ABSTRACT
Peptide-based self-assembly has been used to produce a wide range of nanostructures. While most of these systems involve self-assembly of α-peptides, more recently ß-peptides have also been shown to undergo supramolecular self-assembly, and have been used to produce materials for applications in tissue engineering, cell culture and drug delivery. In order to engineer new materials with specific structure and function, theoretical molecular modelling can provide significant insights into the collective balance of non-covalent interactions that drive the self-assembly and determine the structure of the resultant supramolecular materials under different conditions. However, this approach has only recently become feasible for peptide-based self-assembled nanomaterials, particularly those that incorporate non α-amino acids. This perspective provides an overview of the challenges associated with computational modelling of the self-assembly of ß-peptides and the recent success using a combination of experimental and computational techniques to provide insights into the self-assembly mechanisms and fully atomistic models of these new biocompatible materials.
ABSTRACT
It is now evident that the cell manipulates lipid composition to regulate different processes such as membrane protein insertion, assembly and function. Moreover, changes in membrane structure and properties, lipid homeostasis during growth and differentiation with associated changes in cell size and shape, and responses to external stress have been related to drug resistance across mammalian species and a range of microorganisms. While it is well known that the biomembrane is a fluid self-assembled nanostructure, the link between the lipid components and the structural properties of the lipid bilayer are not well understood. This perspective aims to address this topic with a view to a more detailed understanding of the factors that regulate bilayer structure and flexibility. We describe a selection of recent studies that address the dynamic nature of bacterial lipid diversity and membrane properties in response to stress conditions. This emerging area has important implications for a broad range of cellular processes and may open new avenues of drug design for selective cell targeting.
ABSTRACT
ß-Peptides have great potential as novel biomaterials and therapeutic agents, due to their unique ability to self-assemble into low dimensional nanostructures, and their resistance to enzymatic degradation in vivo. However, the self-assembly mechanisms of ß-peptides, which possess increased flexibility due to the extra backbone methylene groups present within the constituent ß-amino acids, are not well understood due to inherent difficulties of observing their bottom-up growth pathway experimentally. A computational approach is presented for the bottom-up modelling of the self-assembled lipidated ß3-peptides, from monomers, to oligomers, to supramolecular low-dimensional nanostructures, in all-atom detail. The approach is applied to elucidate the self-assembly mechanisms of recently discovered, distinct structural morphologies of low dimensional nanomaterials, assembled from lipidated ß3-peptide monomers. The resultant structures of the nanobelts and the twisted fibrils are stable throughout subsequent unrestrained all-atom molecular dynamics simulations, and these assemblies display good agreement with the structural features obtained from X-ray fiber diffraction and atomic force microscopy data. This is the first reported, fully-atomistic model of a lipidated ß3-peptide-based nanomaterial, and the computational approach developed here, in combination with experimental fiber diffraction analysis and atomic force microscopy, will be useful in elucidating the atomic scale structure of self-assembled peptide-based and other supramolecular nanomaterials.
Subject(s)
Molecular Dynamics Simulation , Nanostructures , Peptides , Nanostructures/chemistry , Peptides/chemistry , Lipids/chemistry , Microscopy, Atomic ForceABSTRACT
The intrinsic pathway of apoptosis is regulated by the Bcl-2 family of proteins. Inhibition of the anti-apoptotic members represents a strategy to induce apoptotic cell death in cancer cells. We have measured the membrane binding properties of a series of peptides, including modified α/ß-peptides, designed to exhibit enhanced membrane permeability to allow cell entry and improved access for engagement of Bcl-2 family members. The peptide cargo is based on the pro-apoptotic protein Bim, which interacts with all anti-apoptotic proteins to initiate apoptosis. The α/ß-peptides contained cyclic ß-amino acid residues designed to increase their stability and membrane-permeability. Dual polarisation interferometry was used to study the binding of each peptide to two different model membrane systems designed to mimic either the plasma membrane or the outer mitochondrial membrane. The impact of each peptide on the model membrane structure was also investigated, and the results demonstrated that the modified peptides had increased affinity for the mitochondrial membrane and significantly altered the structure of the bilayer. The results also showed that the presence of an RRR motif significantly enhanced the ability of the peptides to bind to and insert into the mitochondrial membrane mimic, and provide insights into the role of selective membrane targeting of peptides.
