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The ternary complex (eIF2·GTP·Met-tRNA

The growth hormone secretagogue receptor (GHSR) is a G protein-coupled receptor that regulates essential physiological functions. In particular, activation of GHSR in response to its endogenous agonist ghrelin promotes food intake and blood glucose increase. Therefore, compounds aimed at blocking GHSR signaling constitute potential options against obesity-related metabolic disorders. We have previously developed potent ligands of GHSR based on a triazole scaffold. Here, we report a new 3,4,5-trisubstituted 1,2,4-triazole compound, named JMV 6616, that potently blocks GHSR activity in vitro and in vivo. Specifically, in HEK293T cells JMV 6616 behaves as an inverse agonist since it binds to GHSR and inhibits its ghrelin-independent signaling. Accordingly, using purified labeled GHSR assembled into lipid nanodiscs we found that JMV 6616 decreases GHSR-catalyzed G protein activation and stabilizes an inactive receptor conformation. Importantly, JMV 6616 also acts on native GHSR since it blocks the insulinostatic effect of ghrelin in pancreatic islets. In mice, JMV 6616 inhibits blood glucose-raising effects of ghrelin treatment and the orexigenic actions of acute ghrelin administration. Together, our data suggest that this triazole-derived modulator of GHSR holds promise to mitigate several pathological features associated with eating and metabolic disorders.

Ghrelin is a peptide hormone mainly secreted from gastrointestinal tract that acts via the growth hormone secretagogue receptor (GHSR), which is highly expressed in the brain. Strikingly, the accessibility of ghrelin to the brain seems to be limited and restricted to few brain areas. Previous studies in mice have shown that ghrelin can access the brain via the blood-cerebrospinal fluid (CSF) barrier, an interface constituted by the choroid plexus and the hypothalamic tanycytes. Here, we performed a variety of in vivo and in vitro studies to test the hypothesis that the transport of ghrelin across the blood-CSF barrier occurs in a GHSR-dependent manner. In vivo, we found that the uptake of systemically administered fluorescent ghrelin in the choroid plexus epithelial (CPE) cells and in hypothalamic tanycytes depends on the presence of GHSR. Also, we detected lower levels of CSF ghrelin after a systemic ghrelin injection in GHSR-deficient mice, as compared to WT mice. In vitro, the internalization of fluorescent ghrelin was reduced in explants of choroid plexus from GHSR-deficient mice, and unaffected in primary cultures of hypothalamic tanycytes derived from GHSR-deficient mice. Finally, we found that the GHSR mRNA is detected in a pool of CPE cells, but is nearly undetectable in hypothalamic tanycytes with current approaches. Thus, our results suggest that circulating ghrelin crosses the blood-CSF barrier mainly by a mechanism that involves the GHSR, and also possibly via a GHSR-independent mechanism.

The growth hormone secretagogue receptor (GHSR) signals in response to ghrelin, but also acts via ligand-independent mechanisms that include either constitutive activation or interaction with other G protein-coupled receptors, such as the dopamine 2 receptor (D2R). A key target of GHSR in neurons is voltage-gated calcium channels type 2.2 (CaV2.2). Recently, the liver-expressed antimicrobial peptide 2 (LEAP2) was recognized as a novel GHSR ligand, but the mechanism of action of LEAP2 on GHSR is not well understood. Here, we investigated the role of LEAP2 on the canonical and non-canonical modes of action of GHSR on CaV2.2 function. Using a heterologous expression system and patch-clamp recordings, we found that LEAP2 impairs the reduction of CaV2.2 currents induced by ghrelin-evoked and constitutive GHSR activities, acting as a GHSR antagonist and inverse agonist, respectively. We also found that LEAP2 prevents GHSR from modulating the effects of D2R signaling on CaV2.2 currents, and that the GHSRbinding N-terminal region LEAP2 underlies these effects. Using purified labeled receptors assembled into lipid nanodiscs and Forster Resonance Energy Transfer (FRET) assessments, we found that the N-terminal region of LEAP2 stabilizes an inactive conformation of GHSR that is dissociated from Gq protein and, consequently, reverses the effect of GHSR on D2R-dependent Gi activation. Thus, our results provide critical molecular insights into the mechanism mediating LEAP2 modulation of GHSR., Instituto Multidisciplinario de Biología Celular

