Jean-Louis Soulier

New insights into the human 5-HT4 receptor binding site: exploration of a hydrophobic pocket

A body of evidences suggests that a hydrophobic pocket of the human 5‐HT4 receptor contributes to the high affinity of some bulky 5‐HT4 ligands. A thorough study of this pocket was performed using mutagenesis and molecular modeling. Ligand binding or competition studies with selected bulky ligands (RS39604, RS100235, [3H]GR113808 and ML11411) and small ligands (5‐HT and ML10375) were carried out on wild‐type and mutant receptors (W7.40A/F, Y7.43F, R3.28L) transiently transfected in COS‐7 cells. The functional activity of the mutated receptors was evaluated by measuring the ability of 5‐HT to stimulate adenylyl cyclase. For W7.40F mutation, no changes in the affinity of studied ligands and in the functional activity of the mutant receptor were observed, in contrary to W7.40A mutation, which abolished both binding of ligands and 5‐HT‐induced cAMP production. Mutation R3.28L revealed a totally silent receptor with a basal level of cAMP production similar to the mock control despite its ability to product cAMP in the presence of 5‐HT. Moreover, a one order loss of affinity of RS39604 and a 45‐fold increase of ML11411 affinity were observed. Mutation Y7.43F modified the affinity of GR113808, which displays a 13‐fold lower affinity for the mutant than for the wild‐type receptor. In conclusion, in the hydrophobic pocket, two polar amino acids are able to interact through hydrogen bonds with bulky ligands depending on their chemical properties. Moreover, these experimental data may validate the proposed new three‐dimensional model of the human 5‐HT4 receptor.

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 Alzheimers 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.

Sugar-based peptidomimetics inhibit amyloid β-peptide aggregation

Alzheimers disease is characterized by the oligomerization and amyloid fibril formation of amyloid β-peptide (Aβ). We describe a novel class of small water-soluble Aβ 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 β-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 Aβ 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 Aβ 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.

Toward the First Nonpeptidic Molecular Tong Inhibitor of Wild-Type and Mutated HIV-1 Protease Dimerization

Herein we describe the synthesis and HIV‐1 protease (PR) inhibitory activity of 16 new peptidomimetic molecular tongs with a naphthalene scaffold. Their peptidic character was progressively decreased. Two of these molecules exhibited the best dimerization inhibition activity toward HIV‐1 wild‐type and multimutated ANAM‐11 proteases obtained to date for this class of molecules (∼40 nM for wild‐type PR and 100 nM for ANAM‐11 PR). Although the peptidic character of one molecular tong was completely suppressed, the mechanism of inhibition and inhibitory potency toward both proteases were maintained.

α- and β-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 β-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.

Designed Glycopeptidomimetics Disrupt Protein–Protein Interactions Mediating Amyloid β-Peptide Aggregation and Restore Neuroblastoma Cell Viability

How anti-Alzheimers 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 Aβ1-42 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 Aβ1-42. 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 Aβ1-42 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 β-sheet breaker element provides valuable insight for the future structure-based design of inhibitors of Aβ1-42 aggregation.

Carbonylhydrazide-Based Molecular Tongs Inhibit Wild-Type and Mutated HIV-1 Protease Dimerization

We have designed and synthesized new molecular tongs based on a rigid naphthalene scaffold and evaluated their antidimer activity on HIV-1 protease (PR). We inserted carbonylhydrazide and oligohydrazide (azatide) fragments into their peptidomimetic arms to reduce hydrophobicity and increase metabolic stability. These fragments are designed to disrupt the protein-protein interactions by reproducing the hydrogen bond pattern found in the antiparallel β-sheet formed between the N- and C-ends of the two monomers in the native PR. Kinetic analyses and fluorescent probe binding studies showed that several molecular tongs can inhibit PR dimerization. The best nonpeptidic molecular tongs to date were obtained with an inhibition constant K(id) of 50 nM for PR and 80 nM for the multimutated protease ANAM-11. The PR inhibition was selective, the aspartic proteases renin and pepsin were not inhibited.

Introduction of polar groups on the naphthalene scaffold of molecular tongs inhibiting wild-type and mutated HIV-1 protease dimerization

A new series of naphthalene-based molecular tongs containing polar groups at the 3-position of the naphthalene scaffold was synthesized and its anti-dimerization activity was evaluated against HIV-1 protease. The polar groups were introduced mainly via metal-catalysed cross coupling reactions such as the Buchwald–Hartwig amination, the Sonogashira reaction and the Beller cyanation. Kinetic analyses showed that by introducing specific polar substituents on the naphthalene scaffold the inhibitory activity of molecular tongs against wild-type and mutated HIV-1 proteases is maintained, while increasing their water solubility.

The use of 4,4,4-trifluorothreonine to stabilize extended peptide structures and mimic β-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 β-strands than their natural Ser and Thr pentapeptide analogues. The proof of concept that these fluorinated β-strand mimics are able to disrupt protein–protein interactions involving β-sheet structures is provided. The CF3-threonine containing pentapeptides interact with the amyloid peptide Aβ1-42 in order to reduce the protein–protein interactions mediating its aggregation process.