RESUMO
Some antimicrobial peptides (AMPs) have potent bactericidal activity and are being considered as potential alternatives to classical antibiotics. In response to an infection, such AMPs are often produced in animals alongside other peptides with low or no perceivable antimicrobial activity, whose role is unclear. Here we show that six AMPs from the Winter Flounder (WF) act in synergy against a range of bacterial pathogens and provide mechanistic insights into how this increases the cooperativity of the dose-dependent bactericidal activity and potency that enable therapy. Only two WF AMPs have potent antimicrobial activity when used alone but we find a series of two-way combinations, involving peptides which otherwise have low or no activity, yield potent antimicrobial activity. Weakly active WF AMPs modulate the membrane interactions of the more potent WF AMPs and enable therapy in a model of Acinetobacter baumannii burn wound infection. The observed synergy and emergent behaviour may explain the evolutionary benefits of producing a family of related peptides and are attractive properties to consider when developing AMPs towards clinical applications.
RESUMO
Antimicrobial peptides (AMPs) are a potential alternative to classical antibiotics that are yet to achieve a therapeutic breakthrough for treatment of systemic infections. The antibacterial potency of pleurocidin, an AMP from Winter Flounder, is linked to its ability to cross bacterial plasma membranes and seek intracellular targets while also causing membrane damage. Here we describe modification strategies that generate pleurocidin analogues with substantially improved, broad spectrum, antibacterial properties, which are effective in murine models of bacterial lung infection. Increasing peptide-lipid intermolecular hydrogen bonding capabilities enhances conformational flexibility, associated with membrane translocation, but also membrane damage and potency, most notably against Gram-positive bacteria. This negates their ability to metabolically adapt to the AMP threat. An analogue comprising D-amino acids was well tolerated at an intravenous dose of 15 mg/kg and similarly effective as vancomycin in reducing EMRSA-15 lung CFU. This highlights the therapeutic potential of systemically delivered, bactericidal AMPs.
Assuntos
Antibacterianos/farmacologia , Proteínas de Peixes/farmacologia , Pneumopatias/tratamento farmacológico , Proteínas Citotóxicas Formadoras de Poros/farmacologia , Animais , Antibacterianos/química , Antibacterianos/uso terapêutico , Modelos Animais de Doenças , Proteínas de Peixes/química , Proteínas de Peixes/uso terapêutico , Células HEK293 , Células HeLa , Humanos , Ligação de Hidrogênio , Pneumopatias/microbiologia , Masculino , Membranas Artificiais , Camundongos , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/uso terapêutico , Conformação ProteicaRESUMO
Frogs such as Rana temporaria and Litoria aurea secrete numerous closely related antimicrobial peptides (AMPs) as an effective chemical dermal defence. Damage or penetration of the bacterial plasma membrane is considered essential for AMP activity and such properties are commonly ascribed to their ability to form secondary amphipathic, α-helix conformations in membrane mimicking milieu. Nevertheless, despite the high similarity in physical properties and preference for adopting such conformations, the spectrum of activity and potency of AMPs often varies considerably. Hence distinguishing apparently similar AMPs according to their behaviour in, and effects on, model membranes will inform understanding of primary-sequence-specific antimicrobial mechanisms. Here we use a combination of molecular dynamics simulations, circular dichroism and patch-clamp to investigate the basis for differing anti-bacterial activities in representative AMPs from each species; temporin L and aurein 2.5. Despite adopting near identical, α-helix conformations in the steady-state in a variety of membrane models, these two AMPs can be distinguished both in vitro and in silico based on their dynamic interactions with model membranes, notably their differing conformational flexibility at the N-terminus, ability to form higher order aggregates and the characteristics of induced ion conductance. Taken together, these differences provide an explanation of the greater potency and broader antibacterial spectrum of activity of temporin L over aurein 2.5. Consequently, while the secondary amphipathic, α-helix conformation is a key determinant of the ability of a cationic AMP to penetrate and disrupt the bacterial plasma membrane, the exact mechanism, potency and spectrum of activity is determined by precise structural and dynamic contributions from specific residues in each AMP sequence.