Liver-expressed antimicrobial peptide 2 (LEAP2) was recently recognized as an endogenous ligand for the growth hormone secretagogue receptor (GHSR), which also is a receptor for the hormone ghrelin. LEAP2 blocks ghrelin-induced activation of GHSR and inhibits GHSR constitutive activity. Since fluorescence-based imaging and pharmacological analyses to investigate the biology of GHSR require reliable probes, we developed a novel fluorescent GHSR ligand based on the N-terminal LEAP2 sequence, hereafter named F-LEAP2. In vitro, F-LEAP2 displayed binding affinity and inverse agonism to GHSR similar to LEAP2. In a heterologous expression system, F-LEAP2 labeling was specifically observed in the surface of GHSR-expressing cells, in contrast to fluorescent ghrelin labeling that was mainly observed inside the GHSR-expressing cells. In mice, centrally-injected F-LEAP2 reduced ghrelin-induced food intake, in a similar fashion to LEAP2, and specifically labeled cells in GHSR-expressing brain areas. Thus, F-LEAP2 represents a valuable tool to study the biology of GHSR in vitro and in vivo.

Plasmodium falciparum is responsible for most of the cases of malaria and its resistance to established antimalarial drugs is a major issue. Thus, new chemotherapies are needed to fight the emerging multi-drug resistance of P. falciparum malaria, like choline analogues targeting plasmodial phospholipidic metabolism. Here we describe the synthesis of amidoxime derivatives as prodrug candidates of reverse-benzamidines and hybrid compounds able to mimic choline, as well as the design of a new series of asymmetrical bis-cationic compounds. Bioconversion studies were conducted on amidoximes in asymmetrical series and showed that amidoxime prodrug strategy could be applied on C-alkylamidine moieties, like benzamidines and that N-substituents did not alter the bioconversion of amidoximes. The antimalarial activity of the three series of compounds was evaluated in vitro against P. falciparum and in vivo against P. vinckei petteri in mice.

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International audience; The ghrelin receptor or growth hormone secretagogue receptor (GHSR) is a G-protein-coupled receptor that controls growth hormone and insulin secretion, food intake, and reward-seeking behaviors. Liver-expressed antimicrobial peptide 2 (LEAP2) was recently described as an endogenous antagonist of GHSR. Here, we present a study aimed at delineating the structural determinants required for LEAP2 activity toward GHSR. We demonstrate that the entire sequence of LEAP2 is not necessary for its actions. Indeed, the N-terminal part alone confers receptor binding and activity to LEAP2. We found that both LEAP2 and its N-terminal part behave as inverse agonists of GHSR and as competitive antagonists of ghrelin-induced inositol phosphate production and calcium mobilization. Accordingly, the N-terminal region of LEAP2 is able to inhibit ghrelin-induced food intake in mice. These data demonstrate an unexpected pharmacological activity for LEAP2 that is likely to have an important role in the control of ghrelin response under normal and pathological conditions.

The growth hormone secretagogue receptor (GHSR) and dopamine receptor (D2R) have been shown to oligomerize in hypothalamic neurons with a significant effect on dopamine signaling, but the molecular processes underlying this effect are still obscure. We used here the purified GHSR and D2R to establish that these two receptors assemble in a lipid environment as a tetrameric complex composed of two each of the receptors. This complex further recruits G proteins to give rise to an assembly with only two G protein trimers bound to a receptor tetramer. We further demonstrate that receptor heteromerization directly impacts on dopamine-mediated Gi protein activation by modulating the conformation of its α-subunit. Indeed, association to the purified GHSR:D2R heteromer triggers a different active conformation of Gαi that is linked to a higher rate of GTP binding and a faster dissociation from the heteromeric receptor. This is an additional mechanism to expand the repertoire of GPCR signaling modulation that could have implications for the control of dopamine signaling in normal and physiopathological conditions.

A series of 5-substituted thieno[3,2-e][1,4]diazepin-2-ones was synthesized in four steps from methyl 3-aminothiophene-2-carboxylate. After the coupling of 3-aminothiophene with α-amino acids, the key final step that involves an uncatalysed Pictet–Spengler reaction allowed the cyclization of the seven-membered diazepinone ring. The reaction was first optimized and then exemplified in three different series (phenylalanine, alanine and proline) that led to 24 target diazepinones, which includes 19 optically pure diastereomers.

