The novel nonapeptide acein targets angiotensin converting enzyme in the brain and induces dopamine release.

BACKGROUND AND PURPOSE: Using an in-house bioinformatics programme, we identified and synthesized a novel nonapeptide, H-Pro-Pro-Thr-Thr-Thr-Lys-Phe-Ala-Ala-OH. Here, we have studied its biological activity, in vitro and in vivo, and have identified its target in the brain. EXPERIMENTAL APPROACH: The affinity of the peptide was characterized using purified whole brain and striatal membranes from guinea pigs and rats . Its effect on behaviour in rats following intra-striatal injection of the peptide was investigated. A photoaffinity UV cross-linking approach combined with subsequent affinity purification of the ligand covalently bound to its receptor allowed identification of its target. KEY RESULTS: The peptide bound with high affinity to a single class of binding sites, specifically localized in the striatum and substantia nigra of brains from guinea pigs and rats. When injected within the striatum of rats, the peptide stimulated in vitro and in vivo dopamine release and induced dopamine-like motor effects. We purified the target of the peptide, a ~151 kDa protein that was identified by MS/MS as angiotensin converting enzyme (ACE I). Therefore, we decided to name the peptide acein. CONCLUSION AND IMPLICATIONS: The synthetic nonapeptide acein interacted with high affinity with brain membrane-bound ACE. This interaction occurs at a different site from the active site involved in the well-known peptidase activity, without modifying the peptidase activity. Acein, in vitro and in vivo, significantly increased stimulated release of dopamine from the brain. These results suggest a more important role for brain ACE than initially suspected.

Involvement of tryptophan W276 and of two surrounding amino acid residues in the high constitutive activity of the ghrelin receptor GHS-R1a.

The human ghrelin receptor (GHS-R1a) is known to display a high level of signaling in the absence of ligand. The Trp276, located in the fully conserved CWXP motif of G protein-coupled receptors, is believed to function as a rotameric switch in these receptors. A comparative modelling of GHS-R1a with the motilin receptor, the most related G protein-coupled receptor to GHS-R1a known to date, but characterized by a very low ligand-independent signaling level, revealed that only two surrounding residues of Trp276, that are Val131 and Ile134, were different from these receptors. We mutated them at once in GHS-R1a to create a "motilin receptor-like" environment of Trp276 in order to study the consequences on GHS-R1a activation. We studied the pharmacological properties of the W276A, V131L-I134M GHS-R1a mutants. Basal as well as maximal ghrelin-induced signaling was assessed both by inositol-phosphate accumulation and SRE pathways. As compared to the wild type receptor, the SRE-luciferase assay displayed a markedly impaired basal activity for W276A whereas that of V131L-I134M was, strikingly, two fold increased. Nevertheless, the efficacy of ghrelin to bind or to stimulate mutant receptors remained unchanged. It is concluded that Trp276, Val131 and Ile134 have a significant impact on constitutive signaling of GHS-R1a, V131L-I134M being the first example of a GHS-R1a mutant with a higher basal activity than the wild type receptor.

Activation of the ghrelin receptor is described by a privileged collective motion: a model for constitutive and agonist-induced activation of a sub-class A G-protein coupled receptor (GPCR).

