Composition and Conformation of Hetero- versus Homo-Fluorinated Triazolamers Influence their Activity on Islet Amyloid Polypeptide Aggregation.

Novel fluorinated foldamers based on aminomethyl-1,4-triazolyl-difluoroacetic acid (1,4-Tz-CF2) units were synthesized and their conformational behaviour was studied by NMR and molecular dynamics. Their activity on the aggregation of the human islet amyloid polypeptide (hIAPP) amyloid protein was evaluated by fluorescence spectroscopy and mass spectrometry. The fluorine labelling of these foldamers allowed the analysis of their interaction with the target protein. We demonstrated that the preferred extended conformation of homotriazolamers of 1,4-Tz-CF2 unit increases the aggregation of hIAPP, while the hairpin-like conformation of more flexible heterotriazolamers containing two 1,4-Tz-CF2 units mixed with natural amino acids from the hIAPP sequence reduces it, and more efficiently than the parent natural peptide. The longer heterotriazolamers having three 1,4-Tz-CF2 units adopting more folded hairpin-like and ladder-like structures similar to short multi-stranded ß-sheets have no effect. This work demonstrates that a good balance between the structuring and flexibility of these foldamers is necessary to allow efficient interaction with the target protein.

Proteolytically Stable Diaza-Peptide Foldamers Mimic Helical Hot Spots of Protein-Protein Interactions and Act as Natural Chaperones.

A novel class of peptidomimetic foldamers based on diaza-peptide units are reported. Circular dichroism, attenuated total reflection -Fourier transform infrared, NMR, and molecular dynamics studies demonstrate that unlike the natural parent nonapeptide, the specific incorporation of one diaza-peptide unit at the N-terminus allows helical folding in water, which is further reinforced by the introduction of a second unit at the C-terminus. The ability of these foldamers to resist proteolysis, to mimic the small helical hot spot of transthyretin-amyloid ß (Aß) cross-interaction, and to decrease pathological Aß aggregation demonstrates that the introduction of diaza-peptide units is a valid approach for designing mimics or inhibitors of protein-protein interaction and other therapeutic peptidomimetics. This study also reveals that small peptide foldamers can play the same role as physiological chaperone proteins and opens a new way to design inhibitors of amyloid protein aggregation, a hallmark of more than 20 serious human diseases such as Alzheimer's disease.

5-Fluoro-1,2,3-triazole motif in peptides and its electronic properties.

The 5-fluoro triazole amino acid scaffold prepared by halogen exchange has been incorporated into peptides. From the X-ray diffraction of the 5-fluoro triazole motif, the main observation was an important localization on one side of the negative potential surface. The fluorine atom reveals a cylindrical shape in its deformation electron density.

Two consecutive aza-amino acids in peptides promote stable ß-turn formation in water.

Studies on the synthetic methodologies and the structural propensity of peptides containing consecutive aza-amino acids are still in their infancy. Here, details of the synthesis and conformational analysis of tripeptides containing two consecutive aza-amino acids are provided. The demonstration that the type I ß-turn folding is induced, even in aqueous media, by the introduction of one or two lateral chains on the diaza-peptide unit is of particular importance for the design of peptidomimetics of biological interest.

Introducing the Chiral Constrained α-Trifluoromethylalanine in Aib Foldamers to Control, Quantify and Assign the Helical Screw-Sense.

Oligomers of α-aminoisobutyric acid (Aib) are achiral peptides that adopt 310 helical structures with equal population of left- and right-handed conformers. The screw-sense preference of the helical chain may be controlled by a single chiral residue located at one terminus. 1 H and 19 F NMR, X-ray crystallography and circular dichroism studies on new Aib oligomers show that the incorporation of a chiral quaternary α-trifluoromethylalanine at their N-terminus induces a reversal of the screw-sense preference of the 310 -helix compared to that of a non-fluorinated analogue having an l-α-methyl valine residue. This work demonstrates that, among the many particular properties of introducing a trifluoromethyl group into foldamers, its stereo-electronic properties are of major interest to control the helical screw sense. Its use as an easy-to-handle 19 F NMR probe to reliably determine both the magnitude of the screw-sense preference and its sign assignment is also of remarkable interest.

