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Gastrin-releasing peptide receptor (GRPR) is overexpressed in human prostate cancer and is being used as a target for molecular imaging. In this study, we report on the direct comparison of 3 novel GRPR-targeted radiolabeled tracers: (AlF)-F-18-JMV5132, Ga-68-JMV5132, and Ga-68-JMV4168 (JMV5132 is NODA-MPAA-beta Ala-beta Ala-[H-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2], JMV4168 is DOTA-beta Ala-beta Ala-[H-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2], and NODA-MPAA is 2-[4-(carboxymethyl)-7-{[4-(carboxymethyl) phenyl]methyl}-1,4,7-triazacyclononan-1-yl] acetic acid). Methods: The GRPR antagonist JMV594 (H-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2) was conjugated to NODA-MPAA for labeling with (AlF)-F-18. JMV5132 was radiolabeled with Ga-68 and F-18, and JMV4168 was labeled with Ga-68 for comparison. The inhibitory concentration of 50% values for binding GRPR of JMV4168, JMV5132, Ga-nat-JMV4168, and Ga-nat-JMV5132 were determined in a competition-binding assay using GRPR-overexpressing PC-3 tumors. The tumor-targeting characteristics of the compounds were assessed in mice bearing subcutaneous PC-3 xenografts. Small-animal PET/CT images were acquired, and tracer biodistribution was determined by ex vivo measurements. Results: JMV5132 was labeled with F-18 in a novel 1-pot, 1-step procedure within 20 min, without need for further purification and resulting in a specific activity of 35 MBq/nmol. Inhibitory concentration of 50% values (in nM) for GRPR binding of JMV5132, JMV4168, Ga-nat-JMV5132, Ga-nat-JMV4168, and (AlF)-F-nat-JMV5132 were 6.8 (95% confidence intervals [CIs], 4.6-10.0), 13.2 (95% CIs, 5.9-29.3), 3.0 (95% CIs, 1.5-6.0), 3.2 (95% CIs, 1.8-5.9), and 10.0 (95% CIs, 6.3-16.0), respectively. In mice with subcutaneous PC-3 xenografts, all tracers cleared rapidly from the blood, exclusively via the kidneys for Ga-68-JMV4168 and partially hepatobiliary for Ga-68-JMV5132 and (AlF)-F-18-JMV5132. Two hours after injection, the uptake of Ga-68-JMV4168, Ga-68-JMV5132, and (AlF)-F-18-JMV5132 in PC-3 tumors was 5.96 +/- 1.39, 5.24 +/- 0.29, 5.30 +/- 0.98 (percentage injected dose per gram), respectively. GRPR specificity was confirmed by significantly reduced tumor uptake of the 3 tracers after coinjection of a 100-fold excess of unlabeled JMV4168 or JMV5132. Small-animal PET/CT clearly visualized PC-3 tumors, with the highest resolution observed for Al18F-JMV5132. Conclusion: JMV5132 could be rapidly and efficiently labeled with F-18. (AlF)-F-18-JMV5132, Ga-68-JMV5132, and Ga-68-JMV4168 all showed comparable high and specific accumulation in GRPR-positive PC-3 tumors. These new PET tracers are promising candidates for future clinical translation.

International audience; The dynamic character of G protein-coupled receptors is essential to their function. However, the details of how ligands stabilize a particular conformation to selectively activate a signaling pathway and how signaling proteins affect this conformational repertoire remain unclear. Using a prototypical peptide-activated class A G protein-coupled receptor (GPCR), the ghrelin receptor, reconstituted as a monomer into lipid discs and labeled with a fluorescent conformational reporter, we demonstrate that ligand efficacy and functional selectivity are directly related to different receptor conformations. Of importance, our data bring direct evidence that distinct effector proteins affect the conformational landscape of the ghrelin receptor in different ways. Whereas G proteins affect the balance between active and inactive receptor substates in favor of the active state, agonist-induced arrestin recruitment is accompanied by a marked change in the structural features of the receptor that adopt a conformation different from that observed in the absence of arrestin. In contrast to G proteins and arrestins, μ-AP2 has no significant effect on the organization of the transmembrane core of the receptor. Such a modulation of a GPCR conformational landscape by pharmacologically distinct ligands and effectors provides insights into the structural bases that decisively affect ligand efficacy and subsequent biological responses. This is also likely to have major implications for the design of drugs activating specific GPCR-associated signaling pathways.