ABSTRACT
Small tripeptides composed entirely of ß3-amino acids have been shown to self-assemble into fibres following acylation of the N-terminus. Given the use of Fmoc as a strategy to initiate self-assembly in α-peptides, we hypothesized that the acyl cap can be replaced by an Fmoc without perturbation to the self-assembly and enable simpler synthetic protocols. We therefore replaced the N-acyl cap for an Fmoc group and herein we show that these Fmoc-protected ß3-peptides produce regular spherical particles, rather than fibrous structures, that are stable and capable of encapsulating cargo. We then demonstrated that these particles were able to deliver cargo to cells without any obvious signs of cytotoxicity. This is the first description of such regular nanoparticles derived from Fmoc-protected ß3-peptides.
ABSTRACT
Self-assembling lipopeptide hydrogels have been widely developed for the delivery of therapeutics due to their rapid gelation, injectability, and highly controlled physicochemical properties. Lipopeptides are also known for their membrane-associating and cell penetrating properties, which may impact on their application in cell-encapsulation. Self-assembling lipidated-ß3-peptide materials developed in our laboratory have previously been used in cell culture as 2D substrates, thus as a continuation of this work we aimed to encapsulate cells in 3D by forming a hydrogel. We therefore assessed the self-assembling lipidated-ß3-peptides for cell-penetrating properties in mesenchymal stems cells (MSC) using fluorescence microscopy and membrane association with surface plasmon resonance spectroscopy (SPR). The results demonstrated that lipidated ß3-peptides penetrate the MSC plasma membrane and localise to the mitochondrial network. While self-assembling lipopeptide hydrogels have shown tremendous potential for delivery of therapeutics, further optimisation may be required to minimise the membrane uptake of the lipidated-ß3-peptides for cell encapsulation applications.
Subject(s)
Cell Culture Techniques , Lipopeptides , Biological Transport , Cell Membrane , HydrogelsABSTRACT
Stimulation of the angiotensin II type 2 receptor (AT2R) evokes protective effects in various cardiovascular diseases. Thus, this study aimed to investigate the effects of AT2R stimulation, with or without AT1R blockade, in a model of hypertension with concomitant type 1 diabetes mellitus (T1DM). Spontaneously hypertensive rats (SHRs) were given either citrate or a single dose of streptozotocin (STZ; 55 mg/kg, i.p.) to induce diabetes. After 4 weeks of diabetes, animals were administered either a vehicle (saline), AT2R agonist, ß-Pro7Ang III (0.1 mg/kg/day via osmotic mini-pump), AT1R blocker, candesartan (2 mg/kg/day via drinking water), or a combination of both for a further 8 weeks. ß-Pro7Ang III treatment had no effect on blood pressure, but attenuated the significant increase in cardiac interstitial collagen and protein expression of fibrotic and inflammatory markers, and superoxide levels that was evident in diabetic SHRs. These effects were not observed with candesartan, despite its blood pressure lowering effects. Although ß-Pro7Ang III had no effect on aortic fibrosis, it significantly attenuated MCP-1 protein expression and superoxide levels when compared to both the non-diabetic and diabetic SHRs, to a similar extent as candesartan. In both the heart and vasculature, the effects of ß-Pro7Ang III in combination with candesartan were similar to those of ß-Pro7Ang III alone, and superior to candesartan alone. It was concluded that in hypertension with concomitant diabetes, AT2R stimulation with a novel ligand alone, or in combination with AT1R blockade, improved the cardiac and vascular structural changes that were strongly associated with inflammation and oxidative stress, independent of blood pressure regulation.
