Rochdi Bouhelal

The 7 TM G-protein-coupled receptor target family

Chemical biology approaches have a long history in the exploration of the G‐protein‐coupled receptor (GPCR) family, which represents the largest and most important group of targets for therapeutics. The analysis of the human genome revealed a significant number of new members with unknown physiological function which are today the focus of many reverse pharmacology drug‐discovery programs. As the seven hydrophobic transmembrane segments are a defining common structural feature of these receptors, and as signaling through heterotrimeric Gu2005proteins is not demonstrated in all cases, these proteins are also referred to as seven transmembrane (7u2009TM) or serpentine receptors. This review summarizes important historic milestones of GPCR research, from the beginning, when pharmacology was mainly descriptive, to the age of modern molecular biology, with the cloning of the first receptor and now the availability of the entire human GPCR repertoire at the sequence and protein level. It shows how GPCR‐directed drug discovery was initially based on the careful testing of a few specifically made chemical compounds and is today pursued with modern drug‐discovery approaches, including combinatorial library design, structural biology, molecular informatics, and advanced screening technologies for the identification of new compounds that activate or inhibit GPCRs specifically. Such compounds, in conjunction with other new technologies, allow us to study the role of receptors in physiology and medicine, and will hopefully result in novel therapies. We also outline how basic research on the signaling and regulatory mechanisms of GPCRs is advancing, leading to the discovery of new GPCR‐interacting proteins and thus opening new perspectives for drug development. Practical examples from GPCR expression studies, HTS (high‐throughput screening), and the design of monoamine‐related GPCR‐focused combinatorial libraries illustrate ongoing GPCR chemical biology research. Finally, we outline future progress that may relate today’s discoveries to the development of new medicines.

Study of the calcium dynamics of the human α4β2, α3β4 and α1β1γδ nicotinic acetylcholine receptors

Abstract. In this study three major subtypes of nicotinic acetylcholine receptors were characterized pharmacologically using the calcium influx through the ion channel as a robust functional assay system. Human α3β4 receptors and α4β2 receptors were cloned and stably expressed in HEK293 cells. [125I]epibatidine saturation binding yielded a Bmax of 4420±840xa0fmol/mg protein for the α4β2 receptor (n=4) and 518±15xa0fmol/mg protein for the α3β4 receptor (n=4). As a source for muscle type of nicotinic receptor, the TE671 cell line was used which expresses endogenously the human fetal α1β1γδ subtype of nicotinic receptor. Stimulation of these nicotinic receptor subtypes in the different cell lines led to calcium transients that peaked 5–10xa0s after agonist application and declined thereafter. Eleven agonists were tested in this study and their efficacy and potency at the three nicotinic receptor subtypes were determined (epibatidine, ABT594, anatoxin, ABT418, nicotine, DMPP, cytisine, ABT089, choline, GTS21, AAR17779). This pharmacological characterization of agonist-induced elevation of intracellular free Ca2+ revealed a distinct rank order of agonist potency for each receptor subtype. Epibatidine showed at all three subtypes the highest potency and was a full agonist. The agonist-elicited response could be blocked by co-incubation of different antagonists from which mecamylamine did not display a strong subtype specificity. These data illustrate that the assessment of calcium transients upon receptor stimulation is a powerful tool for rapid examination of the functional properties of nicotinic receptors.

Cloning, expression and pharmacological characterisation of the mouse somatostatin sst5 receptor

