L-4-thiazolylalanine (Protinol), a novel non-proteinogenic amino acid, demonstrates epidermal and dermal efficacy with clinically observable benefits.

OBJECTIVE: Facial skin undergoes major structural and functional changes as a result of intrinsic and extrinsic factors. The goal of the current work is to demonstrate L-4-thiazolylalaine (L4, Protinol), a non-proteinogenic amino acid shown to stimulate the production of dermal proteins by fibroblasts, is an alternative efficacious topical ingredient for visible signs of ageing. METHODS: In vitro studies using 3D human skin tissue models were performed to show changes in protein and gene expression of key dermal markers in samples treated with 0.3% L4 compared to vehicle control. In vivo evaluation of skin turnover was measured in volunteers after treatment with L4 compared to retinol. Skin biopsies (n = 30) were taken to investigate epidermal and dermal changes in cases treated with L4 and compared to retinol. Finally, a clinical evaluation (n = 28) was conducted to assess the efficacy of L4 over a base formulation using various ageing parameters within a population of women 46-66 years old with mild-to-moderate wrinkles. RESULTS: In vitro studies on 3D tissues displayed significant changes in the dermal matrix via an increase in HA and pro-collagen I production and a decrease in the expression of inflammatory genes. In vivo biopsy studies demonstrated that L4 and retinol independently increased epidermal thickness and collagen remodelling significantly more compared with the base formula. Clinical evaluation showed firmer and smoother skin at day 28 post-treatment with L4 over the vehicle control without causing side effects such as redness or irritation. CONCLUSION: L4 is a novel, multi-functional ingredient which offers a superior alternative to currently available technologies for improving epidermal and dermal parameters that change during ageing and photodamage.

OBJECTIF: La peau du visage est sujet à des changements majeurs structuraux et fonctionnels dus à des facteurs intrinsèques et extrinsèques. Dans cette étude, nous montrons que l'acide aminé non-protéinogène L-4-thiazolylalanine (L4, Protinol) est une alternative intéressante pour une application topique. MÉTHODES: Des modèles 3D de peaux ont été utilisés pour mesurer les changements d'expressions géniques et protéiques de marqueurs clés du derme à partir d'échantillons traités avec L4 comparés à une condition contrôle. In vivo, après un traitement L4, le renouvellement cutané a été mesuré chez les volontaires et comparé à un traitement au rétinol. Des biopsies de peaux (n = 30) traitées soit à L4 soit au rétinol ont été isolées afin d'évaluer les changements au niveau du derme et de l'épiderme. Pour finir, une étude clinique (n = 28) a été menée pour évaluer l'efficacité de L4 par rapport à une formulation de base en utilisant différents paramètres de vieillissement au sein d'une population de femmes de 46 à 66 ans présentant des rides légères à modérées. RÉSULTATS: Les études in vitro sur tissues 3D ont montré des changements dans la matrice du derme avec une augmentation de la production d'acide hyaluronique et de procollagène I et une diminution d'expression de gènes pro-inflammatoires. Les études menées in vivo sur biopsies ont démontré que L4 et rétinol augmentaient indépendamment tous deux significativement l'épaisseur de l'épiderme et le remodelage du collagène par rapport à leur base seule. Pour finir, une peau plus ferme et plus lisse a été mesurée cliniquement après 28 jours de traitement L4 par rapport au véhicule et cela sans effets indésirables tels que rougeur et irritation. CONCLUSION: L4 est un ingrédient, innovant et multifonctionnel. Il offre une sérieuse alternative aux technologies actuellement disponibles dans les traitements contre le vieillissement de la peau ou le photodommage.

Development of delivery carriers for plasmid DNA by conjugation of a helical template to oligoarginine.

