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Design of Peptidic Foldamer Helices: A Stereochemical Patterning Approach

Assembly language: The programmed sequences of stereochemical building blocks lead to novel biomimetic helices. The rational design approach offers new possibilities for creating periodic secondary structures.

Controlled in situ preparation of Aβ(1–42) oligomers from the isopeptide “iso-Aβ(1–42)”, physicochemical and biological characterization

Beta-amyloid (A beta) peptides play a crucial role in the pathology of the neurodegeneration in Alzheimers disease (AD). Biological experiments (both in vitro and animal model studies of AD) require synthetic A beta peptides of standard quality, aggregation grade, neurotoxicity and water solubility. The synthesis of A beta peptides has been difficult, owing to their hydrophobic character, poor solubility and high tendency for aggregation. Recently an isopeptide precursor (iso-A beta(1-42)) was synthesized by Fmoc-chemistry and transformed at neutral pH to A beta(1-42) by O-->N acyl migration in a short period of time. We prepared the same precursor peptide using Boc-chemistry and studied the transformation to A beta(1-42) by acyl migration. The peptide conformation and aggregation processes were studied by several methods (circular dichroism, atomic force and transmission electron microscopy, dynamic light scattering). The biological activity of the synthetic A beta(1-42) was measured by ex vivo (long-term potentiation studies in rat hippocampal slices) and in vivo experiments (spatial learning of rats). It was proven that O-->N acyl migration of the precursor isopeptide results in a water soluble oligomeric mixture of neurotoxic A beta(1-42). These oligomers are formed in situ just before the biological experiments and their aggregation grade could be standardized.

Foldameric α/β-peptide analogs of the β-sheet-forming antiangiogenic anginex: structure and bioactivity.

The principles of β-sheet folding and design for α-peptidic sequences are well established, while those for sheet mimetics containing homologated amino acid building blocks are still under investigation. To reveal the structure-function relations of β-amino-acid-containing foldamers, we followed a top-down approach to study a series of α/β-peptidic analogs of anginex, a β-sheet-forming antiangiogenic peptide. Eight anginex analogs were developed by systematic α → β(3) substitutions and analyzed by using NMR and CD spectroscopy. The foldamers retained the β-sheet tendency, though with a decreased folding propensity. β-Sheet formation could be induced by a micellar environment, similarly to that of the parent peptide. The destructuring effect was higher when the α → β(3) exchange was located in the β-sheet core. Analysis of the β-sheet stability versus substitution pattern and the local conformational bias of the bulky β(3)V and β(3)I residues revealed that a mismatch between the H-bonding preferences of the α- and β-residues played a minor role in the structure-breaking effect. Temperature-dependent CD and NMR measurements showed that the hydrophobic stabilization was scaled-down for the α/β-peptides. Analysis of the biological activity of the foldamer peptides showed that four anginex derivatives dose-dependently inhibited the proliferation of a mouse endothelial cell line. The α → β(3) substitution strategy applied in this work can be a useful approach to the construction of bioactive β-sheet mimetics with a reduced aggregation tendency and improved pharmacokinetic properties.