ß-Hairpin Peptide Mimics Decrease Human Islet Amyloid Polypeptide (hIAPP) Aggregation.

Amyloid diseases are degenerative pathologies, highly prevalent today because they are closely related to aging, that have in common the erroneous folding of intrinsically disordered proteins (IDPs) which aggregate and lead to cell death. Type 2 Diabetes involves a peptide called human islet amyloid polypeptide (hIAPP), which undergoes a conformational change, triggering the aggregation process leading to amyloid aggregates and fibers rich in ß-sheets mainly found in the pancreas of all diabetic patients. Inhibiting the aggregation of amyloid proteins has emerged as a relevant therapeutic approach and we have recently developed the design of acyclic flexible hairpins based on peptidic recognition sequences of the amyloid ß peptide (Aß1-42) as a successful strategy to inhibit its aggregation involved in Alzheimer's disease. The present work reports the extension of our strategy to hIAPP aggregation inhibitors. The design, synthesis, conformational analyses, and biophysical evaluations of dynamic ß-hairpin like structures built on a piperidine-pyrrolidine ß-turn inducer are described. By linking to this ß-turn inducer three different arms (i) pentapeptide, (ii) tripeptide, and (iii) α/aza/aza/pseudotripeptide, we demonstrate that the careful selection of the peptide-based arms from the sequence of hIAPP allowed to selectively modulate its aggregation, while the peptide character can be decreased. Biophysical assays combining, Thioflavin-T fluorescence, transmission electronic microscopy, capillary electrophoresis, and mass spectrometry showed that the designed compounds inhibit both the oligomerization and the fibrillization of hIAPP. They are also capable to decrease the aggregation process in the presence of membrane models and to strongly delay the membrane-leakage induced by hIAPP. More generally, this work provides the proof of concept that our rational design is a versatile and relevant strategy for developing efficient and selective inhibitors of aggregation of amyloidogenic proteins.

α-Synuclein: An All-Inclusive Trip Around its Structure, Influencing Factors and Applied Techniques.

Alpha-synuclein (αSyn) is a highly expressed and conserved protein, typically found in the presynaptic terminals of neurons. The misfolding and aggregation of αSyn into amyloid fibrils is a pathogenic hallmark of several neurodegenerative diseases called synucleinopathies, such as Parkinson's disease. Since αSyn is an Intrinsically Disordered Protein, the characterization of its structure remains very challenging. Moreover, the mechanisms by which the structural conversion of monomeric αSyn into oligomers and finally into fibrils takes place is still far to be completely understood. Over the years, various studies have provided insights into the possible pathways that αSyn could follow to misfold and acquire oligomeric and fibrillar forms. In addition, it has been observed that αSyn structure can be influenced by different parameters, such as mutations in its sequence, the biological environment (e.g., lipids, endogenous small molecules and proteins), the interaction with exogenous compounds (e.g., drugs, diet components, heavy metals). Herein, we review the structural features of αSyn (wild-type and disease-mutated) that have been elucidated up to present by both experimental and computational techniques in different environmental and biological conditions. We believe that this gathering of current knowledge will further facilitate studies on αSyn, helping the planning of future experiments on the interactions of this protein with targeting molecules especially taking into consideration the environmental conditions.

Peptidotriazolamers Inhibit Aß(1-42) Oligomerization and Cross a Blood-Brain-Barrier Model.

In peptidotriazolamers every second peptide bond is replaced by a 1H-1,2,3-triazole. Such foldamers are expected to bridge the gap in molecular weight between small-molecule drugs and protein-based drugs. Amyloid ß (Aß) aggregates play an important role in Alzheimer's disease. We studied the impact of amide bond replacements by 1,4-disubstituted 1H-1,2,3-triazoles on the inhibitory activity of the aggregation "hot spots" K16 LVFF20 and G39 VVIA42 in Aß(1-42). We found that peptidotriazolamers act as modulators of the Aß(1-42) oligomerization. Some peptidotriazolamers are able to interfere with the formation of toxic early Aß oligomers, depending on the position of the triazoles, which is also supported by computational studies. Preliminary in vitro results demonstrate that a highly active peptidotriazolamer is also able to cross the blood-brain-barrier.

