antagonistas & inibidores , Endotoxinas/imunologia , Bactérias Gram-Negativas/imunologia , Bactérias Gram-Negativas/fisiologia , Infecções por Bactérias Gram-Negativas/imunologia , Humanos , Peptídeos/imunologia , Sepse/imunologiaRESUMO
An analysis of the interaction of the NK-lysin derived peptide NK-2 and of analogs thereof with bacterial lipopolysaccharide (LPS, endotoxin) was performed to determine the most important biophysical parameters for an effective LPS neutralization. We used microcalorimetry, FTIR spectroscopy, Zeta potential measurements, and small-angle X-ray scattering to analyze the peptide:LPS binding enthalpy, the accessible LPS surface charge, the fluidity of the LPS hydrocarbon chains, their phase transition enthalpy change, the aggregate structure of LPS, and how these parameters are modulated by the peptides. We conclude that (i) a high peptide:LPS binding affinity, which is facilitated by electrostatic and hydrophobic interactions and which leads to a positive Zeta potential, (ii) the formation of peptide-enriched domains, which destabilize the lipid packing, demonstrated by a drastic decrease of phase transition enthalpy change of LPS, and (iii) the multilamellarization of the LPS aggregate structure are crucial for an effective endotoxin neutralization by cationic peptides.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Endotoxinas/antagonistas & inibidores , Peptídeos/química , Peptídeos/farmacologia , Sequência de Aminoácidos , Sítios de Ligação , Endotoxinas/química , Endotoxinas/imunologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Dados de Sequência Molecular , Transição de Fase , Proteolipídeos/química , Salmonella enterica/imunologia , Espalhamento a Baixo Ângulo , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios XRESUMO
Lipopolysaccharides (LPS, endotoxins) belong to the strongest elicitors of the mammalian immune system due to the induction of a series of cytokines such as tumor-necrosis-factor-alpha (TNFalpha) in immunocompetent cells like mononuclear cells. Since the effects of LPS on human health may be pathologically at too high concentrations (e.g., septic shock syndrome), it is of uttermost importance to have a reliable assay for measuring the concentrations of endotoxins in vitro and in vivo (human body fluids). The activation of the clotting cascade from the horseshoe crab (Limulus polyphemus), the Limulus amoebocyte lysate test (LAL), has been the standard and most sensitive assay to detect bacterial endotoxins. However, there are restrictions with this test. It was found in some clinical trials that the results from the LAL test did not correlate with the presence of bacteremia due to Gram-negative organisms or with the mortality but correlated with the presence of fungal bloodstream infections. This resulted from the fact that the LAL assay does not only respond to bacterial endotoxins but is activated also by (1-->3)-beta-D-glucan. Furthermore, in extensive studies the structural requirements for activation of the LAL test were analyzed, and it was found that the LAL activity correlated with pyrogenicity but not with activation of the complement cascade. Furthermore, there was no correlation of the LAL activity with cytokine expression (for example tumor-necrosis-factor-alpha and interleulkins-1 and 6) in mononuclear cells when the 4/2 acyl chain pattern of enterobacterial lipid A was changed, or when the cytokine production induced by LPS from various different species in the whole blood assay was compared with the response from the LAL test. To clarify the questions raised by the different experimental findings, data from literature are summarized to get a more closer insight where the Limulus test confidentially monitors the endotoxicity of LPS and other compounds and where this is not the case, and which are the decisive epitopes for recognition of the LPS molecules. These data are very crucial for example in clinical tests, whether the LAL assay can reliably describe the effectivity of an antibacterial therapy.
