Staphylococcus aureus, a bacterium resistant to multiple drugs, is a significant cause of illness and death worldwide. Antimicrobial peptides (AMPs) provide an excellent potential strategy to cope with this threat. Recently, we characterized a derivative of the frog-skin AMP esculentin-1a, Esc(1-21) (1) that is endowed with potent activity against Gram-negative bacteria but poor efficacy against Gram-positive strains. In this study, three analogues of peptide 1 were designed by replacing Gly8 with α-aminoisobutyric acid (Aib), Pro, and dPro (2-4, respectively). The single substitution Gly8 → Aib8 in peptide 2 makes it active against the planktonic form of Gram-positive bacterial strains, especially Staphylococcus aureus, including multidrug-resistant clinical isolates, with an improved biostability without resulting in cytotoxicity to mammalian cells. Moreover, peptide 2 showed a higher antibiofilm activity than peptide 1 against both reference and clinical isolates of S. aureus. Peptide 2 was also able to induce rapid bacterial killing, suggesting a membrane-perturbing mechanism of action. Structural analysis of the most active peptide 2 evidenced that the improved biological activity of peptide 2 is the consequence of a combination of higher biostability, higher α helical content, and ability to reduce membrane fluidity and to adopt a distorted helix, bent in correspondence of Aib8. Overall, this study has shown how a strategic single amino acid substitution is sufficient to enlarge the spectrum of activity of the original peptide 1, and improve its biological properties for therapeutic purposes, thus paving the way to optimize AMPs for the development of new broad-spectrum anti-infective agents.

The fibrillation of therapeutic peptides can present significant quality concerns and poses challenges for manufacturing and storage. A fundamental understanding of the mechanisms of fibrillation is critical for the rational design of fibrillation-resistant peptide drugs and can accelerate product development by guiding the selection of solution-stable candidates and formulations. The studies reported here investigated the effects of structural modifications on the fibrillation of a 29-residue peptide (PepA) and two sequence modified variants (PepB, PepC). The C-terminus of PepA was amidated, whereas both PepB and PepC retained the carboxylate, and Ser16 in PepA and PepB was substituted with a helix-stabilizing residue, α-aminoisobutyric acid (Aib), in PepC. In thermal denaturation studies by far-UV CD spectroscopy and fibrillation kinetic studies by fluorescence and turbidity measurements, PepA and PepB showed heat-induced conformational changes and were found to form fibrils, whereas PepC did not fibrillate and showed only minor changes in the CD signal. Pulsed hydrogen-deuterium exchange mass spectrometry (HDX-MS) showed a high degree of protection from HD exchange in mature PepA fibrils and its proteolytic fragments, indicating that most of the sequence had been incorporated into the fibril structure and occurred nearly simultaneously throughout the sequence. The effects of the net peptide charge and formulation pH on fibrillation kinetics were investigated. In real-time stability studies of two formulations of PepA at pH\'s 7.4 and 8.0, analytical methods detected significant changes in the stability of the formulations at different time points during the study, which were not observed during accelerated studies. Additionally, PepA samples were withdrawn from real-time stability and subjected to additional stress (40 °C, continuous shaking) to induce fibrillation; an approach that successfully amplified oligomers or prefibrillar species previously undetected in a thioflavin T assay. Taken together, these studies present an approach to differentiate and characterize fibrillation risk in structurally related peptides under accelerated and real-time conditions, providing a model for rapid, iterative structural design to optimize the stability of therapeutic peptides.