Introducing a second chiral center on our previously described 1,2,4-triazole, allowed us to increase diversity and elongate the 'C-terminal part' of the molecule. Therefore, we were able to explore mimics of the substance P analogs described as inverse agonists. Some compounds presented affinities in the nanomolar range and potent biological activities, while one exhibited a partial inverse agonist behavior similar to a Substance P analog.

A series of 20 optically pure 3,4-dihydro-5H-pyrido[1',2':1,2]imidazo[4,5-d][1,3]diazepin-5-ones which form a new family of azaheterocycle-fused [1,3]diazepines were synthesized in four steps with 17-66% overall yields. The key step consists of a selective C-acylation reaction of easily accessible 2-aminoimidazo[1,2-a]pyridine at C-3.

Symmetrical N,N′-diarylureas: 1,3-bis(3,4-dichlorophenyl)-, 1,3-bis[4-chloro-3-(trifluoromethyl)phenyl]- and 1,3-bis[3,5-bis(trifluoromethyl)phenyl]urea, were identified as potent activators of the eIF2α kinase heme regulated inhibitor. They reduce the abundance of the eIF2·GTP· tRNA i Met ternary complex and inhibit cancer cell proliferation. An optimization process was undertaken to improve their solubility while preserving their biological activity. Non-symmetrical hybrid ureas were generated by combining one of the hydrophobic phenyl moieties present in the symmetrical ureas with the polar 3-hydroxy-tolyl moiety. O-alkylation of the later added potentially solubilizing charge bearing groups. The new non-symmetrical N,N′-diarylureas were characterized by ternary complex reporter gene and cell proliferation assays, demonstrating good bioactivities. A representative sample of these compounds potently induced phosphorylation of eIF2α and expression of CHOP at the protein and mRNA levels. These inhibitors of translation initiation may become leads for the development of potent, non-toxic, and target specific anti-cancer agents.

A new series of dissymmetric hemifluorocarbon bolaamphiphiles were designed to investigate their main characteristics as a non-viral gene transfer carrier. The dissymmetric functionalization of diiodoperfluorooctane led to bolaamphiphile molecules composed of a partially fluorocarbon core end-capped with a glycoside and an ammonium salt. The physical–chemical results, cytotoxic effect, gene complexation and transfection efficiency were analyzed and functional relationships were addressed. The study clearly showed that the chemical structure of bolaamphiphiles influences the complexation with DNA, gene transfer, and cytotoxicity. Two different polar head groups (histidine and lysine) have been tested for their efficiency to complex DNA. The impact of the side chain of bolaamphiphile on the interaction with DNA was also investigated. Good transfection activity was demonstrated for partially fluorinated bolaamphiphile having lysine head group. Weak cell cytotoxicity, positive surface charge of bolaplexes, well-defined structure, and protection from the DNAses seem to be essential characteristics for efficient transfection.

International audience; The G protein-coupled receptor GHS-R1a mediates ghrelin-induced growth hormone secretion, food intake, and reward-seeking behaviors. GHS-R1a signals through Gq, Gi/o, G13, and arrestin. Biasing GHS-R1a signaling with specific ligands may lead to the development of more selective drugs to treat obesity or addiction with minimal side effects. To delineate ligand selectivity at GHS-R1a signaling, we analyzed in detail the efficacy of a panel of synthetic ligands activating the different pathways associated with GHS-R1a in HEK293T cells. Besides β-arrestin2 recruitment and ERK1/2 phosphorylation, we monitored activation of a large panel of G protein subtypes using a bioluminescence resonance energy transfer-based assay with G protein-activation biosensors. We first found that unlike full agonists, Gq partial agonists were unable to trigger β-arrestin2 recruitment and ERK1/2 phosphorylation. Using G protein-activation biosensors, we then demonstrated that ghrelin promoted activation of Gq, Gi1, Gi2, Gi3, Goa, Gob, and G13 but not Gs and G12. Besides, we identified some GHS-R1a ligands that preferentially activated Gq and antagonized ghrelin-mediated Gi/Go activation. Finally, we unambiguously demonstrated that in addition to Gq, GHS-R1a also promoted constitutive activation of G13. Importantly, we identified some ligands that were selective inverse agonists toward Gq but not of G13. This demonstrates that bias at GHS-R1a signaling can occur not only with regard to agonism but also to inverse agonism. Our data, combined with other in vivo studies, may facilitate the design of drugs selectively targeting individual signaling pathways to treat only the therapeutically relevant function.