Assuntos
Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Membrana Celular/efeitos dos fármacos , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Transporte de Íons , Simulação de Dinâmica Molecular , Conformação Proteica em alfa-Hélice , Lipossomas Unilamelares/químicaRESUMO
Antimicrobial peptides (AMPs) are a potential source of new molecules to counter the increase in antimicrobial resistant infections but a better understanding of their properties is required to understand their native function and for effective translation as therapeutics. Details of the mechanism of their interaction with the bacterial plasma membrane are desired since damage or penetration of this structure is considered essential for AMPs activity. Relatively modest modifications to AMPs primary sequence can induce substantial changes in potency and/or spectrum of activity but, hitherto, have not been predicted to substantially alter the mechanism of interaction with the bacterial plasma membrane. Here we use a combination of molecular dynamics simulations, circular dichroism, solid-state NMR and patch clamp to investigate the extent to which temporin B and its analogues can be distinguished both in vitro and in silico on the basis of their interactions with model membranes. Enhancing the hydrophobicity of the N-terminus and cationicity of the C-terminus in temporin B improves its membrane activity and potency against both Gram-negative and Gram-positive bacteria. In contrast, enhancing the cationicity of the N-terminus abrogates its ability to trigger channel conductance and renders it ineffective against Gram-positive bacteria while nevertheless enhancing its potency against Escherichia coli. Our findings suggest even closely related AMPs may target the same bacterium with fundamentally differing mechanisms of action.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Membrana Celular/metabolismo , Sequência de Aminoácidos , Membrana Celular/efeitos dos fármacos , Condutividade Elétrica , Bicamadas Lipídicas/química , Micelas , Testes de Sensibilidade Microbiana , Simulação de Dinâmica Molecular , Conformação Proteica , Dodecilsulfato de Sódio , Relação Estrutura-AtividadeRESUMO
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.
RESUMO
In many species the capacity to accurately differentiate the energy density (kcal/g) of foods is critical because it greatly improves efficiency in foraging. In modern humans this ability remains intact and is expressed in a selective preference for types of fruit and vegetables that contain more calories. However, humans evolved consuming these low energy-dense foods (typically < 1.75 kcal/g) and it remains unclear whether they can also discriminate more energy-dense foods that now feature in modern Western diets. In two experiment participants (both N = 40) completed four tasks that assessed the 'value' of different sets of 22 foods that ranged in energy density (0.1 kcal/g-5.3 kcal/g and range 0.1 kcal/g to 6.2 kcal/g in Experiment 1 and 2, respectively). In Experiment 1 three measures (expected fullness, calorie estimation, and food choice), and in foods less than approximately 1.5 kcal/g (typically fruits and vegetables), the relationship between perceived value and energy density is linear. Above this, we observed clear compressive functions, indicating relative and progressive undervaluation of higher energy-dense foods. The fourth task (rated liking) failed to provide evidence for any relationship with energy density. In Experiment 2 the same pattern was replicated in measures of expected fullness, and in two different assessments of subjective calorie content. Consistent with the concept of 'evolutionary discordance,' this work indicates that modern human physiology is poorly adapted to evaluate foods that have a historically unusual (high) energy density. This has implications both for our understanding of how 'modern' energy-dense foods affect choice and energy intake, and for strategies aimed at removing calories from highly energy-rich foods.