Subject(s)
Diabetes Mellitus , Hypertension , Animals , Rats , Benzimidazoles/pharmacology , Benzimidazoles/therapeutic use , Hypertension/complications , Hypertension/drug therapy , Rats, Inbred SHR , Receptor, Angiotensin, Type 1/metabolism , Superoxides , Cardiotonic AgentsABSTRACT
BACKGROUND AND PURPOSE: This study investigated the reno-protective effects of a highly selective AT2R agonist peptide, ß-Pro7Ang III in a mouse model of acute kidney injury (AKI). METHODS: C57BL/6 J mice underwent either sham surgery or unilateral kidney ischemia-reperfusion injury (IRI) for 40 min. IRI mice were treated with either ß-Pro7Ang III or perindopril and at 7 days post-surgery the kidneys analysed for histopathology and the development of fibrosis and matrix metalloproteinase (MMP)-2 and -9 activity. The association of the therapeutic effects of ß-Pro7Ang III with macrophage number and phenotype was determined in vivo and in vitro. KEY RESULTS: Decreased kidney tubular injury, interstitial matrix expansion and reduced interstitial immune cell infiltration in IRI mice receiving ß-Pro7Ang III treatment was observed at day 7, compared to IRI mice without treatment. This correlated to reduced collagen accumulation and MMP-2 activity in IRI mice following ß-Pro7Ang III treatment. FACS analysis showed a reduced number and proportion of CD45+CD11b+F4/80+ macrophages in IRI kidneys in response to ß-Pro7Ang III, correlating with a significant increase in M2 macrophage markers and decreased M1 markers at day 3 and 7 post-IR injury, respectively. In vitro analysis of cultured THP-1 cells showed that ß-Pro7Ang III attenuated lipopolysaccharide (LPS)-induced tumour necrosis factor-α (TNF-α) and interleukin (IL)- 6 production but increased IL-10 secretion, compared to LPS alone. CONCLUSION: Administration of ß-Pro7Ang III via mini-pump improved kidney structure and reduced interstitial collagen accumulation, in parallel with an alteration of macrophage phenotype and anti-inflammatory cytokine release, therefore mitigating the downstream progression of ischemic AKI.
Subject(s)
Acute Kidney Injury , Reperfusion Injury , Mice , Animals , Lipopolysaccharides/pharmacology , Mice, Inbred C57BL , Kidney , Acute Kidney Injury/drug therapy , Acute Kidney Injury/prevention & control , Collagen/pharmacology , Reperfusion Injury/genetics , ReperfusionABSTRACT
Determining the porosity of hydrogels is an important component of material characterisation. While scanning electron microscopy (SEM) is a widely used method to study hydrogel nanoarchitecture, it is well-established that SEM sample preparation methods can alter the structure of hydrogels. Herein we describe the impact of sample preparation on the SEM analysis of self-assembling ß-peptide hydrogels. Three methods of hydrogel preparation for SEM were compared, and each method preserved distinctly different nanoarchitecture, specifically, different levels of fibre alignment and porosity. Comparison of conventional SEM preparation and our hybrid method, which comprises high pressure freezing, freeze substitution without fixative and critical point drying, showed a high degree of similarity at the nanometre scale and diverging architecture at the micron scale. This study quantified the impact of chemical fixation versus high pressure freezing on self-assembling ß3-peptide hydrogels, demonstrated the effect of sample preparation on fibre alignment and porosity, and presents a novel hybrid preparation method where chemical fixation can be avoided when conventional SEM is desired.
Subject(s)
Hydrogels , Peptides , Hydrogels/chemistry , Microscopy, Electron, Scanning , FreezingABSTRACT
A high salt (HS) diet is associated with an increased risk for cardiovascular diseases (CVDs) and fibrosis is a key contributor to the organ dysfunction involved in CVDs. The activation of the renin angiotensin type 2 receptor (AT2R) has been considered as organ protective in many CVDs. However, there are limited AT2R-selective agonists available. Our first reported ß-substituted angiotensin III peptide, ß-Pro7-AngIII, showed high selectivity for the AT2R. In the current study, we examine the potential anti-fibrotic and anti-inflammatory effects of this novel AT2R-selective peptide on HS-induced organ damage. FVB/N mice fed with a 5% HS diet for 8 weeks developed cardiac and renal fibrosis and inflammation, which were associated with increased TGF-ß1 levels in heart, kidney and plasma. Four weeks' treatment (from weeks 5-8) with ß-Pro7-AngIII inhibited the HS-induced cardiac and renal fibrosis and inflammation. These protective effects were accompanied by reduced local and systemic TGF-ß1 as well as reduced cardiac myofibroblast differentiation. Importantly, the anti-fibrotic and anti-inflammatory effects caused by ß-Pro7-AngIII were attenuated by the AT2R antagonist PD123319. These results demonstrate, for the first time, the cardio- and reno-protective roles of the AT2R-selective ß-Pro7-AngIII, highlighting it as an important therapeutic that can target the AT2R to treat end-organ damage.