In the genetic modification of plant cells, the mitochondrion is an important target in addition to the nucleus and plastid. However, gene delivery into the mitochondria of plant cells has yet to be established by conventional methods, such as particle bombardment, because of the small size and high mobility of mitochondria. To develop an efficient mitochondria-targeting signal (MTS) that functions in plant cells, we designed the artificial peptide (LURL)3 and its analogues, which periodically feature hydrophobic α-aminoisobutyric acid (Aib, U) and cationic arginine (R), considering the consensus motif recognized by the mitochondrial import receptor Tom20. Circular dichroism measurements and molecular dynamics simulation studies revealed that (LURL)3 had a propensity to form a stable α-helix in 0.1 M phosphate buffer solution containing 1.0 wt % sodium dodecyl sulfate. After internalization into plant cells via particle bombardment, (LURL)3 revealed highly selective accumulation in the mitochondria, whereas its analogue (LARL)3 was predominantly located in the vacuoles in addition to mitochondria. The high selectivity of (LURL)3 can be attributed to the incorporation of Aib, which promotes the hydrophobic interaction between the MTS and Tom20 by increasing the hydrophobicity and helicity of (LURL)3. The present study provided a prospective mitochondrial targeting system using the simple design of artificial peptides.

The synthetic peptide Z-Gly-Aib-Gly-Aib-Gly-Aib-OtBu was crystallized from a mixture of ethyl acetate and n-hexane. The crystals belong to the centrosymmetric space group Pbca. There are three molecules in the asymmetric unit. The three molecules differ mainly in the Z-group conformation. The first Gly residue adopts a fully extended conformation, residues 2 and 3 lie in the left-handed helical region, residues 4 and 5 in the right-handed helical region, and residue 6 again in the left-handed helical region of the Ramachandran plot. There are only two of four possible intramolecular hydrogen bonds formed, namely, between Aib4 and Gly1 forming a β-turn of type III\' and between Aib6 and Gly3 forming a β-turn of type I. The inverted molecules (by space group symmetry) lie in the regions with opposite handedness and form β-turns of type III and I\'. In contrast to all known long synthetic and naturally occurring Aib-containing peptides that fold as 310 - or α-helix, Z-(Gly-Aib)3 -OtBu folds in a quite flat structure from which only the protecting groups bulge out.

Cell-penetrating peptides (CPPs) have been widely utilized as efficient molecular tools for the delivery of bioactive cargoes such as peptides, proteins, and genetic material. However, to improve their versatility as tools in biological environments, the resistance of CPPs to enzymatic degradation and their structural stability must be improved to achieve long-term efficacy. Here we designed and synthesized novel artificial CPPs, poly(LysAibXaa), containing periodic α-aminoisobutyric acid (Aib) and l-lysine by chemoenzymatic polymerization. Poly(LysAibAla) tended to form 310- and α-helical structures under the amphiphilic cell-membrane-mimicking environment. Poly(LysAibXaa) exhibited long-term internalization and thus high accumulation in live cells, which is attributed to the improvement in the resistance to proteolytic digestion as a result of the incorporation of Aib residues into the peptide backbone. We presented a simple molecular design and synthesis of efficient CPPs applicable to both human and plant cells with long-term stability and negligible cytotoxicity.

Arginine (Arg)-rich cell-penetrating peptides (CPPs) are promising tools for plasmid DNA (pDNA) delivery. α-Aminoisobutyric acid (Aib) is known to stabilize peptide helical secondary structures and has been used as a building block for foldamers. In the current study, we prepared Aib CPP foldamers, (Arg-Arg-Aib)n (n = 1-6) and examined their pDNA transfection abilities. Transfection efficiencies of peptide/pDNA complexes are dependent on peptide chain length, with longer peptides (n ≥ 4) exhibiting better transfection abilities than an Arg nonapeptide. Furthermore, Aib CPP foldamers achieve prolonged transfection abilities compared with commercially available transfection reagents, which is probably because of the high resistance of the peptides to enzymatic degradation, thereby protecting the encapsulated pDNA for a long period. The obtained results demonstrated promising features of Aib CPP foldamers as pDNA delivery carriers.