Assuntos
Comportamento de Escolha , Ingestão de Energia , Preferências Alimentares , Valor Nutritivo , Adulto , Índice de Massa Corporal , Feminino , Frutas , Comportamentos Relacionados com a Saúde , Humanos , Masculino , Saciação , VerdurasRESUMO
The interaction of antimicrobial peptides (AMPs) with the inner membrane of Gram-negative bacteria is a key determinant of their abilities to exert diverse bactericidal effects. Here we present a molecular level understanding of the initial target membrane interaction for two cationic α-helical AMPs that share structural similarities but have a ten-fold difference in antibacterial potency towards Gram-negative bacteria. The binding and insertion from solution of pleurocidin or magainin 2 to membranes representing the inner membrane of Gram-negative bacteria, comprising a mixture of 128 anionic and 384 zwitterionic lipids, is monitored over 100 ns in all atom molecular dynamics simulations. The effects of the membrane interaction on both the peptide and lipid constituents are considered and compared with new and published experimental data obtained in the steady state. While both magainin 2 and pleurocidin are capable of disrupting bacterial membranes, the greater potency of pleurocidin is linked to its ability to penetrate within the bacterial cell. We show that pleurocidin displays much greater conformational flexibility when compared with magainin 2, resists self-association at the membrane surface and penetrates further into the hydrophobic core of the lipid bilayer. Conformational flexibility is therefore revealed as a key feature required of apparently α-helical cationic AMPs for enhanced antibacterial potency.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Membrana Celular/metabolismo , Bactérias Gram-Negativas/metabolismo , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/farmacologia , Membrana Celular/efeitos dos fármacos , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Ontologia Genética , Bactérias Gram-Negativas/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Lipídeos de Membrana/química , Simulação de Dinâmica Molecular , Ligação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Transcriptoma/genéticaRESUMO
Systematic phytochemical investigations of the underground rhizome of Pteridium aquilinum (L.) Kuhn (Dennstaedtiaceae) afforded thirty-five pterosins and pterosides. By detailed analysis of one- and two-dimensional nuclear magnetic resonance spectroscopy, circular dichroism (CD) and high-resolution mass spectrometric data, thirteen previously undescribed pterosins and pterosides have been identified. Interestingly, for the first time 12-O-ß-D-glucopyranoside substituted pterosins, rhedynosides C and D, and the sulfate-containing pterosin, rhedynosin H, alongside the two known compounds, histiopterosin A and (2S)-pteroside A2, were isolated from the rhizomes of subsp. aquilinum of bracken. In addition, six-membered cyclic ether pterosins and pterosides, rhedynosin A and rhedynoside A, are the first examples of this type of pterosin-sesquiterpenoid. Additionally, the three previously reported compounds (rhedynosin I, (2S)-2-hydroxymethylpterosin E and (2S)-12-hydroxypterosin A) were obtained for the first time from plants as opposed to mammalian metabolic products. Single crystal X-ray diffraction analysis was applied to the previously undescribed compounds (2R)-rhedynoside B, (2R)-pteroside B and (2S)-pteroside K, yielding the first crystal structures for pterosides, and three known pterosins, (2S)-pterosin A, trans-pterosin C and cis-pterosin C. Rhedynosin C is the only example of the cyclic lactone pterosins with a keto group at position C-14. Six selected pterosins ((2S)-pterosin A, (2R)-pterosin B and trans-pterosin C) and associated glycosides ((2S)-pteroside A, (2R)-pteroside B and pteroside Z) were assessed for their anti-diabetic activity using an intestinal glucose uptake assay; all were found to be inactive at 300 µM.
Assuntos
Glicosídeos/isolamento & purificação , Indanos/isolamento & purificação , Pteridium/química , Rizoma/química , Sesquiterpenos/isolamento & purificação , Animais , Diabetes Mellitus/tratamento farmacológico , Glicosídeos/química , Indanos/química , Ressonância Magnética Nuclear Biomolecular , Sesquiterpenos/química , Sesquiterpenos/farmacologiaRESUMO
Zinc α2 glycoprotein (ZAG) is an adipokine with a class I MHC protein fold and is associated with obesity and diabetes. Although its intrinsic ligand remains unknown, ZAG binds the dansylated C11 fatty acid 11-(dansylamino)undecanoic acid (DAUDA) in the groove between the α1 and α2 domains. The surface of ZAG has approximately 15 weak zinc-binding sites deemed responsible for precipitation from human plasma. In the present study the functional significance of these metal sites was investigated. Analytical ultracentrifugation (AUC) and CD showed that zinc, but not other divalent metals, causes ZAG to oligomerize in solution. Thus ZAG dimers and trimers were observed in the presence of 1 and 2 mM zinc. Molecular modelling of X-ray scattering curves and sedimentation coefficients indicated a progressive stacking of ZAG monomers, suggesting that the ZAG groove may be occluded in these. Using fluorescence-detected sedimentation velocity, these ZAG-zinc oligomers were again observed in the presence of the fluorescent boron dipyrromethene fatty acid C16-BODIPY (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-hexadecanoic acid). Fluorescence spectroscopy confirmed that ZAG binds C16-BODIPY. ZAG binding to C16-BODIPY, but not to DAUDA, was reduced by increased zinc concentrations. We conclude that the lipid-binding groove in ZAG contains at least two distinct fatty acid-binding sites for DAUDA and C16-BODIPY, similar to the multiple lipid binding seen in the structurally related immune protein CD1c. In addition, because high concentrations of zinc occur in the pancreas, the perturbation of these multiple lipid-binding sites by zinc may be significant in Type 2 diabetes where dysregulation of ZAG and zinc homoeostasis occurs.
