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.

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-HT(4) receptor contributes to the high affinity of some bulky 5-HT(4) 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, [(3)H]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-HT(4) receptor.

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.