Author: Lubomir Vezenkov

Agonism, Antagonism, and Inverse Agonism Bias at the Ghrelin Receptor Signaling

Journal of Biological Chemistry, 2015, Volume: 290, Issue: 45, Pages: 27021-27039, DOI: 10.1074/jbc.M115.659250

C. M’Kadmi, J.-P. Leyris, L. Onfroy, C. Gales, A. Sauliere, D. Gagne,  M. Damian, S. Mary, M. Maingot, S. Denoyelle, P. Verdie, J.-A. Fehrentz, J. Martinez, J.-L. Baneres, J. Marie


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 assocd. 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 addn. 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.

Triazole GHS-R1a antagonists JMV4208 and JMV3002 attenuate food intake, body weight, and adipose tissue mass in mice

Molecular and Cellular Endocrinology, 2014, Volume: 393, Issue: 1-2, Pages: 120-128,  DOI: 10.1016/j.mce.2014.06.003

M. Holubova, V. Nagelova, Z. Lacinova, M. Haluzik, D. Sykora, A. Moulin, A. L. Blayo, J. A. Fehrentz, J. Martinez, A. Stofkova, J. Jurcovicova, B. Zelezna, L. Maletinska


The only peripherally released orexigenic hormone, ghrelin, plays a key role in food intake and body wt. regulation.  Antagonizing the ghrelin receptor, GHS-R1a, represents a promising approach for anti-obesity therapy.  In our study, two novel GHS-R1a antagonists JMV4208 and JMV3002, which are trisubstituted 1,2,4-triazoles, decreased food intake in fasted lean mice in a dose-dependent manner, with ED50 values of 5.25 and 2.05 mg/kg, resp.  Both compds. were stable in mouse blood, with half-lives of 90 min (JMV4208) and 60 min (JMV3002), and disappeared from the blood 8 h after administration.  Fourteen days of treatment with the ghrelin antagonists (20 mg/kg twice a day) decreased food intake, body wt. and adipose tissue mass in mice with diet-induced obesity (DIO).  These results are likely attributable to an impact on food intake redn. and an attenuated expression of the lipogenesis-promoting enzymes (acetyl-CoA carboxylase 1 in s.c. fat and fatty acid synthase in s.c. and i.p. fat).  The decrease in fat mass neg. impacted circulating leptin levels.  These data suggest that JMV4208 and JMV3002 could be useful therapeutic agents for the treatment of obesity.

Silaproline Helical Mimetics Selectively Form an All-trans PPII Helix

Chemistry – A European Journal, 2014, Volume: 20, Issue: 44, Pages: 14240-14244, DOI: 10.1002/chem.201404820

C. Martin, B. Legrand, A. Lebrun, D. Berthomieu, J. Martinez, F. Cavelier


The polyproline II helix (PPII) is increasingly recognized as an important element in peptide and protein structures.  The discovery of pertinent PPII peptidomimetics is of great interest to tune phys. properties of the targeted structure.  A series of silaproline oligomers from dimer to pentamer were synthesized.  CD studies, NMR spectroscopy and mol. modeling revealed that the ribbon preferentially populates the polyproline type II secondary structure in both [D]chloroform and [D4]MeOH.  The characteristics of this new lipophilic PPII-like helix were detd.

Unprecedented Chain-Length-Dependent Conformational Conversion Between 11/9 and 18/16 Helix in α/β-Hybrid Peptides

Angewandte Chemie, 2014, Volume: 53, Issue: 48, Pages: 13131-13135, DOI: 10.1002/anie.201407329

B. Legrand, C. Andre, L. Moulat, E. Wenger, C. Didierjean, E. Aubert, M. Averlant-Petit, J. Martinez, M. Calmes, M. Amblard


α,β-Hybrid oligomers of varying lengths with alternating proteogenic α-amino acid and the rigid β2,3,3-trisubstituted bicyclic amino acid ABOC residues were studied using both x-ray crystal and NMR soln. structures.  While only an 11/9 helix was obtained in the solid state regardless of the length of the oligomers, conformational polymorphism as a chain-length-dependent phenomenon was obsd. in soln.  Consistent with DFT calcns., short oligomers adopted an 11/9 helix, whereas an 18/16 helix was favored for longer oligomers in soln.  A rapid interconversion between the 11/9 helix and the 18/16 helix occurred for oligomers of intermediate length.

