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Human ACE is a central component of the renin-angiotensin system and a major therapeutic target for cardiovascular diseases. The somatic form of the enzyme (sACE) comprises two homologous metallopeptidase domains (N and C), each bearing a zinc active site with similar but distinct substrate and inhibitor specificities. In this study, we present the biological activity of silacaptopril, a silylated analogue of captopril, and its binding affinity towards ACE. Based on the recently determined crystal structures of both the ACE domains, a series of docking calculations were carried out in order to study the structural characteristics and the binding properties of silacaptopril and its analogues with ACE.

In the course of our study concerning gastrin and cholecystokinin (CCK) receptors, we have synthesized and characterized a new labeled gastrin ligand, 125I-BH-[Leu15]-gastrin-(5-17) [(3-[125I]iodo-4-hydroxyphenyl)-propionyl-[Leu15]-gastrin-(5-17)]. Binding of 125I-BH-[Leu15]-gastrin-(5-17) to isolated canine fundic mucosal cells was specific, saturable and of high affinity. 125I-BH-[Leu15]-gastrin- (5-17) and 125I-BH-CCK-8[(3-[125I]iodo-4-hydroxyphenyl)-propionyl-CCK-8] interact with isolated canine fundic mucosal cells with small differences in maximal binding capacities and affinities, 3800 +/- 900 binding sites/cell (Kd = 0.52 +/- 0.23 nM) and 6200 +/- 1100 binding sites/cell (Kd = 0.31 +/- 0.18 nM), respectively. The relative order of potencies for gastrin and CCK analogs in displacing 125I-BH-[Leu15]-gastrin-(5-17) binding correlated well with those obtained using 125I-BH-CCK-8. Selective CCK/gastrin antagonists L-364,718 (MK-329) and L-365,260 also inhibited 125I-BH-[Leu15]-gastrin-(5-17) binding. These results indicate that 125I-BH-[Leu15]-gastrin-(5-17) binds to gastrin receptors in isolated canine fundic mucosal cells. We have also characterized 125I-BH-[Leu15]-gastrin-(5-17) binding to the human Jurkat lymphoblastic cell line (Jurkat cells) known to express the CCK-B/gastrin receptor. Saturation experiments have shown that both 125I-BH-[Leu15]-gastrin-(5-17) and 125I-BH-CCK-8 interact with a single class of high-affinity binding sites in the Jurkat cell line.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

1 The growth hormone secretagogue receptor 1a (GHSR-1a) is a G-protein coupled receptor, involved in the biological actions of ghrelin by triggering inositol phosphates and calcium intracellular second messengers. It has also been reported that ghrelin could activate the 44- and 42-kDa extracellular signal-regulated protein kinases (ERK1/2) in different cell lines, but it is not clear whether this regulation is GHSR-1a dependent or not. 2 To provide direct evidence for the coupling of GHSR-1a to ERK1/2 activation, this pathway has been studied in a heterologous expression system. 3 Thus, in Chinese hamster ovary (CHO) cells we showed that ghrelin induced, via the human GHSR-1a, a transient and dose-dependent activation of ERK1/2 leading to activation of the transcriptional factor Elk1. 4 We then investigated the precise mechanisms involved in GHSR-1a-mediated ERK1/2 activation using various specific inhibitors and dominant-negative mutants and found that internalization of GHSR-1a was not necessary. Our results also indicate that phospholipase C (PLC) was involved in GHSR-1a-mediated ERK1/2 activation, however, pathways like tyrosine kinases, including Src, and phosphoinositide 3-kinases were not found to be involved. GHSR-1a-mediated ERK1/2 activation was abolished both by a general protein kinase C (PKC) inhibitor, Go6983, and by PKC depletion using overnight pretreatment with phorbol ester. Moreover, the calcium chelator, BAPTA-AM, and the inhibitor of conventional PKCs, Go6976, had no effect on the GHSR-1a-mediated ERK1/2 activation, suggesting the involvement of novel PKC isoforms (ɛ, δ), but not conventional or atypical PKCs. Further analyses suggest that PKCɛ is required for the activation of ERK1/2. 5 Taken together, these data suggest that ghrelin, through GHSR-1a, activates the Elk1 transcriptional factor and ERK1/2 by a PLC- and PKCɛ-dependent pathway. British Journal of Pharmacology (2006) 148, 350–365. doi:10.1038/sj.bjp.0706727