Assuntos
Citocinas/metabolismo , Teste do Limulus/métodos , Acilação , Citocinas/análise , Endotoxinas/análise , Endotoxinas/toxicidade , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/análise , Lipopolissacarídeos/toxicidadeRESUMO
Aggregation of alpha-synuclein is a key event in several neurodegenerative diseases, including Parkinson disease. Recent findings suggest that oligomers represent the principal toxic aggregate species. Using confocal single-molecule fluorescence techniques, such as scanning for intensely fluorescent targets (SIFT) and atomic force microscopy, we monitored alpha-synuclein oligomer formation at the single particle level. Organic solvents were used to trigger aggregation, which resulted in small oligomers ("intermediate I"). Under these conditions, Fe(3+) at low micromolar concentrations dramatically increased aggregation and induced formation of larger oligomers ("intermediate II"). Both oligomer species were on-pathway to amyloid fibrils and could seed amyloid formation. Notably, only Fe(3+)-induced oligomers were SDS-resistant and could form ion-permeable pores in a planar lipid bilayer, which were inhibited by the oligomer-specific A11 antibody. Moreover, baicalein and N'-benzylidene-benzohydrazide derivatives inhibited oligomer formation. Baicalein also inhibited alpha-synuclein-dependent toxicity in neuronal cells. Our results may provide a potential disease mechanism regarding the role of ferric iron and of toxic oligomer species in Parkinson diseases. Moreover, scanning for intensely fluorescent targets allows high throughput screening for aggregation inhibitors and may provide new approaches for drug development and therapy.
Assuntos
Regulação da Expressão Gênica , Ferro/química , alfa-Sinucleína/química , Benzotiazóis , Eletrofisiologia/métodos , Flavanonas/química , Corantes Fluorescentes/química , Humanos , Bicamadas Lipídicas , Microscopia de Força Atômica , Microscopia Confocal , Modelos Biológicos , Doença de Parkinson/metabolismo , Ligação Proteica , Solventes/química , Tiazóis/químicaRESUMO
The structural polymorphism of two selected disaccharide glycolipids with a maltose (DMMA) and a melibiose (DMME) carbohydrate headgroup linked to dimyristyl alkyl chains were investigated by FTIR-spectroscopy, differential scanning calorimetry (DSC), small-angle X-ray scattering (SAXS) and film-balance measurements. The compounds displayed thermotropic multilamellar phases. In the gel phase, DMMA formed also a crystalline phase of orthorhombic symmetry, and DMME an interdigitated phase. The gel to liquid crystalline phase transition temperature T(c) of DMMA depended on the storage and hydration conditions, a precooled sample having a T(c) around 45 degrees C, and a freshly prepared sample around 33 degrees C. In contrast, the phase transition temperature for the gel to liquid crystalline phase of DMME was always found at 24 degrees C. Surface pressure isotherms of the lipids on water and buffer showed that DMMA covers only a small surface area (approximately 35A(2)) whereas DMME requires 50 A(2) of space on the surface. Films of DMMA can be compressed up to a maximum compressibility Pi(max) of 54 mN m(-1) whereas the tilted DMME forms less stable films with Pi(max) of 34 mN m(-1). These different structural characteristics reflect the different conformations of the disaccharide head groups. The presence of the alpha1-->4 linked maltose head group in DMMA and an alpha1-->6 linked melibiose head group in DMME induces geometrical structures ranging from a slightly wedge-shaped towards a more tilted structure, and as a consequence of Israelachvilis packing model, to the formation of different phases. In addition, the structural constraints of DMME allow the formation of a phase with interdigitated hydrocarbon chains.