Preclinical comparison of Al18F- and 68Ga-labeled gastrin-releasing peptide receptor antagonists for PET imaging of prostate cancer

Journal of Nuclear Medicine, 2014, Volume: 55, Issue: 12, Pages: 2050-2056,  DOI: 10.2967/jnumed.114.141143

K. l. L. S. Chatalic, G. M. Franssen, W. M. van Weerden, W. J. McBride,  P. Laverman, E. de Blois, B. Hajjaj, L. Brunel, D. M. Goldenberg, J.-A. Fehrentz,  J. Martinez, O. C. Boerman, M. de Jong,


Gastrin-releasing peptide receptor (GRPR) is overexpressed in human prostate cancer and is being used as a target for mol. imaging.  In this study, we report on the direct comparison of 3 novel GRPR-targeted radiolabeled tracers: Al18F-JMV5132, 68Ga-JMV5132, and 68Ga-JMV4168 (JMV5132 is NODA-MPAA-βAla-βAla-[H-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2], JMV4168 is DOTA-βAla-βAla-[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 Al18F.  JMV5132 was radiolabeled with 68Ga and 18F, and JMV4168 was labeled with 68Ga for comparison.  The inhibitory concn. of 50% values for binding GRPR of JMV4168, JMV5132, natGa-JMV4168, and natGa-JMV5132 were detd. in a competition-binding assay using GRPR-overexpressing PC-3 tumors.  The tumor-targeting characteristics of the compds. were assessed in mice bearing s.c. PC-3 xenografts.  Small-animal PET/CT images were acquired, and tracer biodistribution was detd. by ex vivo measurements.  Results: JMV5132 was labeled with 18F in a novel 1-pot, 1-step procedure within 20 min, without need for further purifn. and resulting in a specific activity of 35 MBq/nmol.  Inhibitory concn. of 50% values (in nM) for GRPR binding of JMV5132, JMV4168, natGa-JMV5132, natGa-JMV4168, and AlnatF-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), resp.  In mice with s.c. PC-3 xenografts, all tracers cleared rapidly from the blood, exclusively via the kidneys for 68Ga-JMV4168 and partially hepatobiliary for 68Ga-JMV5132 and Al18F-JMV5132.  Two hours after injection, the uptake of 68Ga-JMV4168, 68Ga-JMV5132, and Al18F-JMV5132 in PC-3 tumors was 5.96 ± 1.39, 5.24 ± 0.29, 5.30 ± 0.98 (percentage injected dose per g), resp.  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 resoln. obsd. for Al18F-JMV5132.  Conclusion: JMV5132 could be rapidly and efficiently labeled with 18F.  Al18F-JMV5132, 68Ga-JMV5132, and 68Ga-JMV4168 all showed comparable high and specific accumulation in GRPR-pos. PC-3 tumors.  These new PET tracers are promising candidates for future clin. translation.

Imidazopyridine-fused [1,3]-diazepinones: Synthesis and antiproliferative activity

European Journal of Medicinal Chemistry, 2014, Volume: 75, Pages: 382-390,  DOI: 10.1016/j.ejmech.2014.01.044

A. Gallud, O. Vaillant, L.T. Maillard, D. Arama, J. Dubois, M. Maynadier, V. Lisowski, M. Garcia, J. Martinez, N. Masurier


A series of 15 pyrido-imidazo-1,3-diazepin-5-ones and pyrido-1,3-diazepine-2,5-diones were synthesized and their anticancer activities were evaluated.  Among tested compds. on a cell lines panel, compd. I presents the best growth inhibition activity on 21 cell lines with a cytotoxic effect on MDA-MB-435 melanoma cells.  This compd. led to deep cell morphol. changes and revealed to be an inhibitor of the Hepatocyte progenitor kinase-like kinase (HGK), which is known to be implicated in the migration, adhesion and invasion of various tumor cells.

