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.

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.

Designed Glycopeptidomimetics Disrupt Protein-Protein Interactions Mediating Amyloid ß-Peptide Aggregation and Restore Neuroblastoma Cell Viability.

How anti-Alzheimer's drug candidates that reduce amyloid 1-42 peptide fibrillization interact with the most neurotoxic species is far from being understood. We report herein the capacity of sugar-based peptidomimetics to inhibit both Aß1-42 early oligomerization and fibrillization. A wide range of bio- and physicochemical techniques, such as a new capillary electrophoresis method, nuclear magnetic resonance, and surface plasmon resonance, were used to identify how these new molecules can delay the aggregation of Aß1-42. We demonstrate that these molecules interact with soluble oligomers in order to maintain the presence of nontoxic monomers and to prevent fibrillization. These compounds totally suppress the toxicity of Aß1-42 toward SH-SY5Y neuroblastoma cells, even at substoichiometric concentrations. Furthermore, demonstration that the best molecule combines hydrophobic moieties, hydrogen bond donors and acceptors, ammonium groups, and a hydrophilic ß-sheet breaker element provides valuable insight for the future structure-based design of inhibitors of Aß1-42 aggregation.

α- and ß-hydrazino acid-based pseudopeptides inhibit the chymotrypsin-like activity of the eukaryotic 20S proteasome.

We describe the synthesis of a library of new pseudopeptides and their inhibitory activity of the rabbit 20S proteasome chymotrypsin-like (ChT-L) activity. We replaced a natural α-amino acid by an α- or a ß-hydrazino acid and obtained inhibitors of proteasome up to a submicromolar range (0.7 µM for molecule 24b). Structural variations influenced the inhibition of the ChT-L activity. Models of inhibitor/20S proteasome complexes corroborated the inhibition efficacies obtained by kinetic studies.

Sugar-based peptidomimetics inhibit amyloid ß-peptide aggregation.

Alzheimer's disease is characterized by the oligomerization and amyloid fibril formation of amyloid ß-peptide (Aß). We describe a novel class of small water-soluble Aß binding peptidomimetics based on two hydrophobic Ala-Val and Val-Leu dipeptides linked to a D-glucopyranosyl scaffold through aminoalkyl and carboxyethyl links in C1 and C6 positions. These compounds combine the targeting of hydrophobic recognition interfaces with an original hydrophilic sugar ß-breakage strategy. These molecules were shown, by fluorescence thioflavin-T assays, to dramatically slow down the kinetics of amyloid fibril formation even at a low peptidomimetics to Aß ratio of 0.1:1. Electron microscopy images revealed that the peptidomimetics efficiently reduced the amount of typical amyloid fibrils. NMR saturation transfer difference experiments indicated that these molecules interact with Aß aggregated species through their hydrophobic amino acid residues. This inhibition effect was found to be sequence-specific since these molecules did not alter the kinetics of aggregation of another amyloid peptide, IAPP, involved in type 2 diabetes mellitus.

Design, synthesis, and biological evaluation of new 5-HT4 receptor agonists: application as amyloid cascade modulators and potential therapeutic utility in Alzheimer's disease.

Serotonin 5-HT(4) receptor (5-HT(4)R) agonists are of particular interest for the treatment of Alzheimer's disease because of their ability to ameliorate cognitive deficits and to modulate production of amyloid beta-protein (Abeta). However, despite the range of 5-HT(4)R agonists synthesized to date, potent and selective 5-HT(4)R agonists are still lacking. In the present study, two libraries of molecules based on the scaffold of ML10302, a highly specific and partial 5-HT(4)R agonist, were efficiently prepared by parallel supported synthesis and their binding affinities and agonist activities evaluated. Furthermore, we showed that, in vivo, the two best candidates exhibited neuroprotective activity by increasing the level of the soluble form of the amyloid precursor protein (sAPPalpha) in the cortex and hippocampus of mice. Interestingly, one of these compounds could also inhibit Abeta fibril formation in vitro.