International audience; Aims: Ghrelin (GHR) is an orexigenic gut peptide that interacts with ghrelin receptors (GHR-Rs) to modulate brain reinforcement circuits. Systemic GHR infusions augment cocaine stimulated locomotion and conditioned place preference (CPP) in rats, whereas genetic or pharmacological ablation of GHR-Rs has been shown to attenuate the acute locomotor-enhancing effects of nicotine, cocaine, amphetamine and alcohol and to blunt the CPP induced by food, alcohol, amphetamine and cocaine in mice. The stimulant nicotine can induce CPP and like amphetamine and cocaine, repeated administration of nicotine induces locomotor sensitization in rats. A key issue is whether pharmacological antagonismof GHR-Rs would similarly attenuate nicotine-induced locomotor sensitization. Method: To examine the role of GHR-Rs in the behavioral sensitizing effects of nicotine, adult male rats were injected with either 0, 3 or 6 mg/kg of the GHR-R receptor antagonist JMV 2959 (i.p.) and 20 min later with either vehicle or 0.4 mg/kg nicotine hydrogen tartrate (s.c.) on each of 7 consecutive days. Results: Rats treated with nicotine alone showed robust locomotor sensitization, whereas rats pretreated with JMV 2959 showed significantly attenuated nicotine-induced hyperlocomotion. Conclusions: These results suggest that GHR-R activity is required for the induction of locomotor sensitization to nicotine and complement an emerging literature implicating central GHR systems in drug reward/reinforcement.

International audience; Many peripheral substances, including ghrelin, induce neuronal activation in the brain. In the present study, we compared the effect of subcutaneously administered ghrelin and its three stable agonists: Dpr 3ghr ([Dpr(N-octanoyl)3] ghrelin) (Dpr - diaminopropionic acid), YA GHRP-6 (H-Tyr-Ala-His-DTrpAla-Trp-DPhe-Lys-NH2), and JMV1843 (H-Aib-DTrp-D-gTrp-CHO) on the Fos expression in food intakeresponsive brain areas such as the hypothalamic paraventricular (PVN) and arcuate (ARC) nuclei, the nucleus of the solitary tract (NTS), and area postrema (AP) in male C57BL/6 mice. Immunohistochemical analysis showed that acute subcutaneous dose of each substance (5 mg/kg b.w.), which induced a significant food intake increase, elevated Fos protein expression in all brain areas studied. Likewise ghrelin, each agonist tested induced distinct Fos expression overall the PVN. In the ARC, ghrelin and its agonists specifically activated similarly distributed neurons. Fos occurrence extended from the anterior (aARC) to middle (mARC) ARC region. In the latter part of the ARC, the Fos profiles were localized bilaterally, especially in the ventromedial portions of the nucleus. In the NTS, all substances tested also significantly increased the number of Fos profiles in neurons, which also revealed specific location, i.e., in the NTS dorsomedial subnucleus (dmNTS) and the area subpostrema (AsP). In addition, cells located nearby the NTS, in the AP, also revealed a significant increase in number of Fos-activated cells. These results demonstrate for the first time that ghrelin agonists, regardless of their different chemical nature, have a significant and similar activating impact on specific groups of neurons that can be a part of the circuits involved in the food intake regulation. Therefore there is a real potency for ghrelin agonists to treat cachexia and food intake disorders. Thus, likewise JMV1843, the other ghrelin agonists represent substances that might be involved in trials for clinical purposes.

International audience; The diastereoisomeric separation of peptidomimetics of hexarelin, a strong growth hormone secretagogue, in CE has been studied. Highly sulfated-γ-CD was found to be an appropriate selector for the separation of the stereoisomers. However, non-repeatable analyses were obtained on bare fused silica capillary due to the progressive adsorption of the analytes on the capillary wall. Two types of polyelectrolyte coating agents were tested to prevent this phenomenon. Coating with neutral polyethylene oxide was found to be efficient but resulted in a very long analysis time (about 40 min). Coating with cationic poly(diallyldimethylammonium) chloride was found both to prevent analyte adsorption, reduce analysis time and alter separation selectivity. EOF measurement revealed that the highly sulfated-γ-CDs were strongly adsorbed on the poly(diallyldimethylammonium) chloride coating surface yielding a stable strong cathodic EOF, which considerably reduced analysis time (about 12 min). Very good repeatability of analysis was obtained (RSDmigration time

The knowledge of the binding sites of G protein-coupled cholecystokinin receptors represents important insights that may serve to understand their activation processes and to design or optimize ligands. Our aim was to identify the amino acid of the cholecystokinin-A receptor (CCK-AR) binding site in an interaction with the sulfate of CCK, which is crucial for CCK binding and activity. A three-dimensional model of the [CCK-AR-CCK] complex was built. In this model, Arg197 was the best candidate residue for a ionic interaction with the sulfate of CCK. Arg197 was exchanged for a methionine by site-directed mutagenesis. Wild-type and mutated CCK-AR were transiently expressed in COS-7 cells for pharmacological and functional analysis. The mutated receptor on Arg197 did not bind the agonist radioligand 125I-BH-[Thr, Nle]-CCK-9; however, it bound the nonpeptide antagonist [3H]-SR27,897 as the wild-type receptor. The mutant was approximately 1,470- and 3,200-fold less potent than the wild-type CCK-AR to activate G proteins and to induce inositol phosphate production, respectively. This is consistent with the 500-fold lower potency and 800-fold lower affinity of nonsulfated CCK relative to sulfated CCK on the wild-type receptor. These data, together with those showing that the mutated receptor failed to discriminate nonsulfated and sulfated CCK while it retained other pharmacological features of the CCK-AR, strongly support an interaction between Arg197 of the CCK-AR binding site and the sulfate of CCK. In addition, the mutated CCK-AR resembled the low affinity state of the wild-type CCK-AR, suggesting that Arg197-sulfate interaction regulates conformational changes of the CCK-AR that are required for its physiological activation.