Fluorinated Triazole Foldamers: Folded or Extended Conformational Preferences.

The Ministère de l'Enseignement Supérieur et de la Recherche (MESR) is thanked for financial support for José Laxio Arenas. The China Scholarship Council is thanked for financial support for Yaochun Xu. The authors thank Pr. Vadim Soloshonok and TOSOH F-TECH, Inc. for the kind gift of N-terbutyl-sulfinylimine.

Helical γ-Peptide Foldamers as Dual Inhibitors of Amyloid-ß Peptide and Islet Amyloid Polypeptide Oligomerization and Fibrillization.

Type 2 diabetes (T2D) and Alzheimer's disease (AD) belong to the 10 deadliest diseases and are sorely lacking in effective treatments. Both pathologies are part of the degenerative disorders named amyloidoses, which involve the misfolding and the aggregation of amyloid peptides, hIAPP for T2D and Aß1-42 for AD. While hIAPP and Aß1-42 inhibitors have been essentially designed to target ß-sheet-rich structures composing the toxic amyloid oligomers and fibrils of these peptides, the strategy aiming at trapping the non-toxic monomers in their helical native conformation has been rarely explored. We report herein the first example of helical foldamers as dual inhibitors of hIAPP and Aß1-42 aggregation and able to preserve the monomeric species of both amyloid peptides. A foldamer composed of 4-amino(methyl)-1,3-thiazole-5-carboxylic acid (ATC) units, adopting a 9-helix structure reminiscent of 310 helix, was remarkable as demonstrated by biophysical assays combining thioflavin-T fluorescence, transmission electronic microscopy, capillary electrophoresis and mass spectrometry.

Evidence for different in vitro oligomerization behaviors of synthetic hIAPP obtained from different sources.

Type 2 diabetes is characterized by the aggregation of human islet amyloid polypeptide (hIAPP), from monomer to amyloid deposits that are made of insoluble fibrils. Discrepancies concerning the nature of formed species or oligomerization kinetics among reported in vitro studies on hIAPP aggregation process have been highlighted. In this work, we investigated if the sample itself could be at the origin of those observed differences. To this aim, four hIAPP samples obtained from three different sources or suppliers have been analyzed and compared by ThT fluorescence spectroscopy and by two recently developed techniques, capillary electrophoresis (CE), and ESI-IMS-QToF-MS. Lots provided by the same supplier were shown to be very similar whatever the analytical technique used to characterize them. In contrast, several critical differences could be pointed out for hIAPP provided by different suppliers. We demonstrated that in several samples, some oligomerized peptides (e.g., dimer) were already present upon reception. Purity was also different, and the proneness of the peptide solution to form fibrils in vitro within 24 h could vary considerably from one sample source to another but not from lot to lot of the same source. All those results demonstrate that the initial state of conformation, oligomerization, and quality of the hIAPP can greatly impact the aggregation kinetics, and thus the information provided by these in vitro tests. Finally, a careful selection of the peptide batch and source is mandatory to perform relevant in vitro studies on hIAPP oligomerization and to screen new molecules modulating this pathological process. Graphical abstract.

Introducing sequential aza-amino acids units induces repeated ß-turns and helical conformations in peptides.

A major current issue in medicinal chemistry is the design of small peptide analogues resistant to proteolysis and able to adopt preferential conformations, while preserving the selectivity and efficiency of natural peptides. Whereas the introduction of one aza-Gly in peptides has proven numerous biological and structural interest, the conformational effect of sequential aza-Gly or aza-amino acids bearing side chains has not been investigated. In this work, experimental NMR and X-ray data together with in silico conformational studies reveal that the introduction of two consecutive aza-amino acids in pseudotripeptides induces the formation of stable hydrogen-bonded ß-turn structures. Notably, this stabilization effect relies on the presence of side chains on aza-amino acids, as more flexible conformations are observed with aza-Gly residues. Remarkably, a longer aza/aza/α/aza/aza/α pseudohexapeptide containing substituted aza-amino acids adopts repeated ß-turns conformations which interconvert with a fully helical structure mimicking a 310 helix.

