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.

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 and conformational studies of pseudopeptides containing an unsymmetrical triazine scaffold.

Solid-phase synthesis and conformational studies of two pseudopeptides constituted by a triazine scaffold bound to two peptidic arms are described. In this paper, a new scaffold based on unsymmetrical triamino 1,3,5-triazine bearing two alkyl chains has been designed, assisted by molecular modelling, as a mimic of the backbone of the i + 1 and i + 2 residues of a beta-turn. The results confirm the ability of the triazine scaffold to induce extended conformations of the peptidic strands and point out that this scaffold is a good candidate as a template to induce anti-parallel beta-sheet structure.

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.

Two transmembrane Cys residues are involved in 5-HT4 receptor dimerization.

The 5-HT(4) receptor (5-HT(4)R) belongs to the G-protein-coupled receptor (GPCR) family and is of considerable interest for the development of new drugs to treat gastrointestinal diseases and memory disorders. The 5-HT(4)R exists as a constitutive dimer but its molecular determinants are still unknown. Using co-immunoprecipitation and Bioluminescence Resonance Energy Transfer (BRET) techniques, we show here that 5-HT(4)R homodimerization but not 5-HT(4)R-beta(2) adrenergic receptor (beta(2)AR) heterodimerization is largely decreased under reducing conditions suggesting the participation of disulfide bonds in 5-HT(4)R dimerization. Molecular modeling and protein docking experiments identified four cysteine (Cys) residues potentially involved in the dimer interface through intramolecular or intermolecular disulfide bonds. We show that disulfide bridges between Cys112 and Cys145 located within TM3 and TM4, respectively, are of critical importance for 5-HT(4)R dimer formation. Our data suggest that two disulfide bridges between two transmembrane Cys residues are involved in the dimerization interface of a GPCR.

Molecular tongs containing amino acid mimetic fragments: new inhibitors of wild-type and mutated HIV-1 protease dimerization.

We have designed, synthesized, and evaluated the inhibitory activity and metabolic stability of new peptidomimetic molecular tongs based on a naphthalene scaffold for inhibiting HIV-1 protease dimerization. Peptidomimetic motifs were inserted into one peptidic strand to make it resistant to proteolysis. The peptidic character of the molecular tongs can be decreased without changing the way they inhibit dimerization. Mutated HIV-1 proteases are also vulnerable to dimerization inhibitors, and the multimutated protease ANAM-11 is twice as sensitive to the inhibitor compared to wild-type protease. Thus, the metabolic stability of antidimeric molecular tongs can be increased without compromising their ability to inhibit wild-type and mutated HIV-1 proteases in vitro.

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.

Constitutive dimerization of human serotonin 5-HT4 receptors in living cells.

Serotonin 5-HT4 receptor isoforms are G protein-coupled receptors (GPCRs) with distinct pharmacological properties and may represent a valuable target for the treatment of many human disorders. Here, we have explored the process of dimerization of human 5-HT4 receptor (h5-HT4R) by means of co-immunoprecipitation and bioluminescence resonance energy transfer (BRET). Constitutive h5-HT4(d)R dimer was observed in living cells and membrane preparation of CHO and HEK293 cells. 5-HT4R ligands did not influence the constitutive energy transfer of the h5-HT4(d)R splice variant in intact cells and isolated plasma membranes. In addition, we found that h5-HT4(d)R and h5-HT4(g)R which structurally differ in the length of their C-terminal tails were able to form constitutive heterodimers independently of their activation state. Finally, we found that coexpression of h5-HT4R and beta2-adrenergic receptor (beta2AR) led to their heterodimerization. Given the large number of h5-HT4R isoforms which are coexpressed in a same tissue, our results points out the complexity by which this 5-HTR sub-type mediates its biological effects.

New constrained "molecular tongs" designed to dissociate HIV-1 protease dimer.

New "molecular tongs" based on naphthalene and quinoline scaffolds linked to two peptidic strands were synthesized. They were designed to prevent dimerization of HIV-1 protease by targeting the antiparallel beta-sheet involving N- and C-termini of each monomer. Compared to "molecular tongs" previously described (Bouras, A.; Boggetto, N.; Benatalah, Z.; de Rosny, E.; Sicsic, S.; Reboux-Ravaud, M. J. Med. Chem. 1999, 42, 957-962), two main different structural features were introduced: positively charged quinoline as a new scaffold and two peptidic strands displaying different sequences. Seventeen new "molecular tongs" with dipeptidic or tripeptidic strands were synthesized. These molecules were assayed on HIV-1 protease using the Zhang kinetic technique. Eleven molecules behaved as pure dimerization inhibitors, mostly at the submicromolar range. Compared to a naphthalene scaffold, the quinoline one was shown in several cases to favor dimerization inhibition. The simplified hydrophobic Val-Leu-Val-OMe strand was confirmed as particularly favorable. The C-terminal analogue strand Thr-Leu-Asn-OMe was shown to be the best one for inducing dimerization inhibition (K(id) of 80 nM for compound 30). The mechanism of inhibition was ascertained using ANS binding and gel filtration. Experimental results are in agreement with the dissociation of the HIV-1 protease dimeric form in the presence of the synthesized molecular tongs.

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.

Structure-activity relationships of some 3-substituted-4-hydroxycoumarins as HIV-1 protease inhibitors.

The screening of the HIV-1 protease (PR) inhibitory activity (IC-50) of various substituted 3-phenyl-4-hydroxycoumarins, 3-benzyl-4-hydroxycoumarins, 3-phenoxy-4-hydroxy-coumarins, 3-benzenesulfonyl-4-hydroxycoumarins and 3-(7-coumarinyloxy)-4-hydroxycoumarins was performed. The data indicate the importance of substituents at positions 5 and 7 of the coumarin ring on the inhibitory potency of the HIV-1-PR.

Metal-organic compounds: a new approach for drug discovery. N1-(4-methyl-2-pyridyl)-2,3,6-trimethoxybenzamide copper(II) complex as an inhibitor of human immunodeficiency virus 1 protease.

The use of metal-organic complexes is a potentially fruitful approach for the development of novel enzyme inhibitors. They hold the attractive promise of forming stronger attachments with the target by combining the co-ordination ability of metals with the unique stereoelectronic properties of the ligand. We demonstrated that this approach can be successfully used to inhibit the protease of the human immunodeficiency virus (type 1). Several ligands bearing substituents designed to interact with the catalytic site of the enzyme when complexed to Cu(2+) were synthesised. The inhibition pattern of the resulting copper(II) complexes was analysed. We showed that the copper(II) complex of N1-(4-methyl-2-pyridyl)-2,3,6-trimethoxybenzamide (C1) interacts with the active site of the enzyme leading to competitive inhibition. On the other hand, N2-pyridine-amide ligands and oxazinane carboxamide ligand were found to be poor chelators of the cupric ion under the enzymatic assay conditions. In these cases, the observed inhibition was attributed to released cupric ions which react with cysteine residues on the surface of the protease. While unchelated metal cations are not likely to be useful agents, metal chelates such as C1 should be considered as promising lead compounds for the development of targeted drugs.