International audience; A new method for the synthesis of substituted imidazopyridines (2-azaindolizines) starting from carboxylic acid and 2-methylaminopyridine is described. The reaction of the obtained N-2-pyridylmethylamides with Lawesson's reagent generated the target imidazopyridines, along with the corresponding thioamide intermediates. After a simple filtration on alumina, addition of mercury(II) acetate allowed for total conversion of the thio-amides into the imidazopyridines. The reaction conditions, as well as the influence of the substituent in position 3 of the imidazopyridine ring were explored. We also demonstrated that this heterocyclization was racemization free in the presence of a chiral carbon in position alpha to the heterocycle.

International audience; How G protein-coupled receptor conformational dynamics control G protein coupling to trigger signaling is a key but still open question. We addressed this question with a model system composed of the purified ghrelin receptor assembled into lipid discs. Combining receptor labeling through genetic incorporation of unnatural amino acids, lanthanide resonance energy transfer, and normal mode analyses, we directly demonstrate the occurrence of two distinct receptor:Gq assemblies with different geometries whose relative populations parallel the activation state of the receptor. The first of these assemblies is a preassembled complex with the receptor in its basal conformation. This complex is specific of Gq and is not observed with Gi. The second one is an active assembly in which the receptor in its active conformation triggers G proteinactivation. The active complex is present even in the absence of agonist, in a direct relationship with the high constitutive activity of the ghrelin receptor. These data provide direct evidence of a mechanism for ghrelin receptor-mediated Gq signaling in which transition of the receptor from an inactive to an active conformation is accompanied by a rearrangement of a preassembled receptor: G protein complex, ultimately leading to G protein activation and signaling.

International audience; Ghrelin receptor ligands based on a trisubstituted 1,2,4-triazole scaffold were recently synthesized and evaluated for their in vitro affinity for the GHS-R1a receptor and their biological activity. In this study, replacement of the α-aminoisobutyryl (Aib) moiety (a common feature present in numerous growth hormone secretagogues described in the literature) by aromatic and heteroaromatic groups was explored. We found potent antagonists incorporating the picolinic moiety in place of the Aib moiety. In an attempt to increase affinity and activity of our lead compound 2, we explored the modulation of the pyridine ring. Herein we report the design and the structure–activity relationships study of these new ghrelin receptor ligands.

Triazolopyridines are an important class of biologically active heterocyclic compounds. In this letter, we describe a new method for the synthesis of [1,2,4]triazolo[4,3-a]pyridines starting from 2-hydrazinopyridine and carboxylic acids. The resulting acetohydrazides are cyclized in a key step using the Lawesson’s reagent. The reaction conditions were explored, as well as the scope of this reaction concerning the substituent in position 3 of the triazolopyridine ring. We also demonstrated that this heterocyclization is racemization free in the presence of a chiral carbon in position α to the heterocycle.

The functionalization of peptides and proteins by aldehyde groups has become the subject of intensive research since the discovery of the inhibition properties of peptide aldehydes towards various enzymes. Furthermore, peptide aldehydes are of great interest for peptide backbone modification or ligation reactions. This review focuses upon their synthesis, which has been developed following two main strategies. The first strategy consists of prior synthesis of the peptide, followed by the introduction of the aldehyde function. The second possible strategy uses alpha-amino aldehydes as starting materials. After protection of the aldehyde, peptide elongation occurs. At the end of the synthesis, the aldehyde function can be unmasked.

The anchoring of an α-amino-acid derivative by its amine function on to a solid support allows some chemical reactions starting from the carboxylic acid function. This paper describes the preparation of α-amino aldehydes linked to the support by their amine function. This was performed by reduction with LiAlH4 of the corresponding Weinreb amide linked to the resin. The aldehydes obtained were then involved in Wittig or reductive amination reactions. In addition, the linked Weinreb amide was reacted with methylmagnesium bromide to yield the corresponding ketone. After cleavage from the support, the compounds were obtained in good to excellent yields and characterized. Copyright © 2004 European Peptide Society and John Wiley & Sons, Ltd.