Real-Time BODIPY-Binding Assay To Screen Inhibitors of the Early Oligomerization Process of Aß1-42 Peptide.

Misfolding and aggregation of amyloid ß1-42 peptide (Aß1-42) play a central role in the pathogenesis of Alzheimer's disease (AD). Targeting the highly cytotoxic oligomeric species formed during the early stages of the aggregation process represents a promising therapeutic strategy to reduce the toxicity associated with Aß1-42. Currently, the thioflavin T (ThT) assay is the only established spectrofluorometric method to screen aggregation inhibitors. The success of the ThT assay is that it can detect Aß1-42 aggregates with high ß-sheet content, such as protofibrils or fibrils, which appear in the late aggregation steps. Unfortunately, by using the ThT assay, the detection of inhibitors of early soluble oligomers that present a low ß-sheet character is challenging. Herein, a new, facile, and robust boron-dipyrromethene (BODIPY) real-time assay suitable for 96-well plate format, which allows screening of compounds as selective inhibitors of the formation of Aß1-42 oligomers, is reported. These inhibitors decrease the cellular toxicity of Aß1-42, although they fail in the ThT assay. The findings have been confirmed and validated by structural analysis and cell viability assays under comparable experimental conditions. It is demonstrated that the BODIPY assay is a convenient method to screen and discover new candidate compounds that slow down or stop the pathological early oligomerization process and are active in the cellular assay. Therefore, it is a suitable complementary screening method of the current ThT assay.

Peptides and peptidomimetics as inhibitors of protein-protein interactions involving ß-sheet secondary structures.

Protein-protein interactions involving ß-sheet secondary structures have been questioned in many fatal human diseases such as cancer, autoimmune and neurodegenerative diseases. Small selective peptide derivatives and analogues are promising drug candidates for inhibiting this poorly known class of PPIs. In this review, we will highlight the main strategies developed for designing linear and cyclic peptide and peptidomimetic inhibitors of PPIs involving ß-sheet structures. These compounds either do not adopt preferred conformations or can mimic protein secondary structures such as ß-strands, ß-hairpins or α-helices.

A capillary zone electrophoresis method to investigate the oligomerization of the human Islet Amyloid Polypeptide involved in Type 2 Diabetes.

Type 2 diabetes is characterized by the aggregation of human Islet Amyloid Polypeptide (hIAPP) from monomer to large and insoluble fibrils. According to several recent studies, small soluble oligomers are now considered as potential toxic species. No monitoring tool has been to date reported to mimic in vitro the oligomerization process of hIAPP over time, although this would allow selecting candidate compounds that slow down or stop this pathological process. Considering the poor stability of those species and the necessity to monitor in real time, a compatible with the monitoring of hIAPP oligomerization CE method coupled to UV detection was developed. Three groups of hIAPP oligomers/monomers formed during this process could be separated. A polybrene coating was used to avoid adsorption of hIAPP onto capillary walls. Peaks identification was performed using a combination of CE-MS, filtrations and SDS-PAGE. They revealed that one peak is composed of monomer with a very small amount of dimer and trimer, whereas the two others are composed of bigger species higher than 100 kDa. We demonstrated that this real time oligomerization process started from the very initial step, with hIAPP principally as a monomer, until the formation of very big oligomers. This method was shown to be repeatable with RSDs on electrophoretic mobilities and relative peak areas less than 1.6 and 5.8% respectively for the monomer peak. Its application to study the anti-aggregation properties of resveratrol showed that this compound saved more than 30% of the monomeric hIAPP form whereas it almost disappeared without. The method opens new perspectives for the screening of potential drugs for type 2 diabetes.

Binding Modes of a Glycopeptidomimetic Molecule on Aß Protofibrils: Implication for Its Inhibition Mechanism.