Synthesis of specific bivalent probes that functionally interact with 5-HT(4) receptor dimers.

G-protein-coupled receptor dimerization directs the design of new drugs that specifically bind to receptor dimers. Here, we generated a targeted series of homobivalent ligands for serotonin 5-HT(4) receptor (5-HT(4)R) dimers composed of two 5-HT(4)R-specific ML10302 units linked by a spacer. The design of spacers was assisted by molecular modeling using our previously described 5-HT(4)R dimer model. Their syntheses were based on Sonogashira-Linstrumelle coupling methods. All compounds retained high-affinity binding to 5-HT(4)R but lost the agonistic character of the monomeric ML10302 compound. Direct evidence for the functional interaction of both pharmacophores of bivalent ligands with the 5-HT(4)R was obtained using a bioluminescence resonance energy transfer (BRET) based assay that monitors conformational changes within 5-HT(4) dimers. Whereas the monovalent ML10302 was inactive in this assay, several bivalent derivatives dose-dependently increased the BRET signal, indicating that both pharmacophores functionally interact with the 5-HT(4) dimer. These bivalent ligands may serve as a new basis for the synthesis of potential drugs for 5-HT(4)-associated disorders.

Design and synthesis of specific probes for human 5-HT4 receptor dimerization studies.

Recently, human 5-HT4 receptors have been demonstrated to form constitutive dimers in living cells. To evaluate the role of dimerization on the 5-HT4 receptor function, we investigated the conception and the synthesis of bivalent molecules able to influence the dimerization process. Their conception is based on a model of the 5-HT4 receptor dimer derived from protein/protein docking experiments. These bivalent ligands are constituted by two ML10302 units, a specific 5-HT4 ligand, linked through a spacer of different sizes and natures. These synthesized bivalent ligands were evaluated in binding assays and cyclic AMP production on the 5-HT4(e/g) receptor isoform stably transfected in C6 glial cells. Our data showed that bivalent ligands conserved a similar affinity compared to the basal ML10302 unit. Nevertheless, according to the nature and the size of the spacer, the pharmacological profile of ML10302 is more or less conserved. In view of the interest of bivalent ligands for investigating the GPCR dimerization process, these 5-HT4 specific bivalent ligands constitute valuable pharmacological tools for the study of 5-HT4 receptor dimerization.

Synthesis and characterization of the first fluorescent antagonists for human 5-HT4 receptors.

Fluorescent antagonists for human 5-HT(4) receptors were synthesized based on ML10302 1, a potent 5-HT(4) receptor agonist and on piperazine analogue 2. These molecules were derived with three fluorescent moieties, dansyl, naphthalimide, and NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl), through alkyl chains. The synthesized molecules were evaluated in binding assays on the recently cloned human 5-HT(4(e)) receptor isoform stably expressed in C6 glial cells with [(3)H]GR113808 as the radioligand. The affinity values depended upon the basal structure together with the alkyl chain length. The derivatives based on ML10302 were more potent ligands than the derivatives based on piperazine analogue. For ML10302-based ligands, dansyl and NBD derivatives attached through a chain length of one carbon atom 17a and 32, respectively, led to affinities close to the affinity of ML10302. The most potent compounds 17a, 28, and 32 produced an inhibition of the 5-HT stimulated cyclic AMP synthesis in the same cellular system with nanomolar K(b) values. Fluorescent properties of 17a, 28, and 32 were more particularly studied. Interactions of the fluorescent ligand 28 with the h5-HT(4(e)) receptor were indicated using h5-HT(4(e)) receptor transfected C6 glial cell membranes and entire cells. Ligand 28 was also used in fluorescence microscopy experiments in order to label h5-HT(4(e)) receptor transfected C6 glial cells, and subcellular localization of these receptors was more precisely determined using confocal microscopy.