A Foldamer-Dendrimer Conjugate Neutralizes Synaptotoxic β-Amyloid Oligomers

Background and Aims Unnatural self-organizing biomimetic polymers (foldamers) emerged as promising materials for biomolecule recognition and inhibition. Our goal was to construct multivalent foldamer-dendrimer conjugates which wrap the synaptotoxic β-amyloid (Aβ) oligomers with high affinity through their helical foldamer tentacles. Oligomeric Aβ species play pivotal role in Alzheimers disease, therefore recognition and direct inhibition of this undruggable target is a great current challenge. Methods and Results Short helical β-peptide foldamers with designed secondary structures and side chain chemistry patterns were applied as potential recognition segments and their binding to the target was tested with NMR methods (saturation transfer difference and transferred-nuclear Overhauser effect). Helices exhibiting binding in the µM region were coupled to a tetravalent G0-PAMAM dendrimer. In vitro biophysical (isothermal titration calorimetry, dynamic light scattering, transmission electron microscopy and size-exclusion chromatography) and biochemical tests (ELISA and dot blot) indicated the tight binding between the foldamer conjugates and the Aβ oligomers. Moreover, a selective low nM interaction with the low molecular weight fraction of the Aβ oligomers was found. Ex vivo electrophysiological experiments revealed that the new material rescues the long-term potentiation from the toxic Aβ oligomers in mouse hippocampal slices at submicromolar concentration. Conclusions The combination of the foldamer methodology, the fragment-based approach and the multivalent design offers a pathway to unnatural protein mimetics that are capable of specific molecular recognition, and has already resulted in an inhibitor for an extremely difficult target.

In vitro Anti-diabetic Activity and Chemical Characterization of an Apolar Fraction of Morus alba Leaf Water Extract

The tea from the white mulberry (Morus alba L.) leaf is a worldwide known traditional medicine of type II diabetes. Here, we report the investigation of the dichloromethane‐soluble fraction obtained in a 0.24% m/m yield from the hot water extract of mulberry leaves. A significant, dose‐dependent activity was found by means of the 24‐h glucose consumption of fully differentiated adipocytes both in the absence and presence of insulin. The fraction was characterized by HPLC‐DAD, GC‐MS and GC‐FID. The main constituent (40.3% by means of GC‐FID) was isolated and identified as loliolide by EIMS, HRESIMS and NMR spectroscopy. In the active fraction benzyl alcohol, ethyl benzoate, t‐cinnamic acid, p‐hydroxyacetophenone, t‐coniferyl alcohol and synapil alcohol were also identified by GC‐MS and quantified by GC‐FID (0.7, 1.3, 1.5, 2.9, 7.5 and 2.6%, respectively). Copyright

Induced Folding of Protein‐Sized Foldameric β‐Sandwich Models with Core β‐Amino Acid Residues

The mimicry of protein-sized β-sheet structures with unnatural peptidic sequences (foldamers) is a considerable challenge. In this work, the de novo designed betabellin-14 β-sheet has been used as a template, and α→β residue mutations were carried out in the hydrophobic core (positions 12 and 19). β-Residues with diverse structural properties were utilized: Homologous β(3) -amino acids, (1R,2S)-2-aminocyclopentanecarboxylic acid (ACPC), (1R,2S)-2-aminocyclohexanecarboxylic acid (ACHC), (1R,2S)-2-aminocyclohex-3-enecarboxylic acid (ACEC), and (1S,2S,3R,5S)-2-amino-6,6-dimethylbicyclo[3.1.1]heptane-3-carboxylic acid (ABHC). Six α/β-peptidic chains were constructed in both monomeric and disulfide-linked dimeric forms. Structural studies based on circular dichroism spectroscopy, the analysis of NMR chemical shifts, and molecular dynamics simulations revealed that dimerization induced β-sheet formation in the 64-residue foldameric systems. Core replacement with (1R,2S)-ACHC was found to be unique among the β-amino acid building blocks studied because it was simultaneously able to maintain the interstrand hydrogen-bonding network and to fit sterically into the hydrophobic interior of the β-sandwich. The novel β-sandwich model containing 25 % unnatural building blocks afforded protein-like thermal denaturation behavior.

