Paolo Cavanni

Structure-Activity Studies on Neuropeptide S IDENTIFICATION OF THE AMINO ACID RESIDUES CRUCIAL FOR RECEPTOR ACTIVATION

Neuropeptide S (NPS) has been recently recognized as the endogenous ligand for the previous orphan G-protein-coupled receptor GPR154, now referred to as the NPS receptor (NPSR). The NPS-NPSR receptor system regulates important biological functions such as sleeping/wakening, locomotion, anxiety, and food intake. To collect information on the mechanisms of interaction between NPS and its receptor, a classical structure-activity relationship study was performed. Human (h) NPS derivatives obtained by Ala and d-scan and N- and C-terminal truncation were assessed for their ability to stimulate calcium release in HEK293 cells expressing the human recombinant NPSR. The results of this study indicate that (i) the effect of hNPS is mimicked by the fragment hNPS-(1–10); (ii) Phe2, Arg3, and Asn4 are crucial for biological activity; (iii) the sequence Thr8-Gly9-Met10 is important for receptor activation, although with non-stringent chemical requirements; and (iv) the sequence Val6-Gly7 acts as a hinge region between the two above-mentioned domains. However, the stimulatory effect of hNPS given intracerebroventricularly on mouse locomotor activity was not fully mimicked by hNPS-(1–10), suggesting that the C-terminal region of the peptide maintains importance for in vivo activity. In conclusion, this study identified the amino acid residues of this peptide most important for receptor activation.

6-(3,4-Dichlorophenyl)-1-[(Methyloxy)methyl]-3-azabicyclo[4.1.0]heptane: A New Potent and Selective Triple Reuptake Inhibitor

A pharmacophore model for triple reuptake inhibitors and the new class of 1-(aryl)-6-[alkoxyalkyl]-3-azabicyclo[3.1.0]hexanes were recently reported. Further investigation in this area led to the identification of a new series of potent and selective triple reuptake inhibitors endowed with good developability characteristics. Excellent bioavailability and brain penetration are associated with this series of 6-(3,4-dichlorophenyl)-1-[(methyloxy)methyl]-3-azabicyclo[4.1.0]heptanes together with high in vitro potency and selectivity at SERT, NET, and DAT. In vivo microdialysis experiments in different animal models and receptor occupancy studies in rat confirmed that derivative 17 showed an appropriate profile to guarantee further progression of the compound.

Pharmacological analysis of carboxyphenylglycines at metabotropic glutamate receptors

Three carboxyphenylglycine derivatives were examined for their activity on glutamate metabotropic receptors negatively linked to adenylate cyclase. Chinese hamster ovary cells stably expressing mGlu2 and mGlu4 were utilised for this study. A receptor binding analysis was also performed for the main classes of glutamate ionotropic receptors and for the glycine binding site on the NMDA-receptor complex. In mGlu2 expressing cells (S)4-carboxy-3-hydroxyphenylglycine and (S)4-carboxy-phenylglycine antagonized forskolin-stimulated cAMP levels, with EC50 of 21 and 970 microM, respectively, acting as agonists at this receptor subtype, whereas (RS) alpha-methyl-4-carboxyphenylglycine antagonized glutamate response in these cells. None of these compounds showed any agonistic or antagonistic activity on mGlu4 expressing cells. No affinity for the ionotropic receptors (NMDA, AMPA and kainate) and for the glycine site of the NMDA-receptor complex was found using the receptor binding approach, except for (RS)4-carboxy-3-hydroxyphenylglycine which showed a pKi of 5.68 in ((+/-)2-carboxypiperazin-4-yl)propyl-1-phosphonic acid binding for NMDA receptor, although this can be ascribed to the (R) form of the racemic mixture.

Synthesis and pharmacological characterisation of 2,4-Dicarboxy-pyrroles as selective non-Competitive mGluR1 antagonists

Metabotropic glutamate receptors (mGluRs) are an unusual family of G-protein coupled receptor (GPCR), and are characterised by a large extracellular N-terminal domain that contains the glutamate binding site. We have identified a new class of non-competitive metabotropic glutamate receptor 1 (mGluR1) antagonists, 2,4-dicarboxy-pyrroles which are endowed with nanomolar potency. They interact within the 7 transmembrane (7TM) domain of the receptor and show antinociceptive properties when tested in a number of different animal models.

1-(Aryl)-6-[alkoxyalkyl]-3-azabicyclo[3.1.0]hexanes and 6-(aryl)-6-[alkoxyalkyl]-3-azabicyclo[3.1.0]hexanes: a new series of potent and selective triple reuptake inhibitors.

The discovery of new highly potent and selective triple reuptake inhibitors is reported. The new classes of 1-(aryl)-6-[alkoxyalkyl]-3-azabicyclo[3.1.0]hexanes and 6-(aryl)-6-[alkoxyalkyl]-3-azabicyclo[3.1.0]hexanes are described together with detailed SAR. Appropriate decoration of the scaffolds was achieved with the help of a triple reuptake inhibitor pharmacophore model detailed here. Selected derivatives showed good oral bioavailability (>30%) and brain penetration (B/B > 4) in rats associated with high in vitro potency and selectivity at SERT, NET, and DAT. Among these compounds, microdialysis and in vivo experiments confirm that derivative 15 has an appropriate developability profile to be considered for further progression.