Arginine (Arg)-rich peptides can penetrate the cell membrane and deliver nucleic acid-based therapeutics into cells. In this study, a helical template designed with a repeating sequence composed of two l-leucines (l-Leu) and a 2-aminoisobutyric acid (Aib) (l-Leu-l-Leu-Aib) was conjugated to nona-arginine on either the C- or N- terminus, designated as Block 1 and Block 2. Each terminal modification induced helical structure formation and improved the physicochemical properties of peptide/plasmid DNA (pDNA) complexes, resulting in efficient intracellular pDNA delivery. The introduction of a helical template may be effective for the endosomal escape of pDNA and pDNA release from complexes in cells. These results emphasized the potency of a helical template for the development of novel cell-penetrating peptides for pDNA delivery.

Effects of Substituting Disubstituted Amino Acids into the Amphipathic Cell Penetrating Peptide Pep-1.

Amphipathic cell-penetrating peptides based on the pep-1 sequence were synthesized by replacing the three hydrophilic glutamic acid residues with disubstituted, non-proteinogenic, hydrophobic amino acids. These substitutions facilitated maintenance of the peptides' secondary structure in a helical conformation, even in aqueous solution. Stability against enzymatic degradation was improved through the use of disubstituted amino acids. The resultant peptides exhibited high membrane permeability that remained relatively stable during prolonged incubation times. The results of this study indicate that the use of non-proteinogenic amino acids may be an effective approach to improve the cell membrane permeability for existing amphiphilic peptides.

5-Amino-3-methyl-Isoxazole-4-carboxylic Acid as a Novel Unnatural Amino Acid in the Solid Phase Synthesis of α/ß-Mixed Peptides.

The hybrid peptides consisting of α and ß-amino acids show great promise as peptidomimetics that can be used as therapeutic agents. Therefore, the development of new unnatural amino acids and the methods of their incorporation into the peptide chain is an important task. Here, we described our investigation of the possibility of 5-amino-3-methyl-isoxazole-4-carboxylic acid (AMIA) application in the solid phase peptide synthesis. This new unnatural ß-amino acid, presenting various biological activities, was successfully coupled to a resin-bound peptide using different reaction conditions, including classical and ultrasonic agitated solid-phase synthesis. All the synthesized compounds were characterized by tandem mass spectrometry. The obtained results present the possibility of the application of this ß-amino acid in the synthesis of a new class of bioactive peptides.

Discovery of non-proteinogenic amino acids inhibiting biofilm formation by S. aureus and methicillin-resistant S. aureus.

Bacterial biofilms often cause medical complications and there has been a great deal of interest in the discovery of small-molecule agents that can inhibit the formation of biofilms. Among these agents, it has been reported that several d-amino acids, such as d-Leu, d-Trp, d-Tyr, and d-Met, exhibit weak inhibitory activity toward bacterial biofilm formation. In this study, we have screened a library of 332 non-proteinogenic amino acids for new biofilm inhibitory agents and discovered several compounds exhibiting biofilm-inhibitory activity against Gram-positive bacteria. In particular, H-DL-ß-(3,4-dihydroxyphenyl)-dl-Ser-OH (253) showed potent activity against S. aureus, including methicillin-resistant S. aureus.

Impact of non-proteinogenic amino acids in the discovery and development of peptide therapeutics.

With the development of modern chemistry and biology, non-proteinogenic amino acids (NPAAs) have become a powerful tool for developing peptide-based drug candidates. Drug-like properties of peptidic medicines, due to the smaller size and simpler structure compared to large proteins, can be changed fundamentally by introducing NPAAs in its sequence. While peptides composed of natural amino acids can be used as drug candidates, the majority have shown to be less stable in biological conditions. The impact of NPAA incorporation can be extremely beneficial in improving the stability, potency, permeability, and bioavailability of peptide-based therapies. Conversely, undesired effects such as toxicity or immunogenicity should also be considered. The impact of NPAAs in the development of peptide-based therapeutics is reviewed in this article. Further, numerous examples of peptides containing NPAAs are presented to highlight the ongoing development in peptide-based therapeutics.