We recently reported that a glycopeptidomimetic molecule significantly delays the fibrillization process of Aß42 peptide involved in Alzheimer's disease. However, the binding mode of this compound, named 3ß, was not determined at the atomic scale, hindering our understanding of its mechanism of action and impeding structure-based design of new inhibitors. In the present study, we performed molecular docking calculations and molecular dynamics simulations to investigate the most probable structures of 3ß complexed with Aß protofibrils. Our results show that 3ß preferentially binds to an area of the protofibril surface that coincides with the protofibril dimerization interface observed in the solid-state NMR structure 5KK3 from the PDB. Based on these observations, we propose a model of the inhibition mechanism of Aß fibrillization by compound 3ß.

Structure-activity relationships of ß-hairpin mimics as modulators of amyloid ß-peptide aggregation.

Aggregation of amyloid proteins is currently involved in more than 20 serious human diseases that are actually untreated, such as Alzheimer's disease (AD). Despite many efforts made to target the amyloid cascade in AD, finding an aggregation inhibiting compound and especially modulating early oligomerization remains a relevant and challenging strategy. We report herein the first examples of small and non-peptide mimics of acyclic beta-hairpins, showing an ability to delay the fibrillization of amyloid-ß (Aß1-42) peptide and deeply modify its early oligomerization process. Modifications providing better druggability properties such as increased hydrophilicity and reduced peptidic character were performed. We also demonstrate that an appropriate balance between flexibility and stability of the ß-hairpin must be reached to adapt to the different shape of the various aggregated forms of the amyloid peptide. This strategy can be investigated to target other challenging amyloid proteins.

In vitro monitoring of amyloid ß-peptide oligomerization by Electrospray differential mobility analysis: An alternative tool to evaluate Alzheimer's disease drug candidates.

Alzheimer's disease (AD) is a neurodegenerative disorder linked to protein aggregation, like more than twenty other human pathologies. One major protein incriminated in AD is the 42-residue amyloid-ß peptide (Aß1-42) which aggregates to form neurotoxic oligomers and fibrils. While, low molecular weight oligomers have been evidenced as neurotoxic species, only scarce methods raise the challenge to monitor the beginning of the aggregation process, called oligomerization. We propose here an innovative and fast monitoring of the time-dependent Aß1-42 oligomerization pattern by electrospray differential mobility analysis (ES-DMA). We developed a non-denaturing method based on ES-DMA to afford a real-time and direct characterization of the early, metastable and neurotoxic species. This technique provided their size distribution over time. At the beginning of the in vitro oligomerization process of Aß1-42, the size distribution is characterized by two populations with modal diameters around 3.5 and 4nm, corresponding to Monomer and Small oligomers. After few hours, larger species around 10nm are observed. The results were correlated to those obtained by capillary electrophoresis. We also demonstrated the ability of our method to evaluate Aß1-42 kinetics modulators. Thereby, ES-DMA provides new insights on Aß1-42 oligomerization in the presence of sugar-based peptidomimetic analogs which were recently described as modulators of Aß1-42 self-assembly and neurotoxicity inhibitors.

The use of 4,4,4-trifluorothreonine to stabilize extended peptide structures and mimic ß-strands.

Pentapeptides having the sequence R-HN-Ala-Val-X-Val-Leu-OMe, where the central residue X is L-serine, L-threonine, (2S,3R)-L-CF3-threonine and (2S,3S)-L-CF3-threonine were prepared. The capacity of (2S,3S)- and (2S,3R)-CF3-threonine analogues to stabilize an extended structure when introduced in the central position of pentapeptides is demonstrated by NMR conformational studies and molecular dynamics simulations. CF3-threonine containing pentapeptides are more prone to mimic ß-strands than their natural Ser and Thr pentapeptide analogues. The proof of concept that these fluorinated ß-strand mimics are able to disrupt protein-protein interactions involving ß-sheet structures is provided. The CF3-threonine containing pentapeptides interact with the amyloid peptide Aß1-42 in order to reduce the protein-protein interactions mediating its aggregation process.