Assuntos
Proteínas de Transporte/metabolismo , Ácidos Graxos/metabolismo , Glicoproteínas/metabolismo , Zinco/metabolismo , Adipocinas , Sítios de Ligação/fisiologia , Proteínas de Transporte/química , Ácidos Graxos/química , Glicoproteínas/química , Humanos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Zinco/farmacologiaRESUMO
Current understanding in locomotion research is that, for humans, navigating natural environments relies heavily on visual input; in contrast, walking on even ground in man-made obstacle and hazard-free environments is so highly automated that visual information derived from floor patterns should not affect locomotion and in particular have no impact on the direction of travel. The vision literature on motion perception would suggest otherwise; specifically that oblique floor patterns may induce substantial veering away from the intended direction of travel due to the so-called aperture problem. Here, we tested these contrasting predictions by letting participants walk over commonly encountered floor patterns (paving slabs) and investigating participants' ability to walk "straight ahead" for different pattern orientations. We show that, depending on pattern orientation, participants veered considerably over the measured travel distance (up to 8% across trials), in line with predictions derived from the literature on motion perception. We argue that these findings are important to the study of locomotion, and, if also observed in real world environments, might have implications for architectural design.
Assuntos
Materiais de Construção , Percepção de Movimento/fisiologia , Orientação/fisiologia , Desempenho Psicomotor , Percepção Espacial/fisiologia , Caminhada , Fenômenos Biomecânicos , Feminino , Humanos , MasculinoRESUMO
This article reports on the stereochemical aspects of the chemical stability of lactose solutions stored between 25 and 60 °C. The lactose used for the preparation of the aqueous solutions was α-lactose monohydrate with an anomer purity of 96% α and 4% ß based on the supplied certificate of analysis (using a GC analytical protocol), which was further confirmed here by nuclear magnetic resonance (NMR) analysis. Aliquots of lactose solutions were collected at different time points after the solutions were prepared and freeze-dried to remove water and halt epimerization for subsequent analysis by NMR. Epimerization was also monitored by polarimetry and infrared spectroscopy using a specially adapted Fourier transform infrared attenuated total reflectance (FTIR-ATR) method. Hydrolysis was analyzed by ion chromatography. The three different analytical approaches unambiguously showed that the epimerization of lactose in aqueous solution follows first order reversible kinetics between 25 to 60 °C. The overall rate constant was 4.4 × 10(-4) s(-1) ± 0.9 (± standard deviation (SD)) at 25 °C. The forward rate constant was 1.6 times greater than the reverse rate constant, leading to an equilibrium constant of 1.6 ± 0.1 (±SD) at 25 °C. The rate of epimerization for lactose increased with temperature and an Arrhenius plot yielded an activation energy of +52.3 kJ/mol supporting the hypothesis that the mechanism of lactose epimerization involves the formation of extremely short-lived intermediate structures. The main mechanism affecting lactose stability is epimerization, as no permanent hydrolysis or chemical degradation was observed. When preparing aqueous solutions of lactose, immediate storage in an ice bath at 0 °C will allow approximately 3 min (180 s) of analysis time before the anomeric ratio alters significantly (greater than 1%) from the solid state composition of the starting material. In contrast a controlled anomeric composition (~38% α and ~62% ß) will be achieved if an aqueous solution is left to equilibrate for over 4 h at 25 °C, while increasing the temperature up to 60 °C rapidly reduces the required equilibration time.