De Novo Design of Cell-Penetrating Foldamers.

Cell-penetrating peptides (CPPs) have gained much attention as carriers of hydrophilic molecules, such as drugs, peptides, and nucleic acids, into cells. CPPs are mainly composed of cationic amino acid residues, which play an important role in their intracellular uptake via interactions with acidic groups on cell surfaces. In addition, the secondary structures of CPPs also affect their cell-membrane permeability. Based on this knowledge, a variety of cell-penetrating foldamers (oligomers that form organized secondary structures) have been developed to date. In this account, we describe recent attempts to develop cell-penetrating foldamers containing various building blocks, and their application as DDS carriers.

An assay for DNA polymerase ß lyase inhibitors that engage the catalytic nucleophile for binding.

DNA polymerase ß (Pol ß) repairs cellular DNA damage. When such damage is inflicted upon the DNA in tumor cells treated with DNA targeted antitumor agents, Pol ß thus diminishes their efficacy. Accordingly, this enzyme has long been a target for antitumor therapy. Although numerous inhibitors of the lyase activity of the enzyme have been reported, none has yet proven adequate for development as a therapeutic agent. In the present study, we developed a new strategy to identify lyase inhibitors that critically engage the lyase active site primary nucleophile Lys72 as part of the binding interface. This involves a parallel evaluation of the effect of the inhibitors on the wild-type DNA polymerase ß (Pol ß) and Pol ß modified with a lysine analogue at position 72. A model panel of five structurally diverse lyase inhibitors identified in our previous studies (only one of which has been published) with unknown modes of binding were used for testing, and one compound, cis-9,10-epoxyoctadecanoic acid, was found to have the desired characteristics. This finding was further corroborated by in silico docking, demonstrating that the predominant mode of binding of the inhibitor involves an important electrostatic interaction between the oxygen atom of the epoxy group and Nε of the main catalytic nucleophile, Lys72. The strategy, which is designed to identify compounds that engage certain structural elements of the target enzyme, could find broader application for identification of ligands with predetermined sites of binding.

tRNA engineering for manipulating genetic code.

In ribosomal translation, only 20 kinds of proteinogenic amino acids (pAAs), namely 19 l-amino acids and glycine, are exclusively incorporated into polypeptide chain. To overcome this limitation, various methods to introduce non-proteinogenic amino acids (npAAs) other than the 20 pAAs have been developed to date. However, the repertoire of amino acids that can be simultaneously introduced is still limited. Moreover, the efficiency of npAA incorporation is not always sufficient depending on their structures. Fidelity of translation is sometimes low due to misincorporation of competing pAAs and/or undesired translation termination. Here, we provide an overview of efforts to solve these issues, focusing on the engineering of tRNAs.

Efficient synthesis of both diastereomers of ß,γ-diamino acids from phenylalanine and tryptophan.

We synthesized in a few steps both diastereomers of orthogonally protected ß,γ-diamino acids starting from L-phenylalanine or L-tryptophan. These final compounds are interesting building blocks for peptide synthesis and foldamer chemistry. The key step is a Blaise reaction performed under ultrasound conditions.

Crystal structure of anhydrous poly[bis-(µ2-sarcosinato-κ(3) O,N:O')copper(II)].

The title compound, [Cu(C3H6NO2)2] n , is a bis-complex of the anion of sarcosine (N-methyl-glycine). The asymmetric unit consists of a copper(II) ion, located on a center of inversion, and one mol-ecule of the uninegative sarcosinate anion. The copper(II) ion exhibits a typical Jahn-Teller distorted [4 + 2] coordination geometry. The four shorter equatorial bonds are to the nitro-gen and carboxyl-ate O atoms of two sarcosinate anions, and the longer axial bonds are to carboxyl-ate O atoms of neighboring complexes. The overall structure is made up from two chains formed by these longer axial Cu-O bonds, one extending parallel to [011] and the other parallel to [0-11]. Each one-dimensional array is connected by the equatorial bridging moieties to the chains on either side, creating an extended two-dimensional framework parallel to (100). There is a single inter-molecular hydrogen-bonding inter-action within the sheets between the amino NH group and an O atom of an adjacent mol-ecule.