We have shown that gastrin and cholecystokinin octapeptide (CCK-8) are differently coupled to G protein (GTP-binding protein) through type B cholecystokinin receptors in guinea-pig brain membranes and Jurkat cells. Indeed, the gastrin-13 binding affinity is strongly reduced by stable guanyl nucleotides, whereas CCK-8 binding is only slightly affected. In order to determine the structural requirements regulating such coupling, we have synthesized several gastrin and cholecystokinin fragments (sulphated or unsulphated) elongated at the N-terminus of the common C-terminal tetrapeptide. We investigated their interaction with CCKB receptors in guinea pig brain membranes and Jurkat cells and their involvement in the G protein coupling. Their apparent binding affinities to CCKB receptors were measured by inhibition of [125I]Bolton Hunter-CCK-8 (3-[125I]iodo-4-hydroxyphenyl)propionyl-CCK-8) binding in the presence or absence of GTP gamma S (guanosine 5'-O-(3-thio)triphosphate) or aluminum tetrafluoride (AlF4-). Activation of the G proteins by GTP gamma S or AlF4- led to a decrease in binding affinity for the gastrin related peptides, the common CCK-gastrin C-terminal forms, the cholecystokinin hexapeptide and the unsulphated cholecystokinin heptapeptide. Sulphated CCK-7, CCK-8, and cionin apparent binding affinities were not affected. These finding indicated that the sulphated tyrosine in position 7 in CCK (as counted from the C-terminus), provides the cholecystokinin selectivity for the CCKB receptor compared to gastrin. The results are discussed with the aim to better clarify the physiological relevance of gastrin and cholecystokinin toward CCKB receptors and their related intracellular events.

International audience; The growth hormone secretagogue receptor type 1a (GHS-R1a) belongs to class A G-protein-coupled receptors (GPCR). This receptor mediates pleiotropic effects of ghrelin and represents a promising target for dysfunctions of growth hormone secretion and energy homeostasis including obesity. Identification of new compounds which bind GHS-R1a is traditionally achieved using radioactive binding assays. Here we propose a new fluorescence-based assay, called Tag-lite binding assay, based on a fluorescence resonance energy transfer (FRET) process between a terbium cryptate covalently attached to a SNAP-tag fused GHS-R1a (SNAP-GHS-R1a) and a high-affinity red fluorescent ghrelin ligand. The long fluorescence lifetime of the terbium cryptate allows a time-resolved detection of the FRET signal. The assay was made compatible with high-throughput screening by using prelabeled cells in suspension under a 384-well plate format. Ki values for a panel of 14 compounds displaying agonist, antagonist, or inverse agonist properties were determined using both the radioactive and the Tag-lite binding assays performed on the same batches of GHS-R1a-expressing cells. Compound potencies obtained in the two assays were nicely correlated. This study is the first description of a sensitive and reliable nonradioactive binding assay for GHS-R1a in a format amenable to high-throughput screening.

642NO Times Cited:0 Cited References Count:30; 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-1134M 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-1134M being the first example of a GHS-R1a mutant with a higher basal activity than the wild type receptor. (C) 2010 Elsevier B.V. All rights reserved.

International audience; 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 R-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.

270KS Times Cited:0 Cited References Count:14; International audience; The synthesis and structure-activity relationships concerning 3,4,5-trisubstituted 1,2,4-triazoles as ghrelin receptor ligands are described. The importance of the starting aminoacid material as well as its configuration was explored and the (D) Trp residue was found to lead to the best agonist or antagonist compounds. (C) 2007 Elsevier Ltd. All rights reserved.

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.

International audience; It is well known that the amidated C-terminal part of gastrin is crucial for its interaction with the classical seven transmembrane domain receptors CCK-1 or CCK-2. Nevertheless, over the past 10 years, several groups have characterized new binding sites using peptides related to gastrin (particularly glycine-extended forms of gastrin) on various tumoral and nontumoral cell lines. In the present study, we focused on the human astrocytic tumoral cell line U373. Although it has been described that gastrin was able to inhibit the motility of these cells, we were unable to detect any classical CCK/gastrin receptor. On the other hand, by using the radiolabeled C-terminal heptapeptide of gastrin ((125)I-G-7), we evidenced a new binding site that possessed a pharmacological profile different from the classical CCK/gastrin receptors. This new gastrin binding site seemed to be coupled to G proteins and be implicated in c-Fos transcription gene. Moreover, we showed that G-7 was able to induce a strong inhibition of U373 cell migration, a crucial biological effect when we know that astrocytoma cells' migration in brain parenchyma constitutes a major feature of malignancy in astrocytic tumors.It is well known that the amidated C-terminal part of gastrin is crucial for its interaction with the classical seven transmembrane domain receptors CCK-1 or CCK-2. Nevertheless, over the past 10 years, several groups have characterized new binding sites using peptides related to gastrin (particularly glycine-extended forms of gastrin) on various tumoral and nontumoral cell lines. In the present study, we focused on the human astrocytic tumoral cell line U373. Although it has been described that gastrin was able to inhibit the motility of these cells, we were unable to detect any classical CCK/gastrin receptor. On the other hand, by using the radiolabeled C-terminal heptapeptide of gastrin ((125)I-G-7), we evidenced a new binding site that possessed a pharmacological profile different from the classical CCK/gastrin receptors. This new gastrin binding site seemed to be coupled to G proteins and be implicated in c-Fos transcription gene. Moreover, we showed that G-7 was able to induce a strong inhibition of U373 cell migration, a crucial biological effect when we know that astrocytoma cells' migration in brain parenchyma constitutes a major feature of malignancy in astrocytic tumors.