Assuntos
Lactose/química , Soluções/química , Água/química , Liofilização/métodos , Hidrólise , Cinética , Espectroscopia de Ressonância Magnética/métodos , Estereoisomerismo , TemperaturaRESUMO
This work investigates the effect of combining physical and chemical gelation processes in a biopolymer blend: chitosan and tilapia fish gelatin. Chemical (C) gels are obtained by cross-linking with the microbial enzyme transglutaminase at 37 °C. Hybrid physical-co-chemical (PC) gels are cross-linked at 21 °C, below gelatin gelation temperature. These protocols provide two microenvironments for the gelation process: in C gels, both gelatin and chitosan are present as single strands; in PC gels, cross-linking proceeds within a transient physical gel of gelatin, filled by chitosan strands. The chitosan/gelatin chemical networks generated in PC gels show a consistently higher shear modulus than pure C gels; they are also less turbid than their C gels counterparts, suggesting a more homogeneous network. Finally, chitosan enhances the gels' shear modulus in all gels. Proliferation assays show that MC3T3 cells proliferate in these mixed, hybrid gels and better so on PC gels than in C mixed gels.
Assuntos
Proliferação de Células , Quitosana/química , Gelatina/química , Hidrogéis , Osteoblastos/metabolismo , Transglutaminases/química , Animais , Linhagem Celular , Hidrogéis/síntese química , Hidrogéis/química , Teste de Materiais , Camundongos , Osteoblastos/citologiaRESUMO
Zinc-α2-glycoprotein (ZAG) is an adipokine with an MHC class I-like protein fold. Even though zinc causes ZAG to precipitate from plasma during protein purification, no zinc binding has been identified to date. Using mass spectrometry, we demonstrated that ZAG contains one strongly bound zinc ion, predicted to lie close to the α1 and α2 helical groove. UV, CD and fluorescence spectroscopies detected weak zinc binding to holo-ZAG, which can bind up to 15 zinc ions. Zinc binding to 11-(dansylamino) undecanoic acid was enhanced by holo-ZAG. Zinc binding may be important for ZAG binding to fatty acids and the ß-adrenergic receptor.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , Domínios e Motivos de Interação entre Proteínas , Zinco/metabolismo , Adipocinas , Sítios de Ligação , Ácidos Graxos/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/fisiologia , Estrutura Secundária de Proteína , Receptores Adrenérgicos beta/metabolismo , Especificidade por Substrato , Zinco/químicaRESUMO
Cationic amphipathic pH responsive peptides possess high in vitro and in vivo nucleic acid delivery capabilities and function by forming a non-covalent complex with cargo, protecting it from nucleases, facilitating uptake via endocytosis and responding to endosomal acidification by being released from the complex and inserting into and disordering endosomal membranes. We have designed and synthesised peptides to show how Coulombic interactions between ionizable 2,3-diaminopropionic acid (Dap) side chains can be manipulated to tune the functional pH response of the peptides to afford optimal nucleic acid transfer and have modified the hydrogen bonding capabilities of the Dap side chains in order to reduce cytotoxicity. When compared with benchmark delivery compounds, the peptides are shown to have low toxicity and are highly effective at mediating gene silencing in adherent MCF-7 and A549 cell lines, primary human umbilical vein endothelial cells and both differentiated macrophage-like and suspension monocyte-like THP-1 cells.
Assuntos
Preparações de Ação Retardada/química , RNA Interferente Pequeno/administração & dosagem , beta-Alanina/análogos & derivados , Sequência de Aminoácidos , Linhagem Celular , Linhagem Celular Tumoral , Endocitose , Humanos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacocinética , beta-Alanina/químicaRESUMO
The p53 family of genes and their protein products, namely, p53, p63 and p73, have over one billion years of evolutionary history. Advances in computational biology and genomics are enabling studies of the complexities of the molecular evolution of p53 protein family to decipher the underpinnings of key biological conditions spanning from cancer through to various metabolic and developmental disorders and facilitate the design of personalised medicines. However, a complete understanding of the inherent nature of the thermodynamic and structural stability of the p53 protein family is still lacking. This is due, to a degree, to the lack of comprehensive structural information for a large number of homologous proteins and to an incomplete knowledge of the intrinsic factors responsible for their stability and how these might influence function. Here we investigate the thermal stability, secondary structure and folding properties of the DNA-binding domains (DBDs) of a range of proteins from the p53 family using biophysical methods. While the N- and the C-terminal domains of the p53 family show sequence diversity and are normally targets for post-translational modifications and alternative splicing, the central DBD is highly conserved. Together with data obtained from Molecular Dynamics simulations in solution and with structure based homology modelling, our results provide further insights into the molecular properties of evolutionary related p53 proteins. We identify some marked structural differences within the p53 family, which could account for the divergence in biological functions as well as the subtleties manifested in the oligomerization properties of this family.