Synthesis of (2S,3R)-3-amino-2-hydroxydecanoic acid and its enantiomer: a non-proteinogenic amino acid segment of the linear pentapeptide microginin.

A directed manipulation of the functional groups at C3 and C4 of D-glucose was demonstrated to synthesize naturally occurring (2S,3R)-α-hydroxy-ß-aminodecanoic acid (AHDA, 2a) and its enantiomer 2b. The enantiomer of 2a is the N-terminal part of the natural linear pentapeptide microginin, which is used as an antihypertensive agent.

Plant homocysteine, a methionine precursor and plant's hallmark of metabolic disorders.

Homocysteine (Hcy) is a sulfur-containing non-proteinogenic amino acid, which arises from redox-sensitive methionine metabolism. In plants, Hcy synthesis involves both cystathionine ß-lyase and S-adenosylhomocysteine hydrolase activities. Thus, Hcy itself is crucial for de novo methionine synthesis and S-adenosylmethionine recycling, influencing the formation of ethylene, polyamines, and nicotianamine. Research on mammalian cells has shown biotoxicity of this amino acid, as Hcy accumulation triggers oxidative stress and the associated lipid peroxidation process. In addition, the presence of highly reactive groups induces Hcy and Hcy derivatives to modify proteins by changing their structure and function. Currently, Hcy is recognized as a critical, independent hallmark of many degenerative metabolic diseases. Research results indicate that an enhanced Hcy level is also toxic to yeast and bacteria cells. In contrast, in the case of plants the metabolic status of Hcy remains poorly examined and understood. However, the presence of the toxic Hcy metabolites and Hcy over-accumulation during the development of an infectious disease seem to suggest harmful effects of this amino acid also in plant cells. The review highlights potential implications of Hcy metabolism in plant physiological disorders caused by environmental stresses. Moreover, recent research advances emphasize that recognizing the Hcy mode of action in various plant systems facilitates verification of the potential status of Hcy metabolites as bioindicators of metabolism disorders and thus may constitute an element of broadly understood biomonitoring.

A 1-aminocyclopropane-1-carboxylic-acid (ACC) dipeptide elicits ethylene responses through ACC-oxidase mediated substrate promiscuity.

Plants produce the volatile hormone ethylene to regulate many developmental processes and to deal with (a)biotic stressors. In seed plants, ethylene is synthesized from 1-aminocyclopropane-1-carboxylic acid (ACC) by the dedicated enzyme ACC oxidase (ACO). Ethylene biosynthesis is tightly regulated at the level of ACC through ACC synthesis, conjugation and transport. ACC is a non-proteinogenic amino acid, which also has signaling roles independent from ethylene. In this work, we investigated the biological function of an uncharacterized ACC dipeptide. The custom-synthesized di-ACC molecule can be taken up by Arabidopsis in a similar way as ACC, in part via Lysine Histidine Transporters (e.g., LHT1). Using Nano-Particle Assisted Laser Desoprtion/Ionization (Nano-PALDI) mass-spectrometry imaging, we revealed that externally fed di-ACC predominantly localizes to the vasculature tissue, despite it not being detectable in control hypocotyl segments. Once taken up, the ACC dimer can evoke a triple response phenotype in dark-grown seedlings, reminiscent of ethylene responses induced by ACC itself, albeit less efficiently compared to ACC. Di-ACC does not act via ACC-signaling, but operates via the known ethylene signaling pathway. In vitro ACO activity and molecular docking showed that di-ACC can be used as an alternative substrate by ACO to form ethylene. The promiscuous nature of ACO for the ACC dimer also explains the higher ethylene production rates observed in planta, although this reaction occurred less efficiently compared to ACC. Overall, the ACC dipeptide seems to be transported and converted into ethylene in a similar way as ACC, and is able to augment ethylene production levels and induce subsequent ethylene responses in Arabidopsis.