alpha-amidation of a peptide (which takes place from a glycine-extended precursor) is required to produce biologically active amidated hormones, such as gastrin-releasing peptide (GRP)/Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH(2) (bombesin). It was shown that glycine-extended gastrin mediates mitogenic effects on various cell lines by interacting with a specific receptor, different from the classical CCK(1) or CCK(2) receptors. On the basis of this observation, we have extended the concept of obtaining active glycine-extended forms of others amidated peptides to produce new active analogues. In this study, we have tested the biological behaviour of a synthetic analogue of the glycine-extended bombesin (para-hydroxy-phenyl-propionyl-Gln-Trp-Ala-Val-Gly-His-Leu-Met-Gly-OH or JMV-1458) on various in vitro models. We showed that compound JMV-1458 was able to inhibit specific (3-[125I]iodotyrosyl(15)) GRP ([125I]GRP) binding in rat pancreatic acini and in Swiss 3T3 cells with K(i) values of approximately 10(-8) M. In isolated rat pancreatic acini, we found that JMV-1458 induced inositol phosphates production and amylase secretion in a dose-dependent manner. In Swiss 3T3 cells, the glycine-extended bombesin analogue dose-dependently produced [3H]thymidine incorporation. By using potent GRP/bombesin receptor antagonists, we showed that this synthetic glycine-extended bombesin analogue induces its biological activities via the classical GRP/bombesin receptor.

In the course of our study concerning gastrin and cholecystokinin (CCK) receptors, we synthesized and characterized a labelled gastrin ligand, [125I]BH[Leu15]gastrin-(5–17) (3-(3-[125I]iodo-4-hydroxyphenyl)propionyl[Leu15]gastrin-(5–17)). On isolated canine fundic mucosal cells and human Jurkat lymphoblastic cell line, known to express CCKB/gastrin receptors, the binding experiments performed indicate that [125I]BH[Leu15]gastrin-(5–17) provides a convenient biologically active ligand for cholecystokinin/gastrin receptor studies. We showed in this study that, on guinea-pig brain membranes known to possess CCKB and CCKA receptors, [125I]BH[Leu15]gastrin-(5–17) binds to a single class of high-affinity binding sites in a saturable and specific manner. [125I]BH[Leu15]gastrin-(5–17) interacts with guinea-pig brain membranes with a maximal binding capacity that is about three-fold lower than that of [125I]BHCCK8 (CCK8, the C-terminal octapeptide of cholecystokinin). The apparent affinities of CCK analogoues and selective CCK receptor antagonists L-365,260 and MK-329 for the sites labelled by both probes were in accordance with a CCKB-like profile. Association-dissociation kinetics of [125I]BH[Leu15]gastrin-(5–17) and [125I]BHCCK8 were performed and compared. They showed that [125I]BHCCK8 equilibrated more slowly than [125I]BH[Leu15]gastrin-(5–17). The effects of pH, monovalent and divalent cations on binding of both probes were investigated. The results obtained did not indicate strong differences between [125I]BH[Leu15]gastrin-(5–17) and [125I]BHCCK8 binding. Binding experiments in the presence of stable analogues of GTP showed a different behaviour between [125I]BH[Leu15]gastrin-(5–17) and [125I]BHCCK8. GTPγS strongly decreased [125I]BH[Leu15]gastrin-(5–17) binding whereas it weakly affected [125I]BHCCK8 binding. The 5′-adenylylimidodiphosphate was found to exert a similar effect than GTPγS on gastrin and CCK8 binding. The results of binding studies carried out with [125I]BH[Leu15]gastrin-(5–17) showed that gastrin binds specifically to guinea-pig brain membranes. Furthermore, the different effects of guanyl nucleotides on gastrin and CCK binding strongly suggested that gastrin and CCK trigger a differential G protein coupling through the same binding sites.