Abstract The mouse somatostatin (somatotropin release inhibiting factor, SRIF) sst 5 receptor coding sequence was cloned from a mouse BALB/c genomic library. It shows 97% and 81% homology with the corresponding rat and human receptors, respectively. The msst 5 receptor messenger RNA (mRNA) is present at low levels in the adult mouse brain, with significant expression in a few nuclei only, e.g. in the septum (lateral septal nuclei) or the amygdala (medial amygdaloid nucleus); very few signals were observed in the mesencephalon, metencephalon, and myelencephalon (except the dorsal motor nucleus of the vagus nerve). The msst 5 receptor was stably expressed in the hamster fibroblast cell line CCL39-SRE-Luci, which harbours the luciferase reporter gene driven by the serum responsive element. [ 125 I]LTT-SRIF-28 ([Leu 8 , D-Trp 22 , 125 I-Tyr 25 ]-SRIF-28), [ 125 I]Tyr 10 -CST, [ 125 I]CGP 23996, and [ 125 I]Tyr 3 -octreotide labelled msst 5 receptors with high affinity (pK d values: 11.0, 10.15, 9.75 and 9.43) and in a saturable manner, but defined different Bmax values: 697, 495, 540 and 144 fmoles/mg, respectively. [ 125 I]LTT-SRIF-28-labelled sites displayed the following rank order: SRIF-28> rCST-14> somatuline > CGP-23996= SRIF-14= octreotide, whereas [ 125 I]Tyr 3 -octreotide-labelled sites displayed a different profile: octreotide > SRIF-28> rCST-14= somatuline > SRIF-14> CGP-23996. The pharmacological profiles determined with [ 125 I]LTT-SRIF-28, [ 125 I]CGP 23996 and [ 125 I]Tyr 10 -CST correlated highly significantly (r 2 =0.88–0.99), whereas [ 125 I]Tyr 3 -octreotide binding was rather divergent (r 2 =0.77). Also, human and mouse sst 5 receptor profiles are very different, e.g. r 2 =0.385 for [ 125 I]Tyr 10 -CST and r 2 =0.323 for [ 125 I]LTT-SRIF-28-labelled sites. Somatostatin induces expression of luciferase reporter gene in CCL39-SRE-Luci cells. The profile was consistent with a msst 5 receptor-mediated effect although apparent potency in the luciferase assay was much reduced compared to radioligand binding data: Octreotide = SRIF-28> rCST-14= SRIF-14= CGP-23996. Octreotide, SRIF-28, BIM23052 and D Tyr Cyanamid 154806 behaved as full or nearly full agonists in comparison to SRIF-14, whereas the other compounds had relative efficacies of 40 to 70%. The present study shows that agonists radioligands define apparently different receptor populations in terms of number of sites and pharmacological profile in cells expressing a single recombinant receptor. These variations suggest that the conformation of the ligand receptor complex may vary depending on the agonist. Further, the msst 5 receptor, although primarily coupled to Gi/Go proteins, is able to stimulate luciferase gene expression driven by the serum responsive element. Finally, it is suggested that putative sst 2 selective agonists e.g. octreotide, RC160 or BIM23027 show similar or higher potency at msst 5 receptors than SRIF-14.

Blocking Metabotropic Glutamate Receptor Subtype 7 (mGlu7) via the Venus Flytrap Domain (VFTD) Inhibits Amygdala Plasticity, Stress, and Anxiety-related Behavior

Background: Behavioral genetics identified mGlu7 as a key regulator of brain emotion circuits. Results: An mGlu7-selective, Venus flytrap domain (VFTD)-directed antagonist inhibits fear, synaptic plasticity, stress, and anxiety in rodents. Conclusion: Pharmacological blockers of mGlu7 may represent promising future anxiolytics and antidepressants in man. Significance: The VFTD region of class C GPCRs provides a promising target for computer-assisted drug design. The metabotropic glutamate receptor subtype 7 (mGlu7) is an important presynaptic regulator of neurotransmission in the mammalian CNS. mGlu7 function has been linked to autism, drug abuse, anxiety, and depression. Despite this, it has been difficult to develop specific blockers of native mGlu7 signaling in relevant brain areas such as amygdala and limbic cortex. Here, we present the mGlu7-selective antagonist 7-hydroxy-3-(4-iodophenoxy)-4H-chromen-4-one (XAP044), which inhibits lateral amygdala long term potentiation (LTP) in brain slices from wild type mice with a half-maximal blockade at 88 nm. There was no effect of XAP044 on LTP of mGlu7-deficient mice, indicating that this pharmacological effect is mGlu7-dependent. Unexpectedly and in contrast to all previous mGlu7-selective drugs, XAP044 does not act via the seven-transmembrane region but rather via a binding pocket localized in mGlu7s extracellular Venus flytrap domain, a region generally known for orthosteric agonist binding. This was shown by chimeric receptor studies in recombinant cell line assays. XAP044 demonstrates good brain exposure and wide spectrum anti-stress and antidepressant- and anxiolytic-like efficacy in rodent behavioral paradigms. XAP044 reduces freezing during acquisition of Pavlovian fear and reduces innate anxiety, which is consistent with the phenotypes of mGlu7-deficient mice, the results of mGlu7 siRNA knockdown studies, and the inhibition of amygdala LTP by XAP044. Thus, we present an mGlu7 antagonist with a novel molecular mode of pharmacological action, providing significant application potential in psychiatry. Modeling the selective interaction between XAP044 and mGlu7s Venus flytrap domain, whose three-dimensional structure is already known, will facilitate future drug development supported by computer-assisted drug design.