Assuntos
Dissacarídeos/química , Glicolipídeos/química , Varredura Diferencial de Calorimetria , Espalhamento a Baixo Ângulo , Espectroscopia de Infravermelho com Transformada de Fourier , Lipossomas Unilamelares , Difração de Raios XRESUMO
On the basis of formerly investigated peptides corresponding to the endotoxin-binding domain from LALF [Limulus anti-LPS (lipopolysaccharide) factor], a protein from Limulus polyphemus, we have designed and synthesized peptides of different lengths with the aim of obtaining potential therapeutic agents against septic shock syndrome. For an understanding of the mechanisms of action, we performed a detailed physicochemical and biophysical analysis of the interaction of rough mutant LPS with these peptides by applying FTIR (Fourier-transform infrared) spectroscopy, SAXS (small-angle X-ray scattering), calorimetric techniques [DSC (differential scanning calorimetry) and ITC (isothermal titration calorimetry)] and FFTEM (freeze-fracture transmission electron microscopy). Also, the action of the peptides on bacteria of different origin in microbial assays was investigated. Using FTIR and DSC, our results indicated a strong fluidization of the lipid A acyl chains due to peptide binding, with a decrease in the endothermic melting enthalpy change of the acyl chains down to a complete disappearance in the 1:0.5 to 1:2 [LPS]:[peptide] molar ratio range. Via ITC, it was deduced that the binding is a clearly exothermic process which becomes saturated at a 1:0.5 to 1:2 [LPS]:[peptide] molar ratio range. The results obtained with SAXS indicated a drastic change of the aggregate structures of LPS into a multilamellar stack, which was visualized in electron micrographs as hundreds of lamellar layers. This can be directly correlated with the inhibition of the LPS-induced production of tumour necrosis factor alpha in human mononuclear cells, but not with the action of the peptides on bacteria.
Assuntos
Biofísica , Endotoxinas/farmacologia , Hormônios de Invertebrado/química , Peptídeos Cíclicos/química , Termodinâmica , Sequência de Aminoácidos , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos , Proteínas de Artrópodes , Fenômenos Biofísicos , Calorimetria , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citoproteção/efeitos dos fármacos , Temperatura Alta , Humanos , Hormônios de Invertebrado/farmacologia , Lipopolissacarídeos/farmacologia , Lipossomos , Viabilidade Microbiana/efeitos dos fármacos , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/farmacologia , Transição de Fase , Fosfolipídeos/química , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios XRESUMO
We studied the interaction of several nona-peptide mimotopes of different sequence and Staphylococcal protein A (SpA) with a recombinant human IgG1 antibody using isothermal titration calorimetry (ITC). The amino acid primary structure of the peptides was varied in order to identify the specific antibody-peptide binding sites. Additionally, the influence of temperature and salt concentration was investigated. An attempt was made to elucidate the structural changes upon complex formation using the determined thermodynamic parameters. The amino acid composition of the mimotopes determined their binding affinity. The binding constant K (a) of the mimotopes was in the range 1 x 10(4) to 1 x 10(6) M(-1). The binding constant of SpA was on the average about three orders of magnitude higher than that of the peptides. The binding constant of the peptides and of SpA decreased with temperature and the binding process was connected with negative changes in enthalpy, entropy, and heat capacity. The binding of the mimotopes to the Fab part of the IgG1 antibody and binding of SpA to the Fc part of the IgG1 antibody were mainly driven by hydrophobic effects and associated with a relatively large change in water-accessible surface area. Determinants for a strong/reduced antibody-peptide binding were identified.
Assuntos
Imunoglobulina G/química , Peptídeos/química , Proteína Estafilocócica A/química , Aminoácidos/química , Varredura Diferencial de Calorimetria/métodos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imunoglobulina G/metabolismo , Peptídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteína Estafilocócica A/metabolismoRESUMO
A basic challenge in the treatment of septic patients in critical care units is the release of bacterial pathogenicity factors such as lipopolysaccharide (LPS, endotoxin) from the cell envelope of Gram-negative bacteria due to killing by antibiotics. LPS aggregates may interact with serum and membrane proteins such as LBP (lipopolysaccharide-binding protein) and CD14 leading to the observed strong reaction of the immune system. Thus, an effective treatment of patients infected by Gram-negative bacteria must comprise beside bacterial killing the neutralization of endotoxins. Here, data are summarized for synthetic compounds indicating the stepwise development to very effective LPS-neutralizing agents. These data include synthetic peptides, based on the endotoxin-binding domains of natural binding proteins such as lactoferrin, Limulus anti-LPS factor, NK-lysin, and cathelicidins or based on LPS sequestering polyamines. Many of these compounds could be shown to act not only in vitro, but also in vivo (e.g. in animal models of sepsis), and might be useful in future clinical trials and in sepsis therapy.