The Novel AT2 Receptor Agonist ß-Pro7-AngIII Exerts Cardiac and Renal Anti-Fibrotic and Anti-Inflammatory Effects in High Salt-Fed Mice.

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.

Using conformational constraints at position 6 of Angiotensin II to generate compounds with enhanced AT2R selectivity and proteolytic stability.

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.

Single Peptide Backbone Surrogate Mutations to Regulate Angiotensin GPCR Subtype Selectivity.

Mutating the side-chains of amino acids in a peptide ligand, with unnatural amino acids, aiming to mitigate its short half-life is an established approach. However, it is hypothesized that mutating specific backbone peptide bonds with bioisosters can be exploited not only to enhance the proteolytic stability of parent peptides, but also to tune its receptor subtype selectivity. Towards this end, four [Y]6 -Angiotensin II analogues are synthesized where amide bonds have been replaced by 1,4-disubstituted 1,2,3-triazole isosteres in four different backbone locations. All the analogues possessed enhanced stability in human plasma in comparison with the parent peptide, whereas only two of them achieved enhanced AT2 R/AT1 R subtype selectivity. This diversification has been studied through 2D NMR spectroscopy and unveiled a putative more structured microenvironment for the two selective ligands accompanied with increased number of NOE cross-peaks. The most potent analogue, compound 2, has been explored regarding its neurotrophic potential and resulted in an enhanced neurite growth with respect to the established agent C21.

Renal functional effects of the highly selective AT2R agonist, ß-Pro7 Ang III, in normotensive rats.

Recently, we designed a group of peptides by sequential substitution of the naturally occurring α-amino acid throughout the Ang III peptide sequence with the corresponding ß-amino acid. ß-Amino acid substitution at the proline residue of Ang III (ß-Pro7-Ang III) resulted in a highly selective AT2R ligand, demonstrating remarkable selectivity for the AT2R in both binding and functional studies. To provide additional functional evidence for the suitability of ß-Pro7 Ang III as a novel AT2R agonist, we tested effects of acute systemic administration of ß-Pro7-Ang III on renal hemodynamic and excretory function in anesthetized normotensive male and female rats. We also compared the natriuretic effects of acute intrarenal administration of native Ang III and ß-Pro7-Ang III in the presence of systemic AT1R blockade in anesthetized female rats to allow for the differentiation of systemic versus direct intrarenal natriuretic actions of ß-Pro7-Ang III. In both male and female rats, acute systemic administration of ß-Pro7-Ang III elicited renal vasodilatation and natriuresis. Notably, greater renal vasodilatory effects were observed in female versus male rats at the highest dose of ß-Pro7-Ang III administered. Moreover, intra-renal administration of ß-Pro7-Ang III produced significant natriuretic effects in female rats and, like Ang III, evoked AT2R translocation to the apical plasma membrane in renal proximal tubular cells. Taken together, our findings support the use of ß-Pro7-Ang III as a novel AT2R agonist and experimental tool for exploring AT2R function and its potential as a therapeutic target. Furthermore, our findings provide further evidence of a sex-specific influence of AT2R stimulation on renal function.

Exploring Molecular-Biomembrane Interactions with Surface Plasmon Resonance and Dual Polarization Interferometry Technology: Expanding the Spotlight onto Biomembrane Structure.

The molecular analysis of biomolecular-membrane interactions is central to understanding most cellular systems but has emerged as a complex technical challenge given the complexities of membrane structure and composition across all living cells. We present a review of the application of surface plasmon resonance and dual polarization interferometry-based biosensors to the study of biomembrane-based systems using both planar mono- or bilayers or liposomes. We first describe the optical principals and instrumentation of surface plasmon resonance, including both linear and extraordinary transmission modes and dual polarization interferometry. We then describe the wide range of model membrane systems that have been developed for deposition on the chips surfaces that include planar, polymer cushioned, tethered bilayers, and liposomes. This is followed by a description of the different chemical immobilization or physisorption techniques. The application of this broad range of engineered membrane surfaces to biomolecular-membrane interactions is then overviewed and how the information obtained using these techniques enhance our molecular understanding of membrane-mediated peptide and protein function. We first discuss experiments where SPR alone has been used to characterize membrane binding and describe how these studies yielded novel insight into the molecular events associated with membrane interactions and how they provided a significant impetus to more recent studies that focus on coincident membrane structure changes during binding of peptides and proteins. We then discuss the emerging limitations of not monitoring the effects on membrane structure and how SPR data can be combined with DPI to provide significant new information on how a membrane responds to the binding of peptides and proteins.

The use of hydrogels for cell-based treatment of chronic kidney disease.