Monitoring Gq-coupled receptor response through inositol phosphate quantification with the IP-One assay

Introduction: G-protein-coupled receptors (GPCRs) are transmembrane proteins that play a key role in the signal transduction of extracellular stimuli. GPCRs associate to a complex assembly of intracellular proteins regulating a large variety of signaling pathways. In particular, the production of inositol 1,4,5 triphosphate (IP3) signs the activation of Gq-coupled receptors. However, its very short half-life makes its assessment too challenging for drug screening operations and the monitoring of calcium release, triggered by IP3, has been extensively used as a downstream readout of this signaling pathway. Recently, a new homogeneous time-resolved fluorescence (HTRF) assay, detecting a downstream metabolite of IP3, inositol monophosphate (IP1), has overcome the drawbacks of the IP3 quantification, allowing its use in primary or secondary screening. Areas covered: This review provides an overview of the use of the IP-One assay in screening processes, providing comparisons with results obtained with other existing techniques traditionally used to investigate Gq-coupled receptors. Moreover, the review highlights two key features of the IP-One assay, the discrimination of slow acting compounds and the characterization of inverse agonists, which are impossible to achieve using calcium release. Expert opinion: The IP-One assay is well established to perform screening in the pharmaceutical industry. A number of criteria can be taken into account, including the impact of the sensitivity improvement of the assay, to position the IP-One assay in the different stages of the drug screening process. Moreover, the IP-One assay can be used as a valuable solution to investigate new research concepts such as ligand-biased signaling or receptor heteromerization.

AFQ056/mavoglurant, a novel clinically effective mGluR5 antagonist: identification, SAR and pharmacological characterization.

Here we describe the identification, structure-activity relationship and the initial pharmacological characterization of AFQ056/mavoglurant, a structurally novel, non-competitive mGlu5 receptor antagonist. AFQ056/mavoglurant was identified by chemical derivatization of a lead compound discovered in a HTS campaign. In vitro, AFQ056/mavoglurant had an IC50 of 30 nM in a functional assay with human mGluR5 and was selective over the other mGluR subtypes, iGluRs and a panel of 238 CNS relevant receptors, transporter or enzymes. In vivo, AFQ056/mavoglurant showed an improved pharmacokinetic profile in rat and efficacy in the stress-induced hyperthermia test in mice as compared to the prototypic mGluR5 antagonist MPEP. The efficacy of AFQ056/mavoglurant in humans has been assessed in L-dopa induced dyskinesia in Parkinsons disease and Fragile X syndrome in proof of principle clinical studies.