Three homology models of the human ghrelin receptor (GHS-R1a) have been generated from the available X-ray structures of rhodopsin (RHO model), opsin (OPS model) and beta-2 adrenergic receptor (B2 model). The latter was used as a starting point for combined molecular dynamics simulation (MDS) and full atom normal modes analysis (NMA). A low-frequency normal mode (mode 16) perfectly reproduced the intracellular motions observed between B2 and RHO models; in the opposite direction along the same mode, the generated structures are closer to the OPS model, suggesting a direct link with GHS-R1a activation. This was in agreement with motions of the seven transmembranous segments, increase of the solvent accessibility of the 140-ERY-142 sequence, and flip of the Trp276 (C WLP) residue, some features related to GPCRs activation. According to our model, His280 was proposed to stabilize Trp276 in the active state; this was verified by site-directed mutagenesis and biochemical characterization of the resulting H280A and H280S mutants, which were fully functional but sharing an important decrease of their basal activities. Docking performed with short ghrelin derivatives Gly-Ser-Ser ([octa])-Phe-NH (2) and Gly-Ser-Ser ([octa])-Phe-Leu-NH (2) allowed the identification of a robust position of these peptides in the active site of the receptor. This model was refined by MDS and validated by docking experiments performed on a set of 55 ghrelin receptor ligands based on the 1,2,4- triazole scaffold. Finally, NMA performed on the obtained peptide-receptor complex suggested stabilization of the Trp276 residue and of the whole receptor in the active state, preventing the motion observed along mode 16 computed for the unbound receptor. Our results show that NMA offers a powerful approach to study the conformational diversity and the activation mechanism of GPCRs.

Design, synthesis, and biological evaluation of 1,5-benzothiazepine-4-one derivatives targeting factor VIIa/tissue factor.

The 1,5-benzothiazepine-4-one scaffold was earlier shown to provide efficient protease inhibitors. In this contribution, we describe its use in the design of factor VIIa/tissue factor inhibitors. A series containing a scaffold non-substituted on its aryl part led to compound 20 with an IC(50) of 2.16 microM. Following molecular modelling studies of this compound, a second series was prepared, which necessitated the synthesis of protected 7- or 8-substituted 1,5-benzothiazepine-4-one derivatives.

New trisubstituted 1,2,4-triazole derivatives as potent ghrelin receptor antagonists. 3. Synthesis and pharmacological in vitro and in vivo evaluations.

Ghrelin receptor ligands based on trisubstituted 1,2,4-triazole structure were synthesized and evaluated for their in vitro binding and biological activity. In this study, we explored the replacement of the alpha-aminoisobutyryl moiety by aromatic or heteroaromatic groups. Compounds 5 and 34 acted as potent in vivo antagonists of hexarelin-stimulated food intake. These two compounds did not stimulate growth hormone secretion in rodents and did not antagonize growth hormone secretion induced by hexarelin.

Toward potent ghrelin receptor ligands based on trisubstituted 1,2,4-triazole structure. 2. Synthesis and pharmacological in vitro and in vivo evaluations.

A series of ghrelin receptor ligands based on the trisubstituted 1,2,4-triazole structure were synthesized and evaluated for their in vitro binding and biological activity. In this study, we explored the significance of the aminoisobutyryl (Aib) moiety, a common feature in numerous growth hormone secretagogues described in the literature. Potent agonist and antagonist ligands of the growth hormone secretagogue receptor type 1a (GHS-R1a) were obtained, i.e., compounds 41 (JMV2894) and 17 (JMV3031). The best compounds were evaluated for their in vivo activity on food intake, after sc injection in rodents. Among the tested compounds, few of them were able to stimulate food intake and some others, i.e., compounds 4 (JMV2959), 17, and 52 (JMV3021), acted as potent in vivo antagonist of hexarelin-stimulated food intake. These compounds did not stimulate growth hormone secretion in rats and furthermore did not antagonize growth hormone secretion induced by hexarelin, revealing that it is possible to modulate food intake without altering growth hormone secretion.

On-line synthesis of pseudopeptide library incorporating a benzodiazepinone turn mimic: biological evaluation on MC1 receptors.