We present an up-to-date study on the nature, at the protein level, of various members of the dystrophin complex at the muscle cell membrane by comparing red and white caudal muscles from Torpedo marmorata. Our investigations involved immunodetection approaches and Western blotting analysis. We determined the presence or absence of different molecules belonging to the dystrophin family complex by analyzing their localization and molecular weight. Specific antibodies directed against dystrophin, i.e., DRP2 alpha-dystrobrevin, beta-dystroglycan, alpha-syntrophin, alpha-, beta-, gamma-, and delta-sarcoglycan, and sarcospan, were used. The immunofluorescence study (confocal microscopy) showed differences in positive immunoreactions at the sarcolemmal membrane in these slow-type and fast-type skeletal muscle fibers. Protein extracts from T. marmorata red and white muscles were analyzed by Western blotting and confirmed the presence of dystrophin and associated proteins at the expected molecular weights. Differences were confirmed by comparative immunoprecipitation analysis of enriched membrane preparations with anti-beta-dystroglycan polyclonal antibody. These experiments revealed clear complex or non-complex formation between members of the dystrophin system, depending on the muscle type analyzed. Differences in the potential function of these various dystrophin complexes in fast or slow muscle fibers are discussed in relation to previous data obtained in corresponding mammalian tissues. (J Histochem Cytochem 49:857-865, 2001)

A methodology to modify aspartyl or glutamyl residue side-chains after their incorporation on solid phase synthesis in a peptide sequence was developed. This strategy using the concept of Weinreb amide on the side chain of aspartyl or glutamyl residues allowed reduction of the amide side-chain into aldehyde, the reaction of different groups with this aldehyde function on solid support. The usefulness of this method was proved by the synthesis of H-Phe-[2-(4-ethoxycarbonyl-3-butene)-glycyl]-leucine amide.

The reduction of Weinreb amides was reinvestigated in order to find conditions that will allow the synthesis of protected aspartyl and glutamyl aldehydes derivatives useful for peptide and pseudopeptide syntheses. We have demonstrated that lithium tris(tert-butoxy)aluminium hydride [LiAl(OtBu)3H] and lithium tris[(3-ethyl-3-pentyl)oxy]aluminium hydride (LTEPA) can be used to reduce Weinreb amide derivatives into their corresponding aldehydes. In these conditions, it was possible to synthesize N- and side chain protected aspartyl and glutamyl aldehyde derivatives in fairly good conditions.

Peptides with C-terminal aldehydes (PAs) are of interest due to their inhibitory properties toward numerous classes of proteolytic enzymes. In this paper, we describe and compare two novel approaches for the preparation of PAs by solid phase synthesis, one based on the reduction of the Weinreb amide and the other on the reduction of phenyl esters. A study showed that purification of the PAs by chromatography (silica gel or reversed phase) induced a loss of the optical integrity of the C-terminal residue. Both methods were found to be suitable for the synthesis of PAs which were then used for the preparation of reduced-bond-containing peptides on insoluble polymers. The Weinreb amide approach was preferred for the synthesis of PAs due to an uncontrolled over-reduction of phenyl esters in our hands.

Anchoring of an α-amino-acid amide residue by its amine function to a carbamate resin followed by primary amide Hofmann rearrangement led to a gem -diamino residue linked to the resin. The generated primary amine could be acylated with various carboxylic compounds offering a large variety of molecules. Furthermore, this new solid-phase strategy allowed a reliable synthesis of a gem -diamino monomeric residue which could not be easily obtained in solution due to the limited stability of monocarbamate-protected gem -diaminoalkyl derivatives.

International audience; We report on the synthesis of 1,2,4-triazoles substituted with 2 or 3 amino acid side chains, using silver benzoate as a key reagent for the cyclization step. A complete study of the optical purity retention during the synthetic process leading to these compounds is described. In addition an improved work-up after the addition-cyclization step was also established leading to better yields and metal-free products.

International audience; Starting from N- and C-protected aspartic or glutamic acids, new unnatural α-amino acids containing 3,4,5-trisubstituted 1,2,4-triazole heterocycles on their side chains have been synthesized. Two points of diversity could easily be achieved on the heterocyclic moiety, which could easily be incorporated in peptide sequences to modify or improve their pharmacokinetic properties.

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.

555ZP Times Cited:2 Cited References Count:68; International audience; 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 (CWLP) residue, some features related to GPCRs activation. According to our model, Hs280 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-NH2 and Gly-Ser-Ser([octa])-Phe-Leu-NH2 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, NIMA 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. (C) 2009 Elsevier Ltd. All rights reserved.

International audience

596PA Times Cited:0 Cited References Count:23; We report a new method using silver benzoate instead of mercury salts as a key reagent for the synthesis of 3,4,5-trisubstituted 1,2,4-triazoles. This method allows the introduction of a large variety of substituents in the three positions. We demonstrated that we could introduce one chiral center without any loss of the optical purity. This method is compatible with at least multi-gram scale-up. (C) 2010 Elsevier Ltd. All rights reserved.