Design and Synthesis of Selective and Potent Orally Active S1P5 Agonists

The immunomodulatory drug fingolimod (FTY720, 2-amino-2[2-(4-octylphenyl)ethyl]propane-1,3-diol), derived from a fungal metabolite (ISP-1, myriocin), is phosphorylated in vivo by sphingosine kinases to produce (R)-FTY720-phosphate (FTY720-P). 2] FTY720-P activates sphingosine-1-phosphate (S1P) receptors S1P1, S1P3, S1P4, and S1P5 at low nanomolar concentrations and is inactive toward the S1P2 receptor. The FTY720-P-mediated activation of the S1P1 receptor on lymphocytes induces receptor internalization, which attenuates T-cell response to S1P gradients, preventing their egress from secondary lymphoid tissues. In addition to playing a role in the immune system, all S1P receptors except S1P4 are also found differentially expressed in the central nervous system and on various tumor cell types. 6] Although the precise regulation of these receptors by locally released S1P remains unclear, S1P receptors are thought to play a role in such events as astrocyte migration, oligodendrocyte differentiation, and cell survival and neurogenesis. To assess the relevance of individual S1P receptor subtypes for the activity of FTY720-P, selective agonists are required. Because S1P5 receptors are expressed on oligodendrocytes, and S1P5 receptors are thought to play a role in oligodendrocyte differentiation and survival, we focused on the development of S1P5 agonists. By using a highthroughput screening calcium mobilization assay with GPCR priming and FLIPR technology, we discovered benzamide 1, which has good in vitro potency toward the S1P5 receptor (EC50=270 nm), but has modest selectivity against S1P1 (EC50=3140 nm) and S1P4 (EC50=100 nm). Herein we report our studies of various benzamide modifications carried out to improve the selectivity, bioactivity, pharmacokinetic properties, and ancillary profile of 1, ultimately resulting in the discovery of potent and very selective S1P5 agonists. To guide the optimization process, homology models of all S1P receptors were built from a crystal structure of bovine rhodopsin (PDB ID: 1F88). Docking experiments of 1 into these models revealed a possible location of the binding site, some essential features of the interactions, and indicated potential regions for gaining selectivity and improving potency. In these complexes (Figure 1), 1 adopts a twisted conformation with the aniline ring, ~708 out of the benzamide plane and stabilized by a hydrogen bond between the aniline NH group and the amide carbonyl. In the S1P5 receptor complex, the amide group forms a hydrogen bond with OG1-Thr120. The benzamide phenyl ring lies in a large hydrophobic pocket surrounded by Phe196, Phe201, Phe268, Leu119, Trp264, Leu267, and Leu271. The aniline ring undergoes a T-shaped interaction with Phe116 and hydrophobic contacts with Leu271 and Leu292. The ortho-methyl substituents fill a small pocket formed by Tyr89, Val115, and Leu292 on one side, and sit at the face of Phe196 on the other side. Inspection of sequence alignments (Figure 2) revealed two positions, one in transmembrane (TM) helix TM3 (115, S1P5 sequence) and one in TM5 (192), where S1P5 has smaller residues lining the binding site, thus creating putative pockets. We hypothesized that filling these pockets with atoms from our ligands should lead to high selectivity for the S1P5 receptor. Position 2 on the benzamide core, which was closest to the hypothesized pocket around Val115, was therefore extensively modified. Syntheses of derivative 1A–L (Scheme 1) began with 3-fluorobromobenzene 2, which was converted into acid 3 by reaction with lithium diisopropylamide (LDA) and carbon dioxide. Nucleophilic substitution of the fluorine atom with trimethylaniline at 78 8C yielded 4. This intermediate was then used in various ways. Copper-catalyzed nucleophilic substitution of the bromine atom with various alcohols yielded ethers 5D–N, which were amidated with ammonia using chlorodimethoxytriazine for activation to yield 1D–J. Palladium-catalyzed substitution of the bromine atom in acid 4 with various alkylstannanes yielded 6A–C, which were amidated as described above to yield 1A–C. Alternatively, palladium-catalyzed substitution of the bromine atom with tributyl-(1-ethoxyvinyl)stannane yielded 9, which was cyclized to 1L by reaction with hydrazine. Acid 4 was also amidated with allylamine, using chlorodimethoxytriazine for activation, to yield allylamide 10. Palladiumcatalyzed cyclization of this intermediate led to 1K. All compounds were assayed for S1P5 activation in GTPgS assays, which gave more reliable structure–activity results than the FLIPR assays, at concentrations up to 10 mm. EC50 values were determined for all compounds (Table 1). Disrupting the intramolecular hydrogen bond by introducing small alkyl substituents at position 2 (compounds 1A–C), led to a [a] Dr. H. Mattes, Prof. Dr. K. K. Dev, Dr. R. Bouhelal, Dr. C. Barske, Dr. F. Gasparini, Dr. D. Guerini, Dr. A. K. Mir, Dr. D. Orain, M. Osinde, A. Picard, C. Dubois, E. Tasdelen, S. Haessig Novartis Institute for Biomedical Research WKL-122 4002 Basel (Switzerland) Fax: (+41)61 696 2455 E-mail : henri.mattes@novartis.com [b] Prof. Dr. K. K. Dev Molecular Neuropharmacology, Department of Physiology Trinity College Institute of Neuroscience (TCIN) Medical School Trinity College Dublin, Dublin 2 (Ireland) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.201000253.

1-Alkyl-4-phenyl-6-alkoxy-1H-quinazolin-2-ones: a novel series of potent calcium-sensing receptor antagonists.