Assuntos
Proteína Supressora de Tumor p53/química , Sequência de Aminoácidos , Evolução Molecular , Humanos , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteína Supressora de Tumor p53/genéticaRESUMO
The UV absorption and electronic circular dichroism (ECD) spectra of (R)- and (S)-nicotine and (S)-nornicotine in aqueous solution were measured to a significantly lower wavelength range than previously reported, allowing the identification of four previously unobserved electronic transitions. The ECD spectra of the two enantiomers of nicotine were equal in magnitude and opposite in sign, while the UV absorption spectra were coincidental. In line with previous observations, (S)-nicotine exhibited a negative cotton effect centered on 263 nm with vibronic structure (π-π1 * transition) and a broad, positive ECD signal at around 240 nm associated with the n-π1 * transition. As expected this band disappeared when the pyridyl aromatic moiety was protonated. Four further electronic transitions are reported between 215 and 180 nm; it is proposed the negative maxima around 206 nm is either an n-σ* transition or a charge transfer band resulting from the movement of charge from the pyrrolidyl N lone pair to the pyridyl π* orbital. The pyridyl π-π2* transition may be contained within the negative ECD signal envelope at around 200 nm. Another negative maximum at 188 nm is thought to be the pyridyl π-π3 * transition, while the lowest wavelength end-absorption and positive ECD may be associated with the π-π4 * transition. The UV absorption spectra of (S)-nornicotine was similar to that of (S)-nicotine in the range 280-220 nm and acidification of the aqueous solution enhanced the absorption. The ECD signals of (S)-nornicotine were considerably less intense compared to (S)-nicotine and declined further on acidification; in the far UV region the ECD spectra diverge considerably.
Assuntos
Nicotina/análogos & derivados , Nicotina/química , Espectrofotometria Ultravioleta/métodosRESUMO
The STAT3 transcription factor plays a central role in a wide range of cancer types where it is over-expressed. Previously, phosphorylation of this protein was thought to be a prerequisite for direct binding to DNA. However, we have now shown complete binding of a purified unphosphorylated STAT3 (uSTAT3) core directly to M67 DNA, the high affinity STAT3 target DNA sequence, by a protein electrophoretic mobility shift assay (PEMSA). Binding to M67 DNA was inhibited by addition of increasing concentrations of a phosphotyrosyl peptide. X-ray crystallography demonstrates one mode of binding that is similar to that known for the STAT3 core phosphorylated at Y705.
Assuntos
DNA/química , DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Fator de Transcrição STAT3/química , Fator de Transcrição STAT3/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva , Dicroísmo Circular , Cristalografia por Raios X , DNA/genética , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Fosfopeptídeos/química , Fosfopeptídeos/metabolismo , Fosforilação , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Fator de Transcrição STAT3/genética , Tirosina/química , Tirosina/genética , Tirosina/metabolismoRESUMO
Combined results of theoretical molecular dynamic simulations and in vitro spectroscopic (circular dichroism and fluorescence) studies are presented, providing the atomistic and secondary structure details of the process by which a selected small molecule may destabilize the ß-sheet ordered "amyloid" oligomers formed by the model undecapeptide of amyloid ß-peptide 25-35 [Aß(25-35)]. Aß(25-35) was chosen because it is the shortest fragment capable of forming large ß-sheet fibrils and retaining the toxicity of the full length Aß(1-40/42) peptides. The conformational transition, that leads to the formation of ß-sheet fibrils from soluble unordered structures, was found to depend on the environmental conditions, whereas the presence of myricetin destabilizes the self-assembly and antagonizes this conformational shift. In parallel, we analyzed several molecular dynamics trajectories describing the evolution of five monomer fragments, without inhibitor as well as in the presence of myricetin. Other well-known inhibitors (curcumin and (-)-tetracycline), found to be stronger and weaker Aß(1-42) aggregation inhibitors, respectively, were also studied. The combined in vitro and theoretical studies of the Aß(25-35) self-assembly and its inhibition contribute to understanding the mechanism of action of well-known inhibitors and the peptide amino acid residues involved in the interaction leading to a rational drug design of more potent new molecules able to antagonize the self-assembly process.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides , Curcumina/farmacologia , Flavonoides/farmacologia , Modelos Moleculares , Fragmentos de Peptídeos , Tetraciclina/farmacologia , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/química , Dicroísmo Circular/métodos , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Estrutura Secundária de Proteína , Espectrometria de Fluorescência/métodos , TemperaturaRESUMO