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.

The growing recognition of inhibition of translation initiation as a new and promising paradigm for mechanism-based anti-cancer therapeutics is driving the development of potent, specific, and druggable inhibitors. The 3,3-diaryloxindoles were recently reported as potential inhibitors of the eIF2·GTP·Met-tRNAi(Met) ternary complex assembly and 3-{5-tert-butyl-2-hydroxyphenyl}-3-phenyl-1,3-dihydro-2H-indol-2-one #1181 was identified as the prototypic agent of this chemotype. Herein, we report our continuous effort to further develop this chemotype by exploring the structural latitude toward different polar and hydrophobic substitutions. Many of the novel compounds are more potent than the parent compound in the dual luciferase ternary complex reporter assay, activate downstream effectors of reduced ternary complex abundance, and inhibit cancer cell proliferation in the low μM range. Moreover, some of these compounds are decorated with substituents that are known to endow favorable physicochemical properties and as such are good candidates for evaluation in animal models of human cancer.

Translation initiation factors are over-expressed and/or activated in many human cancers and may contribute to their genesis and/or progression. Removal of physiologic restraints on translation initiation causes malignant transformation. Conversely, restoration of physiological restrains on translation initiation reverts malignant phenotypes. Here, we extensively characterize the anti-cancer activity of two small molecule inhibitors of translation initiation: #1181, which targets the eIF2∙GTP∙Met-tRNA i ternary complex, and 4EGI-1, which targets the eIF4F complex. In vitro, both molecules inhibit translation initiation, abrogate preferentially translation of mRNAs coding for oncogenic proteins, and inhibit proliferation of human cancer cells. In vivo, both #1181 and 4EGI-1 strongly inhibit growth of human breast and melanoma cancer xenografts without any apparent macroscopic- or microscopictoxicity. Mechanistically, #1181 phosphorylates eIF2α while 4EGI-1 disrupts eIF4G/ eIF4E interaction in the tumors excised from mice treated with these agents. These data indicate that inhibition of translation initiation is a new paradigm in cancer therapy.

Some new optically pure title compounds such as (VIII) and (XII) are prepared via selective C-3 acylation.

Translation initiation plays a critical role in cellular homeostasis, proliferation, differentiation and malignant transformation. Consistently, increasing the abundance of the eIF2–GTP–tRNAi Met translation initiation complex transforms normal cells and contributes to cancer initiation and the severity of some anemias. The chemical modifiers of the eIF2–GTP–tRNAi Met ternary complex are therefore invaluable tools for studying its role in the pathobiology of human disorders and for determining whether this complex can be pharmacologically targeted for therapeutic purposes. Using a cell-based assay, we identified N,N9-diarylureas as unique inhibitors of ternary complex accumulation. Direct functional-genetic and biochemical evidence demonstrated that the N,N9-diarylureas activate heme-regulated inhibitor kinase, thereby phosphorylating eIF2a and reducing the abundance of the ternary complex. Using tumor cell proliferation in vitro and tumor growth in vivo as paradigms, we demonstrate that N,N9-diarylureas are potent and specific tools for studying the role of eIF2–GTP–tRNAi Met ternary complex in the pathobiology of human disorders.

International audience; Within the frame of the design of prodrug candidates to deliver a C-alkylamidine antimalarial agent, we showed that specific O-substitutions were needed on the alkylamidoxime structure. Among the newly synthesized molecules, bis-oxadiazolone and bis-O-methylsulfonylamidoxime derivatives induced a complete clearance of parasitemia in mice after oral administration.

The multistep synthesis of a new series of dissymmetric hemifluorocarbon bolaamphiphiles designed for gene transport is described. The dissymmetric functionalization of diiodoperfluoroctane leads to bolaamphiphile molecules composed of a partially fluorocarbon core end-capped with a glycoside and an ammonium salt derived from histidine or lysine. Initial biological results indicate that one of the bolaamphiphile—end-capped with a lysine and a lactobionamide residue—induces a remarkably low cytotoxicity on COS-7 cells and, when self-assembled with DNA plasmid, generates a significant in vitro transfection efficiency without the addition of any fusogenic lipid.