In vitro and in vivo pharmacological characterization of the novel UT receptor ligand [Pen5,DTrp7,Dab8]urotensin II(4–11) (UFP‐803)

The novel urotensin‐II (U‐II) receptor (UT) ligand, [Pen5,DTrp7,Dab8]U‐II(4–11) (UFP‐803), was pharmacologically evaluated and compared with urantide in in vitro and in vivo assays. In the rat isolated aorta, UFP‐803 was inactive alone but, concentration dependently, displaced the contractile response to U‐II to the right, revealing a competitive type of antagonism and a pA2 value of 7.46. In the FLIPR [Ca2+]i assay, performed at room temperature in HEK293hUT and HEK293rUT cells, U‐II increased [Ca2+]i with pEC50 values of 8.11 and 8.48. Urantide and UFP‐803 were inactive as agonists, but antagonized the actions of U‐II by reducing, in a concentration‐dependent manner, the agonist maximal effects with apparent pKB values in the range of 8.45–9.05. In a separate series of experiments performed at 37°C using a cuvette‐based [Ca2+]i assay and CHOhUT cells, urantide mimicked the [Ca2+]i stimulatory effect of U‐II with an intrinsic activity (α) of 0.80, while UFP‐803 displayed a small (α=0.21) but consistent residual agonist activity. When the same experiments were repeated at 22°C (a temperature similar to that in FLIPR experiments), urantide displayed a very small intrinsic activity (α=0.11) and UFP‐803 was completely inactive as an agonist. In vivo in mice, UFP‐803 (10 nmol kg−1) antagonized U‐II (1 nmol kg−1)‐induced increase in plasma extravasation in various vascular beds, while being inactive alone. In conclusion, UFP‐803 is a potent UT receptor ligand which displays competitive/noncompetitive antagonist behavior depending on the assay. While UFP‐803 is less potent than urantide, it displayed reduced residual agonist activity and as such may be a useful pharmacological tool.

2,4-Dicarboxy-pyrroles as selective non-competitive mGluR1 antagonists: further characterization of 3,5-dimethyl pyrrole-2,4-dicarboxylic acid 2-propyl ester 4-(1,2,2-trimethyl-propyl) ester and structure-activity relationships.

Following the disclosure of 3,5-dimethyl pyrrole-2,4-dicarboxylic acid 2-propyl ester 4-(1,2,2-trimethyl-propyl) ester [3,5-dimethyl PPP] as a potent and selective mGluR1 non-competitive antagonist, we report here further in vivo characterization of this important tool and disclose the investigation of the C-5 position, which led to very potent compounds.

Design and Synthesis of Novel Tricyclic Benzoxazines as Potent 5-HT1A/B/D Receptor Antagonists Leading to the Discovery of 6-{2-[4-(2-methyl-5-quinolinyl)-1-piperazinyl]ethyl}-4H-imidazo[5,1-c][1,4]benzoxazine-3-carboxamide (GSK588045)

Bioisoteric replacement of the metabolically labile N-methyl amide group of a series of benzoxazinones with small heterocyclic rings has led to novel series of fused tricyclic benzoxazines which are potent 5-HT(1A/B/D) receptor antagonists with and without concomitant human serotonin transporter (hSerT) activity. Optimizing against multiple parameters in parallel identified 6-{2-[4-(2-methyl-5-quinolinyl)-1-piperazinyl]ethyl}-4H-imidazo[5,1-c][1,4]benzoxazine-3-carboxamide (GSK588045) as a potent 5-HT(1A/B/D) receptor antagonist with a high degree of selectivity over human ether-a-go-go related gene (hERG) potassium channels, favorable pharmacokinetics, and excellent activity in vivo in rodent pharmacodynamic (PD) models. On the basis of its outstanding overall profile, this compound was progressed as a clinical candidate with the ultimate aim to assess its potential as a faster acting antidepressant/anxiolytic with reduced side-effect burden.

Phenylethynyl-pyrrolo[1,2-a]pyrazine: a new potent and selective tool in the mGluR5 antagonists arena.

The synthesis and the structure activity of a new series of pyrrolo[1,2-a]pyrazine is reported. These molecules are potent and selective non-competitive mGluR5 antagonists and may shed new light on the pattern of substitution tolerated by this receptor.

Competitive Antagonism by Phenylglycine Derivatives at Type 1 Metabotropic Glutamate Receptors

The metabotropic glutamate receptors (mGluRs) form a family of G-protein-coupled receptors which consists of at least seven members termed mGluR1-mGluR7. These members are classified into subfamilies according to their sequence similarities, signal transduction mechanisms and agonist selectivities. mGluR1 and mGluR5 are coupled to the phosphoinositide hydrolysis/Ca2+ signal transduction and efficiently respond to quisqualate. In this study, we have stably expressed mGluR1 in Chinese hamster ovary cells on which the activation of the phosphoinositide signal transduction pathway was evaluated by means of two methods and their degree of correspondence was analyzed. These two methods involve the Li(+)-dependent accumulation of [3H]inositol-labeled inositol phosphates or the [3H]cytidine-labeled phospholiponucleotide cytidine diphospho (CDP)- diacylglycerol (DAG). The correlation between the two measures was found to be generally uniform for the different agonists evaluated. However, the levels of CDP-DAG were found to be consistently higher. Furthermore, quisqualate showed a differential activity on the two methods behaving as a partial agonist and as a full agonist on the inositol phosphate and the CDP-DAG responses, respectively. On the same cells the activity of a series of carboxyphenylglycines recently described as possible new tools for investigating the role of mGluRs has been evaluated. Three phenylglycine derivatives were tested and found to be competitive antagonists at this mGluR subtype. They inhibited both the phosphoinositide signal transduction pathway and the release of intracellular Ca2+ induced by quisqualate the most potent agonist at mGluR1. The pharmacological nature of these compounds and their relative potencies in antagonizing mGluR1 activation are described.