A very simple, fast, and sensitive RIA of angiotensin (Ang) II has been developed, based on a monoclonal antibody with high affinity and specificity, making possible the direct measurement of circulating Ang II in human plasma after solid-phase extraction. The purified monoclonal antibody 4D8 has an association constant of 1.3 x 10(11) L/mol with Ang II and a cross-reactivity of < 1% for Ang I. The assay can detect as little as 0.8 fmol of Ang II in 2 mL of plasma and is not influenced by the presence of Ang I. Analytical recoveries between 112% and 116% were obtained for Ang II added to human plasma at physiological concentrations. Comparison of the RIA with a reversed-phase, high-performance liquid chromatographic method followed by RIA to measure Ang II in human plasma samples from normal and hypertensive subjects--and from normotensive subjects before and after an acute inhibition of angiotensin-converting enzyme with captopril (50 mg)--showed a high degree of correlation (r2 = 0.93) between the two methods.

Poly(L-lysine citramide) is a degradable bioresorbable polyanion whose polyamide chains are composed of citric acid and L-lysine building blocks. Its chemical and physicochemical properties were extensively investigated in the past for its interest as polymeric drug carrier. In this work, 4(S)-amino-3-(S)-hydroxy-5-phenylpentanoyl-isoleucyl-phenylalanine methyl ester, a pseudopeptide active against the HIV protease in vitro, was linked to poly(L-lysine citramide) in attempts to promote solubility and cell penetration. Conjugates were characterized by FTIR, NMR, SEC, DLS, amino-acid analyses, and toxicity in mice. They degraded slowly at pH 7.4 and more rapidly at pH 4.5, two pH values selected to mimic extra-cellular fluids and intralysosome medium, respectively. According to capillary zone electrophoresis, degradation did not release the peptide. The phenylalanyl-isoleucyl-phenylalanine methyl ester peptide, inactive against the protease in vitro, was used as negative control. The anti-HIV activities of the carrier, of the conjugates and of model molecules, including a fluorescence-labeled pseudopeptide conjugate, were evaluated comparatively in vitro using two cell lines, namely, CEM-SS and MT-4 cells, infected with HIV-1 LAI and IIIB isolates, respectively. Unexpectedly, all the conjugates showed in vitro antiviral activity independent of peptide release and of inhibition of the HIV protease. According to FACS analysis, the antiviral activity was related to the presence of peptide moieties along the polymer chains and depended on the order by which cells, viruses, and conjugates were presented to each other. Although it was not possible to determine whether the antiviral activity resulted from interactions between conjugates and cells or conjugates and virus or both, the conjugates appeared able to inhibit the binding of the virus to cells in vitro when introduced before cell infection. None of the conjugates exhibited acute toxicity in mice.

432CP Times Cited:2 Cited References Count:12; A new, mild general, and efficient synthesis of N-protected a-amino acid hydrazides is described. This two-step preparation uses N-aminophthalimide as protected hydrazine to prepare N-protected alpha-amino acid hydrazide precursors, and subsequent dephthaloylation with an aminomethyl polystyrene resin yields N-protected alpha-amino acid hydrazides. It has the advantages of avoiding the use of the toxic hydrazine reagent and being compatible with the most commonly used N-protecting groups. This strategy is particularly interesting in the case of N-(9-fluorenylmethoxycarbonyl)protected amino acids. Within the limits of chiral HPLC detection, no epimerization is apparent.

In the present study, we explore the central nervous system mechanism underlying the chronic central effects of ghrelin with respect to increasing body weight and body fat. Specifically, using a recently developed ghrelin receptor antagonist, GHS-R1A (JMV2959), we investigate the role of GHS-R1A in mediating the effects of ghrelin on energy balance and on hypothalamic gene expression. As expected, in adult male rats, chronic central treatment with ghrelin for 14 days, when compared to vehicle-treated control rats, resulted in an increased body weight, lean mass and fat mass (assessed by dual X-ray absorptiometry), dissected white fat pad weight, cumulative food intake, food efficiency, respiratory exchange ratio and a decrease of energy expenditure. Co-administration of the ghrelin receptor antagonist JMV2959 suppressed/blocked the majority of these effects, with the notable exception of ghrelin-induced food intake and food efficiency. The hypothesis emerging from these data, namely that GHS-R1A mediates the chronic effects of ghrelin on fat accumulation, at least partly independent of food intake, is discussed in light of the accompanying data regarding the hypothalamic genes coding for peptides and receptors involved in energy balance regulation, which were found to have altered expression in these studies.

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.

International audience; The 28-amino acid peptide ghrelin is a neuroendocrine hormone synthesized primarily in the stomach. It stimulates growth hormone secretion and appetite, thus promoting food intake and body-weight gain. The pharmacological properties of this peptide are mediated by the growth hormone secretogogue receptor type 1a (GHS-R1a). Given its wide spectrum of biological activities, it is evident that the discovery of ghrelin and its receptor has opened up many perspectives in the fields of neuroendocrine and metabolic research and has had an influence on such fields of internal medicine as gastroenterology, oncology, and cardiology. It is therefore increasingly likely that synthetic, peptidyl, and nonpeptidyl GHS-R1a ligands, acting as agonists, partial agonists, antagonists, or inverse agonists, could have both clinical and therapeutic potential. This review summarizes the various types of GHS-R1a ligands that have been described in the literature and discusses the recent progress made in this research area.