Methodologies for Backbone Macrocyclic Peptide Synthesis Compatible With Screening Technologies.

Backbone macrocyclic structures are often found in diverse bioactive peptides and contribute to greater conformational rigidity, peptidase resistance, and potential membrane permeability compared to their linear counterparts. Therefore, such peptide scaffolds are an attractive platform for drug-discovery endeavors. Recent advances in synthetic methods for backbone macrocyclic peptides have enabled the discovery of novel peptide drug candidates against diverse targets. Here, we overview recent technical advancements in the synthetic methods including 1) enzymatic synthesis, 2) chemical synthesis, 3) split-intein circular ligation of peptides and proteins (SICLOPPS), and 4) in vitro translation system combined with genetic code reprogramming. We also discuss screening methodologies compatible with those synthetic methodologies, such as one-beads one-compound (OBOC) screening compatible with the synthetic method 2, cell-based assay compatible with 3, limiting-dilution PCR and mRNA display compatible with 4.

Cytotoxicity and mitochondrial dysfunction caused by the dietary supplement l-norvaline.

In addition to the 20 protein amino acids that are encoded for protein synthesis, hundreds of other naturally occurring amino acids, known as non-proteinogenic amino acids (NPAAs) exist. It is well known that some NPAAs are toxic through their ability to mimic protein amino acids, either in protein synthesis or in other metabolic pathways, and this property is utilised by some plants to inhibit the growth of other plants or kill herbivores. L-norvaline is an NPAA readily available for purchase as a dietary supplement. In light of previous evidence of l-norvaline's antifungal, antimicrobial and herbicidal activity, we examined the toxicity of l-norvaline to mammalian cells in vitro and showed that l-norvaline decreased cell viability at concentrations as low as 125 µM, caused necrotic cell death and significant changes to mitochondrial morphology and function. Furthermore, toxicity was reduced in the presence of structurally similar 'protein' amino acids, suggesting l-norvaline's cytotoxicity could be attributed to protein amino acid mimicry.

Characterization of the phenylglycine aminotransferase PglE from Streptomyces pristinaespiralis.

l-phenylglycine is a rare non-proteinogenic amino acid, which only occurs in a few natural compounds, such as the streptogramin antibiotics pristinamycin I and virginiamycin S or the bicyclic peptide antibiotic dityromycin. Here we report on the biochemical characterization of the aminotransferase PglE that catalyzes the transamination from phenylglyoxylate to l-phenylglycine, which represents the final reaction step during phenylglycine biosynthesis. Enzyme assays with the purified PglE enzyme revealed that l-phenylalanine is used as an amino group donor for the transamination reaction, leading to the formation of phenylpyruvate, which may re-enter phenylglycine biosynthesis as a precursor. Based on these results, we postulate a novel l-phenylglycine biosynthetic pathway.

Advances in in vitro genetic code reprogramming in 2014-2017.

To date, various genetic code manipulation methods have been developed to introduce non-proteinogenic amino acids into peptides by translation. However, the number of amino acids that can be used simultaneously remains limited even using these methods. Additionally, the scope of amino acid substrates that are compatible with ribosomal translation systems is also limited. For example, difficult substrates such as d-amino acids and ß-amino acids are much less efficiently incorporated into peptides than l-α-amino acids. Here, we focus on three recently developed methodologies that address these issues: (i) artificial division of codon boxes to increase the number of available amino acids, (ii) orthogonal ribosomal translation systems to 'duplicate' the codon table and (iii) development of novel artificial tRNAs that enhance incorporation of difficult amino acid substrates.