Synthesis and reactivity of pyrrolo[3,2-d][1,3]oxazine-2,4-dione. Access to new pyrrolo[3,2-e][1,4]diazepine-2,5-diones

Tetrahedron, 2014, Volume: 70, Issue: 31, Pages: 4631-4639, DOI: 10.1016/j.tet.2014.05.046

J. Malcor, Y. Brouillette, J. Graffion, K. Spielmann, N. Masurier, L. T. Maillard, J. Martinez, V. Lisowski


A convenient synthesis of pyrrolo[3,2-d][1,3]oxazine-2,4-dione is described and its reactivity towards various nucleophiles studied.  The regioselective ring opening of pyrrolo[3,2-d][1,3]oxazine-2,4-dione or its N-alkylated analog in the presence of alanine or proline afforded, resp., imidazolidinedione and 2 N-protected pyrrolo[3,2-e][1,4]diazepines in a one-pot process.  In a last part of this study, an alternative route to produce a library of eight non protected pyrrolo[3,2-e][1,4]diazepine-2,5-diones is described to overcome the limited reactivity of pyrrolo[3,2-d][1,3]oxazine-2,4-dione.

Laser desorption ionization mass spectrometry of peptides on a hybrid CHCA organic-inorganic matrix

Journal of Proteomics, Volume, 2014, 108, Pages: 369-372,  DOI: 10.1016/j.jprot.2014.06.005

C. Fleith, S. Cantel, G. Subra, A. Mehdi, J. Ciccione, J. Martinez, C. Enjalbal


We report applications of new hybrid org.-inorg. silica based materials as laser desorption/ionization (LDI)-promoting surfaces for high-throughput identification of peptides.  The driving force of our work was to design a new material composed of a conventional MALDI matrix covalently attached to silica with a high org./inorg. ratio in order to improve the UV absorption by such LDI hybrid matrixes.  Amorphous CHCA-functionalized silica presenting an org. content up to 1.3 mmol g-1 (around 40% in wt. from TGA and elementary anal. measurements) gave very interesting LDI performances in terms of detection sensitivity as well as relative ionization discrepancy (spectral suppression) through the analyses of small synthetic peptide mixts. (550-1300 Da) taking CHCA and amorphous silica as model matrixes for control expts.

N- and O-acetylation of threonine residues in the context of proteomics

Journal of Proteomics, Volume, 2014, 108, Pages: 369-372,  DOI: 10.1016/j.jprot.2014.06.005

J.-B. Boyer, A. Dedieu, J. Armengaud, P. Verdie, G. Subra, J. Martinez, C. Enjalbal


The detection of post-translational modifications (PTMs) of proteins is a matter of intensive research.  Among all possible pitfalls that may lead to misidentifications, the chem. stability of modified peptides is scarcely questioned.  Global proteomic studies devoted to protein acetylation are becoming popular.  Thus, we were concerned about the intrinsic stability of O-acetylated peptides because of the O-N acyl transfer reactivity occurring when an amino moiety is present in the vicinity of the acylated hydroxyl group.  Here, the behavior of isomeric O- and N-acetylated, N-terminal threonine-contg. peptides was explored in a std. proteomic workflow.  We demonstrated a strong chem. instability of O-acetylation, which prevents its detection.

Ghrelin agonist JMV 1843 increases food intake, body weight and expression of orexigenic neuropeptides in mice

Physiological Research (Prague, Czech Republic), Volume: 62, Issue: 4, Pages: 435-444, ISSN: 0862-8408

M. Holubova, A. Spolcova, Z. Demianova, D. Sykora, J. A. Fehrentz, J. Martinez, A. Stofkova, J. Jurcovicova, J. Drapalova, Z. Lacinova, M. Haluzik, B. Zelezna,  L. Maletinska


Ghrelin and agonists of its receptor GHS-R1a are potential substances for the treatment of cachexia.  In the present study, we investigated the acute and long-term effects of the GHS-R1a agonist JMV 1843 (H-Aib-DTrp-D-gTrp-CHO) on food intake, body wt. and metabolic parameters in lean C57BL/6 male mice.  Addnl., we examd. stability of JMV 1843 in mouse blood serum.  A single s.c. injection of JMV 1843 (0.01-10 mg/kg) increased food intake in fed mice in a dose-dependent manner, up to 5-times relative to the saline-treated group (ED50=1.94 mg/kg at 250 min).  JMV 1843 was stable in mouse serum in vitro for 24 h, but was mostly eliminated from mouse blood after 2 h in vivo.  Ten days of treatment with JMV 1843 (s.c. administration, 10 or 20 mg/kg/day) significantly increased food intake, body wt. and mRNA expression of the orexigenic neuropeptide Y and agouti-related peptide in the medial basal hypothalamus and decreased the expression of uncoupling protein 1 in brown adipose tissue.  Our data suggest that JMV 1843 could have possible future uses in the treatment of cachexia.