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.

WOS: 000247672900005, PubMed ID: 17562564, The increasing clinical importance of drug-resistant fungal and bacterial pathogens has lent additional urgency to microbiological research and new antibacterial compound development. For this purpose, new tent-butyl[1-benzyl-2[(4-aryl-2-thiazolyl)hydrazono]ethyl]carbamate derivatives were synthesized and evaluated for antibacterial activity. The reaction of Boc-L-phenylalaninal with thiosemicarbazide gave the thiosemicarbazone which furnished the title compounds by reaction with phenacyl bromides. The newly synthesized compounds were screened for antibacterial activity and toxicity. While microdilution broth susceptibility assay was used for the antibacterial activity evaluation of the compounds against the strains E. coli (NRRL B-3704), M. luteus (NRRL B-4375), B. cereus (NRRL B-3711), P. aeruginosa (NRRL B-23), and S. fecalis (NRRL B14617), the Artemia sauna 96-well assay was used to determine cytotoxicities of the compounds. Observations obtained from the bioassays showed that some of the compounds are highly active against E. coli, M. luteus, and B. cereus when compared with the control agent and showed low toxicity.

The purpose of this article is to delineate strategic considerations and provide practical procedures to enable non-experts to synthesize peptides with a reasonable chance of success. This article is not encyclopedic but rather devoted to the Fmoc/tBu approach of solid phase peptide synthesis (SPPS), which is now the most commonly used methodology for the production of peptides. The principles of SPPS with a review of linkers and supports currently employed are presented. Basic concepts for the different steps of SPPS such as anchoring, deprotection, coupling reaction and cleavage are all discussed along with the possible problem of aggregation and side-reactions. Essential protocols for the synthesis of fully deprotected peptides are presented including resin handling, coupling, capping, Fmoc-deprotection, final cleavage and disulfide bridge formation.

The purpose of this chapter is to delineate strategic considerations and provide practical procedures to enable non-experts to synthesize peptides with a reasonable chance of success. This chapter focuses on Fmoc chemistry, which is now the most commonly employed strategy for solid phase peptide synthesis (SPPS). Protocols for the synthesis of fully deprotected peptides are presented, together with a review of linkers and supports currently employed for SPPS. The principles and the different steps of SPPS (anchoring, deprotection, coupling reaction, and cleavage) are all discussed, along with their possible side reactions.

A new and easy protocol for the formation of substituted 4,5-dihydro-1,2,4-triazin-6(1H)-ones was developed on solid support. The heterocyclic compounds were formed by nucleophilic reaction of hydrazine on thioamide esters. As cyclization was concomitant with cleavage from the support, substituted 4,5-dihydrotriazinones were obtained in high purity.

Since all organs (i.e. skeletal, cardiac, smooth muscles and sciatic nerve) are never only taken from a single patient, all these tissues were obtained from one cynomolgus monkey, a model closely resembling humans. This work describes an up-to-date reinvestigation of the dystrophin-glycoprotein complex and related molecules in various monkey tissues such those cited above. We used monoclonal and polyclonal antibodies produced in our laboratory, which are directed against dystrophin, utrophin, short-dystrophin products, alpha-dystrobrevin, beta-dystroglycan, alpha-syntrophin, alpha-, beta-, gamma-, delta-, epsilon-sarcoglycan, and sarcospan. For each molecule, we determined their molecular weight and tissue localization. Regardless of the tissue analyzed, at least one dystrophin or utrophin as full-length molecule and one short-dystrophin product or dystrobrevin as proteins belonging to the dystrophin superfamily were found. Beta-dystroglycan, beta and delta sarcoglycans were always detected, while other sarcoglycans varied from all to only three components. Epsilon sarcoglycan appears to be specific to smooth muscle, which is devoid of alpha sarcoglycan. Sarcospan is only absent from sciatic nerve structures. Among the different muscles investigated in this study, short dystrophin products are only present in cardiac muscle. All of these findings are summarized in one table, which highlight in one single animal the variability of the dystrophin-glycoprotein complex components in relation with the organ studied. This statement is important because any attempt to estimate protein restoration needs in each study the knowledge of the expected components that should be considered normal.

A new strategy for the synthesis of lipopeptides has been developed. Using Weinreb (N-methoxy, N-methyl) amide as an aldehyde function precursor on the side chains of Asp or Glu residues, this new strategy avoids the synthesis of a lipidic amino acid residue before its incorporation in the peptide sequence. The aldehyde generated on the solid support can react with ylides leading to unsaturated or saturated side chains or with various nucleophiles to yield non-coded amino acid residues incorporated into the sequence. Copyright © 2002 European Peptide Society and John Wiley & Sons, Ltd.