Subject(s)
Kidney Diseases , Transforming Growth Factor beta1 , Animals , Mice , Transforming Growth Factor beta1/adverse effects , Fibrosis , Kidney Diseases/etiology , Kidney Diseases/chemically induced , Sodium Chloride, Dietary/adverse effects , Sodium Chloride/adverse effects , Inflammation , Anti-Inflammatory Agents/adverse effectsABSTRACT
The mycobacterial cell envelope has spatially resolved inner and outer membrane layers with distinct compositions and membrane properties. However, the functional implication and relevance of this organization remain unknown. Using membrane biophysics and molecular simulations, we reveal a varied interaction profile of these layers with antibiotic Rifabutin, underlined by the structural and chemical makeup of the constituent lipids. The mycobacterial inner membrane displayed the highest partitioning of Rifabutin, which was located exclusively in the lipid head group/interfacial region. In contrast, the drug exhibited specific interaction sites in the head group/interfacial and hydrophobic acyl regions within the outer membrane. Altogether, we show that the design of membrane-active agents that selectively disrupt the mycobacterial outer membrane structure can increase drug uptake and enhance intracellular drug concentrations. Exploiting the mycobacterium-specific membrane-drug interaction profiles, chemotypes consisting of outer membrane-disruptive agents and antitubercular drugs can offer new opportunities for combinational tuberculosis (TB) therapy.
ABSTRACT
As an adaptive survival response to exogenous stress, bacteria undergo dynamic remodelling of their lipid metabolism pathways to alter the composition of their cellular membranes. Here, using Escherichia coli as a well characterised model system, we report the development and application of a 'multi-omics' strategy for comprehensive quantitative analysis of the temporal changes in the lipidome and proteome profiles that occur under exponential growth phase versus stationary growth phase conditions i.e., nutrient depletion stress. Lipidome analysis performed using 'shotgun' direct infusion-based ultra-high resolution accurate mass spectrometry revealed a quantitative decrease in total lipid content under stationary growth phase conditions, along with a significant increase in the mol% composition of total cardiolipin, and an increase in 'odd-numbered' acyl-chain length containing glycerophospholipids. The inclusion of field asymmetry ion mobility spectrometry was shown to enable the enrichment and improved depth of coverage of low-abundance cardiolipins, while ultraviolet photodissociation-tandem mass spectrometry facilitated more complete lipid structural characterisation compared with conventional collision-induced dissociation, including unambiguous assignment of the odd-numbered acyl-chains as containing cyclopropyl modifications. Proteome analysis using data-dependent acquisition nano-liquid chromatography mass spectrometry and tandem mass spectrometry analysis identified 83% of the predicted E. coli lipid metabolism enzymes, which enabled the temporal dependence associated with the expression of key enzymes responsible for the observed adaptive lipid metabolism to be determined, including those involved in phospholipid metabolism (e.g., ClsB and Cfa), fatty acid synthesis (e.g., FabH) and degradation (e.g., FadA/B,D,E,I,J and M), and proteins involved in the oxidative stress response resulting from the generation of reactive oxygen species during ß-oxidation or lipid degradation.
ABSTRACT
A synthetic strategy for conjugating small molecules and peptide-based therapeutics, via a cleavable ester bond, to a lipidated ß3-tripeptide is presented. The drug-loaded ß3-peptide was successfully co-assembled with a functionally inert lipidated ß3-tripeptide to form a hydrogel. Quantitative release of lactose from the hydrogel, by the action of serum esterases, is demonstrated over 28 days. The esterase-mediated sustained release of the bioactive brain-derived neurotrophic factor (BDNF) peptide mimics from the hydrogel resulted in increased neuronal survival and normal neuronal function of peripheral neurons. These studies define a versatile strategy for the facile synthesis and co-assembly of self-assembling ß3-peptide-based hydrogels with the ability to control drug release using endogenous esterases with potential in vivo applications for sustained localized drug delivery.