15N and 13C Group-Selective Techniques Extend the Scope of STD NMR Detection of Weak Host–Guest Interactions and Ligand Screening

Saturation transfer difference (STD) is a valuable tool for studying the binding of small molecules to large biomolecules and for obtaining detailed information on the binding epitopes. Here, we demonstrate that the proposed 15N/13C variants of group‐selective, “GS‐STD” experiments provide a powerful approach to mapping the binding epitope of a ligand even in the absence of efficient spin diffusion within the target protein. Therefore, these experimental variants broaden the scope of STD studies to smaller and/or more‐dynamic targets. The STD spectra obtained in four different experimental setups (selective 1H STD, 15N GS‐STD, 13CAr and 13Caliphatic GS‐STD approaches) revealed that the signal‐intensity pattern of the difference spectra is affected by both the type and the spatial distribution of the excited “transmitter” atoms, as well as by the efficiency of the spin‐diffusion‐mediated magnetization transfer. The performance of the experiments is demonstrated on a system by using the lectin, galectin‐1 and its carbohydrate ligand, lactose.

Ligand-Induced Flocculation of Neurotoxic Fibrillar Aβ(1-42) by Noncovalent Crosslinking

Aggregation of the amyloid‐β (Aβ) peptides has a pivotal role in Alzheimer’s disease (AD). Small molecules and short peptides/peptidomimetics can exert their full protective effects against Aβ within a short time‐frame, but the exact mechanism of action is unclear. Time‐dependent NMR spectroscopic binding and replacement experiments were carried out for peptide LPFFD and thioflavine T (ThT) on neurotoxic fibrillar Aβ(1–42), which revealed transient binding behavior for both compounds, and complex time‐dependent features in the replacement experiments. The results of particle size measurements through the use of diffuse light‐scattering and transmission electron microscopy support the conclusions that the studied ligands induced interfibrillar association on a short timescale, which explains the NMR spectroscopic binding and replacement results. ζ‐Potential measurements revealed a slightly increased electrostatic stability of the Aβ fibrils upon ligand binding; this suggests that the interfibrillar assembly is driven by specific noncovalent cross‐linking interactions. A specific surface and mobility decrease due to the ligand‐induced flocculation of the Aβ fibrils can explain the neuroprotective effects.

Galectin-1–Asialofetuin Interaction Is Inhibited by Peptides Containing the Tyr-Xxx-Tyr Motif Acting on the Glycoprotein

Galectin‐1 (Gal‐1), a ubiquitous β‐galactoside‐binding protein expressed by various normal and pathological tissues, has been implicated in cancer and autoimmune/inflammatory diseases in consequence of its regulatory role in adhesion, cell viability, proliferation, and angiogenesis. The functions of Gal‐1 depend on its affinity for β‐galactoside‐containing glycoconjugates; accordingly, the inhibition of sugar binding blocks its functions, hence promising potential therapeutic tools. The Tyr‐Xxx‐Tyr peptide motifs have been reported to be glycomimetic sequences, mainly on the basis of their inhibitory effect on the Gal‐1–asialofetuin (ASF) interaction. However, the results regarding the efficacy of the Tyr‐Xxx‐Tyr motif as a glycomimetic inhibitor are still controversial. The present STD and trNOE NMR experiments reveal that the Tyr‐Xxx‐Tyr peptides studied do not bind to Gal‐1, whereas their binding to ASF is clearly detected. 15N,1H HSQC titrations with 15N‐labeled Gal‐1 confirm the absence of any peptide–Gal‐1 interaction. These data indicate that the Tyr‐Xxx‐Tyr peptides tested in this work are not glycomimetics as they interact with ASF via an unrevealed molecular linkage.

Competitive inhibition of TRPV1-calmodulin interaction by vanilloids.

There is enormous interest toward vanilloid agonists of the pain receptor TRPV1 in analgesic therapy, but the mechanisms of their sensory neuron‐blocking effects at high or repeated doses are still a matter of debate. Our results have demonstrated that capsaicin and resiniferatoxin form nanomolar complexes with calmodulin, and competitively inhibit TRPV1–calmodulin interaction. These interactions involve the protein recognition interface of calmodulin, which is responsible for all of the cell‐regulatory calmodulin–protein interactions. These results draw attention to a previously unknown vanilloid target, which may contribute to the explanation of the paradoxical pain‐modulating behavior of these important pharmacons.