Delineation of CCK receptor binding sites is a prerequisite for the understanding of the molecular basis for ligand recognition, partial agonism, ligand-induced traffiking of receptor signalling. In the current paper, we illustrate how, in the past 5 years, studies from our laboratory and others have provided new data on the molecular basis of the pharmacology and functioning of CCK1 and CCK2 receptors. Available data on CCK1 and CCK2R binding sites indicate that 1) homologous regions of the two receptors are involved in the binding site of CCK, however, positioning of CCK slightly differs; 2) binding sites of non-peptide agonists/antagonist are buried in the pocket formed by transmembrane helices and overlap that of CCK and 3) residues of the binding sites as well as of conserved motifs such as E/DRY, NPXXY are crucial for receptor activation.

Starting from EP 51389, a potent growth hormone secretagogue (GHS), a new series of GHS has been designed, synthesized and tested. This series was built on a gem-diamino moiety and a structure activity relationship study was performed including N-methylation of the amide bonds. Some analogues exhibited more powerful activity than Hexarelin, they were active per os on dog and have been selected as candidates for further development.

A new method for the synthesis of N-protected allyl- and homoallylglycines was developed from aspartic and glutamic acid derivatives. The carboxylic side-chains of aspartic and glutamic derivatives was first transformed into the Weinreb amide by coupling with N,O-dimethylhydroxylamine and then reduced into the corresponding aldehyde. The latter could react with methyl-triphenylphosphonium bromide to yield the title compounds with 50% total yield.

The functionalization of peptides and proteins by aldehyde or keto groups has become the subject of intensive research since the discovery of the inhibition properties of peptide aldehydes and the advent of protein engineering. The first part of this review focuses upon the tremendous efforts devoted to the solid-phase synthesis of peptide aldehydes as protease inhibitors. The second part describes the utility of the aldehyde or keto functionalities for the site-specific modification of peptides or proteins.

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.

Analogues of bombesin which incorporate dipeptide or turn mimetics have been synthesized. One of them (compound 11) containing a seven-membered lactam ring revealed a good affinity for GRP/BN receptors on rat pancreatic acini (K(i) value of 1.7 +/- 0.4 nM) and on Swiss 3T3 cells (K(i) value of 1.0 +/- 0.2 nM). On the basis of this observation, antagonists containing the same dipeptide mimic were obtained by modification of the C-terminal part of the bombesin analogues. The most potent constrained compounds (15 and 17) were able to antagonize 1 nM bombesin-stimulated amylase secretion from rat pancreatic acini with high potency (K(i) = 21 +/- 3 and 3.3 +/- 1.0 nM, respectively) and 10(-7) M bombesin-stimulated ¿(3)Hthymidine incorporation into Swiss 3T3 cells (K(i) = 7.8 +/- 2. 0 and 0.5 +/- 0.1 nM, respectively).

A new method for the synthesis of N -protected α-amino aldehydes was developed. N -Protected α-amino amides of morpholine were easily prepared and then reduced with LiAlH 4 to produce clean N -protected α-amino aldehydes. This new scheme of synthesis can be used with Boc, Z and Fmoc amino-protecting groups.

Some of the chemistry of amino acids going on in our laboratory (Laboratoire des Amino acides Peptides et Protéines) is described as well as some mass spectrometry methodology for their characterization particularly on solid supports. Several aspects are presented including: (i) the stereoselective synthesis of natural and unnatural amino acids using 2-hydroxypinan-3-one as chiral auxiliary; (ii) the stereoselective synthesis of natural and unnatural amino acids by deracemization of alpha-amino acids via their ketene derivatives; (iii) the synthesis of alpha-aryl-alpha-amino acids via reaction of organometallics with a glycine cation; (iv) the diastereoselective synthesis of glycosyl-alpha-amino acids; (v) the synthesis of beta-amino acids using alpha-aminopyrrolidinopiperazinediones as chiral templates; (vi) the reactivity of urethane-N-protected N-carboxyanhydrides. To characterize natural and non natural amino acids through their immonium ions by mass spectrometry, some methodology is also described.

The cholecystokinin-A receptor (CCK-AR) is a G protein-coupled receptor that mediates important central and peripheral cholecystokinin actions. Residues of the CCK-AR binding site that interact with the C-terminal part of CCK that is endowed with biological activity are still unknown. Here we report on the identification of Arg-336 and Asn-333 of CCK-AR, which interact with the Asp-8 carboxylate and the C-terminal amide of CCK-9, respectively. Identification of the two amino acids was achieved by dynamics-based docking of CCK in a refined three-dimensional model of CCK-AR using, as constraints, previous results that demonstrated that Trp-39/Gln-40 and Met-195/Arg-197 interact with the N terminus and the sulfated tyrosine of CCK, respectively. Arg-336-Asp-8 and Asn-333-amide interactions were pharmacologically assessed by mutational exchange of Arg-336 and Asn-333 in the receptor or reciprocal elimination of the partner chemical functions in CCK. This study also allowed us to demonstrate that (i) the identified interactions are crucial for stabilizing the high affinity phospholipase C-coupled state of the CCK-AR·CCK complex, (ii) Arg-336 and Asn-333 are directly involved in interactions with nonpeptide antagonists SR-27,897 and L-364,718, and (iii) Arg-336 but not Asn-333 is directly involved in the binding of the peptide antagonist JMV 179 and the peptide partial agonist JMV 180. These data will be used to obtain an integrated dynamic view of the molecular processes that link agonist binding to receptor activation.