Subject(s)
Esterases/metabolism , Hydrogels/pharmacology , Neurons/drug effects , Peptides/pharmacology , Animals , Cell Survival/drug effects , Cells, Cultured , Drug Liberation , Esterases/blood , Female , Hydrogels/chemistry , Hydrogels/metabolism , Materials Testing , Molecular Structure , Neurons/metabolism , Peptides/chemistry , Peptides/metabolism , Pregnancy , Rats , Rats, Sprague-DawleyABSTRACT
Parkinson's disease (PD) is a progressive neurological disorder, in which dopaminergic midbrain neurons degenerate, leading to dopamine depletion that is associated with neuronal death. In this Review, we initially describe the pathogenesis of PD and established therapies that unfortunately only delay progression of the disease. With a rapidly escalating incidence in PD, there is an urgent need to develop new therapies that not only halt progression but even reverse degeneration. Biomaterials are playing critical roles in these new therapies which include controlled and site-specific delivery of neurotrophins, increased engraftment of implanted neural stem cells, and redirection of endogenous stem cell populations away from their niche to encourage reparative mechanisms. This Review will therefore cover important design features of biomaterials used in regenerative medicine and tissue engineering strategies targeted at PD.
Subject(s)
Neural Stem Cells , Parkinson Disease , Biocompatible Materials , Dopamine , Dopaminergic Neurons , Humans , Parkinson Disease/therapyABSTRACT
The impact of maculatin 1.1 (Mac1) on the mechanical properties of supported lipid membranes derived from exponential growth phase (EGP) and stationary growth phase (SGP) E. coli lipid extracts was analysed by surface plasmon resonance and atomic force microscopy. Each membrane was analysed by quantitative nanomechanical mapping to derive measurements of the modulus, adhesion and deformation in addition to bilayer height. Image analysis revealed the presence of two domains in the EGP membrane differing in height by 0.4 nm. Three distinct domains were observed in the SGP membrane corresponding to 4.2, 4.7 and 5.4 nm in height. Using surface plasmon resonance, Mac1 was observed to bind strongly to both membranes and then disrupt the membranes as evidenced by a sharp drop in baseline. Atomic force microscopy (AFM) topographic analysis revealed the formation of domains of different height and confirmed that membrane destruction was much faster for the SGP derived bilayer. Moreover, Mac1 selectively disrupted the domain with the lowest thickness, which may correspond to a liquid ordered domain. Overall, the results provide insight into the role of lipid domains in the response of bacteria to antimicrobial peptides.
Subject(s)
Escherichia coli , Peptides , Anti-Bacterial Agents/pharmacology , Lipid Bilayers , Lipids , Microscopy, Atomic ForceABSTRACT
Peptide self-assembly has been exploited to generate a multitude of biomaterials that exhibit biocompatibility due to their similarity to naturally occurring proteins. Previously, we have shown that ß-tripeptides self-assemble despite containing sterically bulky, functional sidechains. Herein, we describe the synthesis of a novel ß-amino acid to allow for the synthesis of a trifunctional ß-tripeptide that remarkably maintains self-assembly and acts as a bioactive neuronal scaffold. These scaffolds show promise for studies involving neuronal cell growth and development.
Subject(s)
Neurons/chemistry , Oligopeptides/chemistry , Tissue Scaffolds/chemistry , Cell ProliferationABSTRACT
The Renin-Angiotensin System (RAS) plays a crucial role in numerous pathological conditions. Two of the critical RAS players, the angiotensin receptors AT1R and AT2R, possess differential functional profiles, although they share high sequence similarity. Although the main focus has been placed on AT1R, several epidemiological studies have evidenced that activation of AT2R could operate as a multimodal therapeutic target for different diseases. Thus, the development of selective AT2R ligands could have a high clinical potential for different therapeutic directions. Furthermore, they could serve as a powerful tool to interrogate the molecular mechanisms that are mediated by AT2R. Based on our recently established high affinity and AT2R selective compound [Y]6-AII we developed several analogues through modifying aminoacids located at positions 6 and 7 with various conformationally constrained analogues to enhance both the selectivity and stability. We report the development of high-affinity AT2R binders, which displayed high selectivity for AT2R versus AT1R. Furthermore, all analogues presented enhanced stability in human plasma with respect to the parent hormone Angiotensin II as also [Y]6-AII.