Thiamine deficiency (TD) results in focal lesions in several regions of the rat brain including the thalamus and inferior colliculus. Since alterations in blood-brain barrier (BBB) integrity may play a role in this damage, we have examined the influence of TD on the unidirectional blood-to-brain transfer constant (Ki) of the low molecular weight species α-aminoisobutyric acid (AIB) in vulnerable and non-vulnerable brain regions at different stages during progression of the disorder, and following its reversal with thiamine. Analysis of the regional distribution of Ki values showed early (day 10) increased transfer of [14C]-AIB across the BBB in the vulnerable medial thalamus as well as the non-vulnerable caudate and hippocampus. At the acute symptomatic stage (day 14), more widespread BBB permeability changes were detected in most areas including the lateral thalamus, inferior colliculus, and non-vulnerable cerebellum and pons. Twenty-four hours following thiamine replenishment, a heterogeneous pattern of increased BBB permeability was observed in which many structures maintained increased uptake of [14C]-AIB. No increase in the [3H]-dextran space, a marker of intravascular volume, was detected in brain regions during the progress of TD, suggesting that BBB permeability to this large tracer was unaffected. These results indicate that BBB opening i) occurs early during TD, ii) is not restricted to vulnerable areas of the brain, iii) is progressive, iv) persists for at least 24 h following treatment with thiamine, and v) is likely selective in nature, depending on the molecular species being transported.

Relaxin-3 is a highly conserved two-chain neuropeptide that acts through its endogenous receptor the Relaxin Family Peptide-3 (RXFP3) receptor. The ligand/receptor system is known to modulate several physiological processes, with changes in food intake and anxiety-levels the most well studied in rodent models. Agonist and antagonist analogues based on the native two-chain peptide are costly to synthesise and not ideal drug leads. Since RXFP3 interacting residues are found in the relaxin B-chain only, this has been the focus of analogue development. The B-chain is unstructured without the A-chain support, but in single-chain variants structure can be induced by dicarba-based helical stapling strategies. Here we investigated whether alternative helical inducing strategies also can enhance structure and activity at RXFP3. Combinations of the helix inducing α-aminoisobutyric acid (Aib) were incorporated into the sequence of the relaxin-3 B-chain. Aib residues at positions 13, 17 and 18 partially reintroduce helicity and activity of the relaxin-3 B-chain, but other positions are generally not suited for modifications. We identify Thr21 as a putative new receptor contact residue important for RXFP3 binding. Cysteine residues were also incorporated into the sequence and cross-linked with dichloroacetone or α, α\'-dibromo-m-xylene. However, in contrast to previously reported dicarba variants, neither were found to promote structure and RXFP3 activity.

The peptide sequence AcNH-TEG-Glu-Aib-Trp-AibAib-Trp-AibAib-Ile-Asp-OH (1), designed to display the WWI epitope found near the C-terminus of gp41, an envelope glycoprotein decorating the surface of the HIV-1 virus, has been synthesized and proved to have a relevant content of helical conformation because of the presence of five α-aminoisobutyric acid (Aib) units. Three copies of it have been connected to a tripodal platform based on 2,4,6-triethylbenzene-1,3,5-trimethylamine. The tripodal template 2 is even more structured than 1 thus suggesting a significant interaction between the three sequences connected to the platform. Preliminary inhibition assays of HIV-mediated cell fusion indicated that while the single peptide 1 is inactive within the concentration range of our assay, when it is conjugated to the tripodal platform, it is moderately active. These promising results suggest that our approach constitute a valid alternative to those reported so far.

The role of the conformationally constrained α-aminoisobutyric acid (Aib) residue in the aggregation and self-assembly properties of oligopeptides is discussed, critically reviewing our recent work in the field. In this connection, three significant case studies are presented: (i) aggregation propensity of Aib homo-oligopeptides of different length; (ii) perturbation of the conformational and aggregation properties of Ala-based pentapeptides by a single Aib versus Ala substitution; and (iii) build up of self-assembled monolayers formed by Aib homo-hexapeptide building blocks. The peptides investigated were all functionalized by a fluorescent probe, that is, a naphthyl group in the first case-study and a pyrenyl group in the other two, with the aim at applying optical spectroscopy techniques and evaluating the relevance of aromatic interactions in the aggregation process. Microscopy techniques at nanometric resolution and results of molecular dynamics simulations are also presented to analyze how the conformational properties of the peptide building blocks would affect the morphology of the peptide aggregates from the nanoscale to the mesoscale. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.