Parathyroid hormone (PTH) is an effective bone anabolic agent. However, only when administered by daily sc injections exposure of short duration is achieved, a prerequisite for an anabolic response. Instead of applying exogenous PTH, mobilization of endogenous stores of the hormone can be envisaged. The secretion of PTH stored in the parathyroid glands is mediated by a calcium sensing receptor (CaSR) a GPCR localized at the cell surface. Antagonists of CaSR (calcilytics) mimic a state of hypocalcaemia and stimulate PTH release to the bloodstream. Screening of the internal compound collection for inhibition of CaSR signaling function afforded 2a. In vitro potency could be improved >1000 fold by optimization of its chemical structure. The binding mode of our compounds was predicted based on molecular modeling and confirmed by testing with mutated receptors. While the compounds readily induced PTH release after iv application a special formulation was needed for oral activity. The required profile was achieved by using microemulsions. Excellent PK/PD correlation was found in rats and dogs. High levels of PTH were reached in plasma within minutes which reverted to baseline in about 1-2 h in both species.

Role of the adrenal gland in the metabolic and cardiovascular effects of 8-OH-DPAT in the rat

In the present study, the role of the adrenal gland in the hypoinsulinaemic and hyperglycaemic effects of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) vis-à-vis the cardiovascular effects were examined using adrenalectomized and sham-operated (with intact adrenals) Sprague-Dawley rats. Intravenous administration of 8-OH-DPAT (150 micrograms/kg) into conscious sham-operated animals caused significant increases in basal plasma glucose levels without any change in basal plasma insulin levels whereas glucose-stimulated plasma insulin levels were markedly inhibited. These changes were associated with significant and sustained falls in blood pressure (BP) and heart rate (HR) preceded by transient (less than 5 min) increases only in BP. In adrenalectomized animals, 8-OH-DPAT-mediated initial vasoconstriction, hyperglycaemia and inhibition of evoked plasma insulin levels were abolished. However, in these animals, 8-OH-DPAT produced falls in BP and HR that were comparable to those observed in sham-operated animals. Plasma corticosterone was increased 15 min after 8-OH-DPAT in sham-operated animals whereas it was undetectable in adrenalectomized animals. The data indicate that unlike the effects of 8-OH-DPAT to decrease BP and HR, the initial increase in BP and its effects on the metabolic parameters are clearly dependent on intact adrenals.

Investigation of the mechanism(s) of 8-OH-DPAT-mediated inhibition of plasma insulin in spontaneously hypertensive rats

1 Effects of the prototype selective 5‐HT1A receptor agonist, 8‐hydroxy‐2‐(di‐n‐dipropylamino)tetralin (8‐OH‐DPAT), were studied on the glycaemia and insulinaemia in conscious spontaneously hypertensive (SH) rats concurrently with blood pressure (BP) and heart rate (HR); underlying mechanism(s) were investigated in anaesthetized and pithed SH rats and in the perfused rat pancreas. 2 Intravenous (i.v.) injections of 8‐OH‐DPAT (150 μg kg−1, i.v.) into fasted conscious but not anaesthetized SH rats increased glycaemia; glucose‐stimulated (i.v. glucose tolerance test) plasma insulin levels were significantly inhibited in both cases without significant changes in glucose tolerance. Metabolic changes were associated with prominent decreases in BP and HR. 3 No inhibitory effect of 8‐OH‐DPAT, 150 μg kg−1 i.v., on glucose‐stimulated plasma insulin was observed in pithed SH rats; in contrast, clonidine (8 μg kg−1 i.v.), produced marked inhibition of insulin levels in association with glucose intolerance. Neither compound decreased BP; rather, pronounced vasopressor effects were observed. 4 In the isolated perfused pancreas of the rat, 8‐OH‐DPAT, at 10−8 and 10−7m, concentrations known to activate 5‐HT1A receptors in vitro, failed to modify glucose‐stimulated insulin release. Inhibition (39 ± 7%) was seen only at a high concentration of 10−6 m. 5 The present data suggest that like the cardiovascular effects of 8‐OH‐DPAT, the inhibition of glucose‐stimulated insulin release is mediated via the central nervous system. However, it is suggested that different mechanisms are involved in the cardiovascular actions and metabolic effects of 8‐OH‐DPAT in the SH rat; the latter are likely to reflect a consequence of activation of the hypothalamic‐adrenal axis.