Subject(s)
Angiotensin II/pharmacology , Receptor, Angiotensin, Type 2/metabolism , Angiotensin II/analogs & derivatives , Angiotensin II/chemistry , Dose-Response Relationship, Drug , Humans , Molecular Conformation , Proteolysis/drug effects , Structure-Activity RelationshipABSTRACT
The novel RNA virus, severe acute respiratory syndrome coronavirus II (SARS-CoV-2), is currently the leading cause of mortality in 2020, having led to over 1.6 million deaths and infecting over 75 million people worldwide by December 2020. While vaccination has started and several clinical trials for a number of vaccines are currently underway, there is a pressing need for a cure for those already infected with the virus. Of particular interest in the design of anti-SARS-CoV-2 therapeutics is the human protein angiotensin converting enzyme II (ACE2) to which this virus adheres before entry into the host cell. The SARS-CoV-2 virion binds to cell-surface bound ACE2 via interactions of the spike protein (s-protein) on the viral surface with ACE2. In this paper, we use all-atom molecular dynamics simulations and binding enthalpy calculations to determine the effect that a bound ACE2 active site inhibitor (MLN-4760) would have on the binding affinity of SARS-CoV-2 s-protein with ACE2. Our analysis indicates that the binding enthalpy could be reduced for s-protein adherence to the active site inhibitor-bound ACE2 protein by as much as 1.48-fold as an upper limit. This weakening of binding strength was observed to be due to the destabilization of the interactions between ACE2 residues Glu-35, Glu-37, Tyr-83, Lys-353, and Arg-393 and the SARS-CoV-2 s-protein receptor binding domain (RBD). The conformational changes were shown to lead to weakening of ACE2 interactions with SARS-CoV-2 s-protein, therefore reducing s-protein binding strength. Further, we observed increased conformational lability of the N-terminal helix and a conformational shift of a significant portion of the ACE2 motifs involved in s-protein binding, which may affect the kinetics of the s-protein binding when the small molecule inhibitor is bound to the ACE2 active site. These observations suggest potential new ways for interfering with the SARS-CoV-2 adhesion by modulating ACE2 conformation through distal active site inhibitor binding.
Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Protease Inhibitors/metabolism , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/antagonists & inhibitors , Binding Sites , COVID-19/pathology , COVID-19/virology , Catalytic Domain , Drug Design , Humans , Hydrogen Bonding , Molecular Dynamics Simulation , Protease Inhibitors/chemistry , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Tertiary , SARS-CoV-2/isolation & purification , Small Molecule Libraries/chemistry , Small Molecule Libraries/metabolism , Spike Glycoprotein, Coronavirus/chemistry , ThermodynamicsABSTRACT
The increasing resistance of pathogenic microbes to antimicrobials and the shortage of antibiotic drug discovery programs threaten the clinical use of antibiotics. This threat calls for the development of new methods for control of drug-resistant microbial pathogens. We have designed, synthesised and characterised an antimicrobial material formed via the self-assembly of a population of two distinct ß-peptide monomers, a lipidated tri-ß-peptide (ß3-peptide) and a novel ß3-peptide conjugated to a glycopeptide antibiotic, vancomycin. The combination of these two building blocks resulted in fibrous assemblies with distinctive structures determined by atomic force microscopy and electron microscopy. These fibres inhibited the growth of methicillin resistant Staphylococcus aureus (MRSA) and associated directly with the bacteria, acting as a peptide nanonet with fibre nucleation sites on the bacteria observed by electron microscopy and confocal microscopy. Our results provide insights into the design of peptide based supramolecular assemblies with antibacterial activity and establish an innovative strategy to develop self-assembled antimicrobial materials for future biomedical application.