Chronic kidney disease (CKD) is a major and growing public health concern with increasing incidence and prevalence worldwide. The therapeutic potential of stem cell therapy, including mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) holds great promise for treatment of CKD. However, there are significant bottlenecks in the clinical translation due to the reduced number of transplanted cells and the duration of their presence at the site of tissue damage. Bioengineered hydrogels may provide a route of cell delivery to enhance treatment efficacy and optimise the targeting effectiveness while minimising any loss of cell function. In this review, we highlight the advances in stem cell therapy targeting kidney disease and discuss the emerging role of hydrogel delivery systems to fully realise the potential of adult stem cells as a regenerative therapy for CKD in humans. MSCs and EPCs mediate kidney repair through distinct paracrine effects. As a delivery system, hydrogels can prolong these paracrine effects by improving retention at the site of injury and protecting the transplanted cells from the harsh inflammatory microenvironment. We also discuss the features of a hydrogel, which may be tuned to optimise the therapeutic potential of encapsulated stem cells, including cell-adhesive epitopes, material stiffness, nanotopography, modes of gelation and degradation and the inclusion of bioactive molecules. This review concludes with a discussion of the challenges to be met for the widespread clinical use of hydrogel delivery system of stem cell therapy for CKD.

Self-assembled nanomaterials based on beta (ß(3)) tetrapeptides.

ß(3)-amino acid based polypeptides offer a unique starting material for the design of self-assembled nanostructures such as fibres and hierarchical dendritic assemblies, due to their well-defined helical geometry in which the peptide side chains align at 120° due to the 3.0-3.1 residue pitch of the helix. In a previous work we have described the head-to-tail self-assembly of N-terminal acetylated ß(3)-peptides into infinite helical nanorods that was achieved by designing a bioinspired supramolecular self-assembly motif. Here we describe the effect of consecutively more polar side chains on the self-assembly characteristics of ß(3)-tetrapeptides Ac-ß (3)Ala-ß(3)Leu-ß(3)Ile-ß(3)Ala (Ac-ß(3)[ALIA]), Ac-ß(3)Ser-ß(3)Leu-ß(3)Ile-ß(3)Ala (Ac-ß(3)[SLIA]) and Ac-ß (3)Lys-ß (3)Leu-ß(3)Ile-ß (3)Glu (Ac-ß(3)[KLIE]). ß(3)-tetrapeptides complete 1 1/3 turns of the helix: thus in the oligomeric form the side chain positions shift 120° with each added monomer, forming a regular periodic pattern along the nanorod. Dynamic light scattering (DLS) measurements confirmed that these peptides self-assemble even in highly polar solvents such as water and DMSO, while diffusion-ordered NMR spectroscopy revealed the presence of a substantial monomeric population. Temperature dependence of the size distribution in DLS measurements suggests a dynamic equilibrium between monomers and oligomers. Solution casting produced distinct fibrillar deposits after evaporating the solvent. In the case of the apolar Ac-ß(3)[ALIA] the longitudinal helix morphology gives rise to geometrically defined (∼70°) junctions between fibres, forming a mesh that opens up possibilities for applications e.g. in tissue scaffolding. The deposits of polar Ac-ß(3)[SLIA] and Ac-ß(3)[KLIE] exhibit fibres in regular parallel alignment over surface areas in the order of 10 µm.

ß-Pro7Ang III is a novel highly selective angiotensin II type 2 receptor (AT2R) agonist, which acts as a vasodepressor agent via the AT2R in conscious spontaneously hypertensive rats.

We have previously shown that individual ß-amino acid substitution in angiotensin (Ang) II reduced Ang II type 1 receptor (AT1R) but not Ang II type 2 receptor (AT2R)-binding and that the heptapeptide Ang III exhibited greater AT2R:AT1R selectivity than Ang II. Therefore, we hypothesized that ß-amino-acid-substituted Ang III peptide analogues would yield highly selective AT2R ligands, which we have tested in binding and functional vascular assays. In competition binding experiments using either AT1R- or AT2R-transfected human embryonic kidney (HEK)-293 cells, novel ß-substituted Ang III analogues lacked appreciable AT1R affinity, whereas most compounds could fully displace (125)I-Sar(1)Ile(8) Ang II from AT2R. The rank order of affinity at AT2R was CGP42112 > Ang III > ß-Pro(7) Ang III=Ang II > ß-Tyr(4) Ang III ≥ PD123319 >> ß-Phe(8) Ang III >> ß Arg(2) Ang III=ß-Val(3) Ang III >> ß-Ile(5) Ang III. The novel analogue ß-Pro(7) Ang III was the most selective AT2R ligand tested, which was >20,000-fold more selective for AT2R than AT1R. IC50 values at AT2R from binding studies correlated with maximum vasorelaxation in mouse aortic rings. Given that ß-Pro(7) Ang III was an AT2R agonist, we compared ß-Pro(7) Ang III and native Ang III for their ability to reduce blood pressure in separate groups of conscious spontaneously hypertensive rats. Whereas Ang III alone increased mean arterial pressure (MAP), ß-Pro(7) Ang III had no effect. During low-level AT1R blockade, both Ang III and ß-Pro(7) Ang III, but not Ang II, lowered MAP (by ∼30 mmHg) at equimolar infusions (150 pmol/kg/min for 4 h) and these depressor effects were abolished by the co-administration of the AT2R antagonist PD123319. Thus, ß-Pro(7) Ang III has remarkable AT2R selectivity determined in binding and functional studies and will be a valuable research tool for insight into AT2R function and for future drug development.