Sulfation of the tyrosine at the seventh position from the C terminus of cholecystokinin (CCK) is crucial for CCK binding to the CCK-A receptor. Using three-dimensional modeling, we identified methionine 195 of the CCK-A receptor as a putative amino acid in interaction with the aromatic ring of the sulfated tyrosine of CCK. We analyzed the role played by the two partners of this interaction. The exchange of Met-195 for a leucine caused a minor decrease (2. 8-fold) on the affinity of the high affinity sites for sulfated CCK-9, a strong drop (73%) of their number, and a 30-fold decrease on the affinity of the low and very low affinity sites for sulfated CCK-9, with no change in their number. The mutation also caused a 54-fold decrease of the potency of the receptor to induce inositol phosphates production. The high affinity sites of the wild-type CCK-A receptor were highly selective (800-fold) toward sulfated versus nonsulfated CCK, whereas low and very low affinity sites were poorly selective (10- and 18-fold). In addition, the M195L mutant bound, and responded to, sulfated CCK analogues with decreased affinities and potencies, whereas it bound and responded to nonsulfated CCK identically to the wild-type receptor. Thus, Met-195 interacts with the aromatic ring of the sulfated tyrosine to correctly position the sulfated group of CCK in the binding site of the receptor. This interaction is essential for CCK-dependent transition of the CCK-A receptor to a high affinity state. Our data should represent an important step toward the identification of the residue(s) of the receptor in interaction with the sulfate moiety of CCK and the understanding of the molecular mechanisms that govern CCK-A receptor activation.

Azay, J., C. Nagain, M. Llinares, C. Devin, J. A. Fehrentz, N. Bernad, C. Roze and J. Martinez. Comparative study of in vitro and vivo activities of bombesin pseudopeptide analogs modified on the C-terminal dipeptide fragment. Peptides 19(1) 57–63, 1998.—Analogs of bombesin in which the peptide bond between the two last amino acid residues were replaced by a pseudopeptide bond mimicking the transition state analog were evaluated. These compounds were able to recognize the bombesin receptor on isolated rat pancreatic acini with high potency (Ki from 0.60 ± 0.27 n M to 4.3 ± 2.3 n M ). Although they were devoid of agonist activity, they were able to antagonize bombesin-induced amylase secretion in this model, with potencies in accordance with their affinities (IC 50 from 1.6 ± 0.3 n M to 10.0 ± 1.7 n M ). When tested in vivo in the anesthetized rat, these bombesin receptor antagonists exhibited high potency in inhibiting bombesin-induced pancreatic secretion (H-DPhe-Gln-Trp-Ala-Val-Gly-His-NH-CH[CH 2 -CH(CH 3 ) 2 ]-CHOH-(CH 2 ) 3 -CH 3 , JMV845, was among the most potent compounds with ED 50 of 7.82 ± 2.89 n M in inhibiting bombesin-induced protein secretion). The results of this study showed that replacing the peptide bond between the two last amino acid residues in bombesin by mimicking the transition state analog resulted in in vitro and in vivo potent bombesin receptor antagonists.

A new strategy for the synthesis of peptide aldehydes on solid support is presented. Reaction of a N-protected aminoaldehyde with carboethoxymethylene triphenylphosphorane yielded an α-β-unsaturated δ-amino derivative. After saponification, the resulting α-β-unsaturated δ-aminoacid was anchored to a solid support by an ester linkage (Merrifield resin) or by an amide linkage (MBHA, Expansin). After elongation of the peptide chain, ozonolysis yielded quite pure C-terminal peptide aldehydes in a good yield with no detectable racemization of the C-terminal residue.

Summary Bombesin receptor antagonists are possible therapeutic agents due to their ability to act as inhibitors of cellular proliferation. On the basis of our hypothesis on the mechanism of action of gastrin associating an activating enzyme system to the receptor and on the results reported in the litterature, we have synthesized bombesin analogues which have been modified in the C-terminal Leu 13 -Leu 14 amide part. We have shown that modification in the C-terminal part of the bombesin strongly affected the biological activity in rat pancreatic acini. The most potent compound which is described here, H-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-Ψ(CH 2 )Leu-NH 2 , was able to recognize the bombesin receptor on rat pancreatic acini (Ki 4.3 nM) and antagonized the bombesin stimulated amylase secretion (Ki 7.7 nM).