The present study describes leucine zipper peptide-lipid hybrid nanoscale vesicles engineered by self-assembled anchoring of the amphiphilic peptide within the lipid bilayer. These hybrid vesicles aim to combine the advantages of traditional temperature-sensitive liposomes (TSL) with the dissociative, unfolding properties of a temperature-sensitive peptide to optimize drug release under mild hyperthermia, while improving in vivo drug retention. The secondary structure of the peptide and its thermal responsiveness after anchoring onto liposomes were studied with circular dichroism. In addition, the lipid-peptide vesicles (Lp-peptide) showed a reduction in bilayer fluidity at the inner core, as observed with DPH anisotropy studies, while the opposite effect was observed with an ANS probe, indicating peptide interactions with both the headgroup region and the hydrophobic core. A model drug molecule, doxorubicin, was successfully encapsulated in the Lp-peptide vesicles at higher than 90% efficiency following the remote loading, pH-gradient methodology. The release of doxorubicin from Lp-peptide hybrids in vitro indicated superior serum stability at physiological temperatures compared to lysolipid-containing temperature-sensitive liposomes (LTSL) without affecting the overall thermo-responsive nature of the vesicles at 42 °C. A similar stabilizing effect was observed in vivo after intravenous administration of the Lp-peptide vesicles by measuring (14)C-doxorubicin blood kinetics that also led to increased tumor accumulation after 24 h. We conclude that Lp-peptide hybrid vesicles present a promising new class of TSL that can offer previously unexplored opportunities for the development of clinically relevant mild hyperthermia-triggered therapeutic modalities.
Assuntos
Preparações de Ação Retardada/química , Doxorrubicina/administração & dosagem , Lipossomos/química , Melanoma/tratamento farmacológico , Nanocápsulas/administração & dosagem , Nanocápsulas/química , Peptídeos/química , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Linhagem Celular Tumoral , Difusão , Doxorrubicina/química , Temperatura Alta , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Nanocápsulas/ultraestrutura , Tamanho da Partícula , Resultado do TratamentoRESUMO
We used a combination of fluorescence, circular dichroism (CD), and NMR spectroscopies in conjunction with size exclusion chromatography to help rationalize the relative antibacterial, antiplasmodial, and cytotoxic activities of a series of proline-free and proline-containing model antimicrobial peptides (AMPs) in terms of their structural properties. When compared with proline-free analogs, proline-containing peptides had greater activity against Gram-negative bacteria, two mammalian cancer cell lines, and intraerythrocytic Plasmodium falciparum, which they were capable of killing without causing hemolysis. In contrast, incorporation of proline did not have a consistent effect on peptide activity against Mycobacterium tuberculosis. In membrane-mimicking environments, structures with high α-helix content were adopted by both proline-free and proline-containing peptides. In solution, AMPs generally adopted disordered structures unless their sequences comprised more hydrophobic amino acids or until coordinating phosphate ions were added. Proline-containing peptides resisted ordering induced by either method. The roles of the angle subtended by positively charged amino acids and the positioning of the proline residues were also investigated. Careful positioning of proline residues in AMP sequences is required to enable the peptide to resist ordering and maintain optimal antibacterial activity, whereas varying the angle subtended by positively charged amino acids can attenuate hemolytic potential albeit with a modest reduction in potency. Maintaining conformational flexibility improves AMP potency and selectivity toward bacterial, plasmodial, and cancerous cells while enabling the targeting of intracellular pathogens.