Design and testing of bicyclic inhibitors of Grb7--are two cycles better than one?

Grb7 is an adapter protein involved in the propagation of signals in cancer cell migration and proliferation, and is thus a target for the development of novel anti-cancer agents. An 11-residue thioether-cyclized peptide known as G7-18NATE has previously been developed, that inhibits Grb7 via specific interactions with its SH2 domain with micromolar affinity. Here we explore whether the peptide binding is enhanced by the addition of a second linkage designed to restrain the peptide in its bound conformation and thus reduce the entropic loss upon binding. The use of an O-ally ser covalent linkage between residue positions 1 and 8 successfully enhanced the affinity, and ITC showed that the entropic loss was reduced. A peptide with thioether-cyclization exchanged for an amide linkage showed reduce affinity, though the formation of a disulfide bond between positions 1 and 8 in this peptide enhanced its binding. This study paves the way for improving the G7-18NATE scaffold for second generation inhibitors of Grb7.

A comparison of fixation methods for SEM analysis of self-assembling peptide hydrogel nanoarchitecture.

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.

The protective effects of a novel AT2 receptor agonist, ß-Pro7Ang III in ischemia-reperfusion kidney injury.

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.

Elucidating the cell penetrating properties of self-assembling ß-peptides.

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.

Novel AT2R agonist, ß-Pro7Ang III, is cardio- and vaso-protective in diabetic spontaneously hypertensive rats.

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.

A switch in N-terminal capping of ß-peptides creates novel self-assembled nanoparticles.

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.

Interaction of the non-phosphorylated peptide G7-18NATE with Grb7-SH2 domain requires phosphate for enhanced affinity and specificity.

Src-homology (SH2) domains are an attractive target for the inhibition of specific signalling pathways but pose the challenge of developing a truly specific inhibitor. The G7-18NATE cyclic peptide is reported to specifically inhibit the growth factor receptor bound protein 7 (Grb7) adapter protein, implicated in the progression of several cancer types, via interactions with its SH2 domain. G7-18NATE effectively inhibits the interaction of Grb7 with ErbB3 and focal adhesion kinase in cell lysates and, with the addition of a cell permeability sequence, inhibits the growth and migration of a number of breast cancer cell lines. It is thus a promising lead in the development of therapeutics targeted to Grb7. Here we investigate the degree to which G7-18NATE is specific for the Grb7-SH2 domain compared with closely related SH2 domains including those of Grb10, Grb14, and Grb2 using surface plasmon resonance. We demonstrate that G7-18NATE binds with micromolar binding affinity to Grb7-SH2 domain (K(D) = 4-6 µm) compared with 50-200 times lower affinity for Grb10, Grb14, and Grb2 but that this specificity depends critically on the presence of phosphate in millimolar concentrations. Other differences in buffer composition, including use of Tris or 2-(N-Morpholino)ethanesulfonic acid or varying the pH, do not impact on the interaction. This suggests that under cellular conditions, G7-18NATE binds with highest affinity to Grb7. In addition, our findings demonstrate that the basis of specificity of G7-18NATE binding to the Grb7-SH2 domain is via other than intrinsic structural features of the protein, representing an unexpected mode of molecular recognition.

Conformational stability studies of a stapled hexa-ß3-peptide library.

A library of 14-helical hexa ß(3)-peptides was synthesized in order to determine the influence of sequence variation as well as staple size and location on conformational stability. From this study we show that appropriately stapled hexa-ß(3)-peptides can allow for a number of variations without significant perturbation of the 14-helix.

Self-assembly of trifunctional tripeptides to form neural scaffolds.

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.

Staphylococcus aureus entanglement in self-assembling ß-peptide nanofibres decorated with vancomycin.

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.

Esterase-Mediated Sustained Release of Peptide-Based Therapeutics from a Self-Assembled Injectable Hydrogel.

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.

Transition of Nano-Architectures Through Self-Assembly of Lipidated ß3-Tripeptide Foldamers.

ß3-peptides consisting exclusively of ß3-amino acids adopt a variety of non-natural helical structures and can self-assemble into well-defined hierarchical structures by axial head-to-tail self-assembly resulting in fibrous materials of varying sizes and shapes. To allow control of fiber morphology, a lipid moiety was introduced within a tri-ß3-peptide sequence at each of the three amino acid positions and the N-terminus to gain finer control over the lateral assembly of fibers. Depending on the position of the lipid, the self-assembled structures formed either twisted ribbon-like fibers or distinctive multilaminar nanobelts. The nanobelt structures were comprised of multiple layers of peptide fibrils as revealed by puncturing the surface of the nanobelts with an AFM probe. This stacking phenomenon was completely inhibited through changes in pH, indicating that the layer stacking was mediated by electrostatic interactions. Thus, the present study is the first to show controlled self-assembly of these fibrous structures, which is governed by the location of the acyl chain in combination with the 3-point H-bonding motif. Overall, the results demonstrate that the nanostructures formed by the ß3-tripeptide foldamers can be tuned via sequential lipidation of N-acetyl ß3-tripeptides which control the lateral interactions between peptide fibrils and provide defined structures with a greater homogeneous population.