Alpha melanocyte stimulating hormone (alpha-MSH) is a widely distributed hormone. This tridecapeptide exhibits various biological activities mediated through different receptors. alpha-MSH binds to the melanocortin-1 receptor (MC1-R), mainly expressed in keratinocytes and melanocytes, inducing melanogenesis and anti-inflammatory processes. The central His-Phe-Arg-Trp tetrapeptide sequence of alpha-MSH is known to form a turn in the bioactive conformation. To find new potent analogs of alpha-MSH, we decided to introduce non-peptide building blocks in the alpha-MSH sequence. Molecular modeling studies showed that two amino acids of the central core sequence could be replaced by the benzodiazepinone building block without loosing the beta-turn conformation. Benzodiazepines are well-known pharmacophores exhibiting a wide scope of biological activities and are described as constrained dipeptide mimics templates. Although numerous synthetic pathways leading to benzodiazepinones have been described in literature, no methodology has 1,4-benzodiazepine-2,5-diones building blocks bearing a free carboxylic acid function and a protected amino function suitable for incorporation into peptide sequences. In this study, we report the synthesis of peptides with a benzodiazepinone moiety obtained directly during the course of solid-phase peptide synthesis (SPPS). This "on-line" strategy leads to the generation of a 54-member pseudo-peptide library of alpha-MSH analogs. After LC/MS purification, binding assays were performed on the MC1 receptor leading to the discovery of several micromolar ligands.

Synthesis and pharmacological in vitro and in vivo evaluations of novel triazole derivatives as ligands of the ghrelin receptor. 1.

A new series of growth hormone secretagogue (GHS) analogues based on the 1,2,4-triazole structure were synthesized and evaluated for their in vitro binding and their ability to stimulate intracellular calcium release to the cloned hGHS-1a ghrelin receptor expressed in LLC PK-1 cells. We have synthesized potent ligands of this receptor, some of them behaving as agonists, partial agonists, or antagonists. Some compounds among the most potent, i.e., agonist 29c (JMV2873), partial agonists including 21b (JMV2810), antagonists 19b (JMV2866) and 19c (JMV2844), were evaluated for their in vivo activity on food intake, after sc injection in rodents. Some compounds were found to stimulate food intake like hexarelin; some others were identified as potent hexarelin antagonists in this assay. Among the tested compounds, 21b was identified as an in vitro ghrelin receptor partial agonist, as well as a potent in vivo antagonist of hexarelin-stimulated food intake in rodents. Compound 21b was without effect on GH release from rat. However, in this series of compounds, it was not possible to find a clear correlation between in vitro and in vivo results.

Cross-interactions of two p38 mitogen-activated protein (MAP) kinase inhibitors and two cholecystokinin (CCK) receptor antagonists with the CCK1 receptor and p38 MAP kinase.

Although SB202190 and SB203580 are described as specific p38 MAP kinase inhibitors, several reports have indicated that other enzymes are also sensitive to SB203580. Using a pharmacological approach, we report for the first time that compounds SB202190 and SB203580 were able to directly and selectively interact with a G-protein-coupled receptor, namely the cholecystokinin receptor subtype CCK1, but not with the CCK2 receptor. We demonstrated that these compounds were non-competitive antagonists of the CCK1 receptor at concentrations typically used to inhibit protein kinases. By chimeric construction of the CCK2 receptor, we determined the involvement of two CCK1 receptor intracellular loops in the binding of SB202190 and SB203580. We also showed that two CCK antagonists, L364,718 and L365,260, were able to regulate p38 mitogen-activated protein (MAP) kinase activity. Using a reporter gene strategy and immunoblotting experiments, we demonstrated that both CCK antagonists inhibited selectively the enzymatic activity of p38 MAP kinase. Kinase assays suggested that this inhibition resulted from a direct interaction with both CCK antagonists. Molecular modeling simulations suggested that this interaction occurs in the ATP binding pocket of p38 MAP kinase. These results suggest that SB202190 and SB203580 bind to the CCK1 receptor and, as such, these compounds should be used with caution in models that express this receptor. We also found that L364,718 and L365,260, two CCK receptor antagonists, directly interacted with p38 MAP kinase and inhibited its activity. These findings suggest that the CCK1 receptor shares structural analogies with the p38 MAP kinase ATP binding site. They open the way to potential design of either a new family of MAP kinase inhibitors from CCK1 receptor ligand structures or new CCK1 receptor ligands based on p38 MAP kinase inhibitor structures.