Reprint of Pharmacological and molecular characterization of the positive allosteric modulators of metabotropic glutamate receptor 2.

The metabotropic glutamate receptor 2 (mGlu2) plays an important role in the presynaptic control of glutamate release and several mGlu2 positive allosteric modulators (PAMs) have been under assessment for their potential as antipsychotics. The binding mode of mGlu2 PAMs is better characterized in functional terms while few data are available on the relationship between allosteric and orthosteric binding sites. Pharmacological studies characterizing binding and effects of two different chemical series of mGlu2 PAMs are therefore carried out here using the radiolabeled mGlu2 agonist 3[H]-LY354740 and mGlu2 PAM 3[H]-2,2,2-TEMPS. A multidimensional approach to the PAM mechanism of action shows that mGlu2 PAMs increase the affinity of 3[H]-LY354740 for the orthosteric site of mGlu2 as well as the number of 3[H]-LY354740 binding sites. 3[H]-2,2,2-TEMPS binding is also enhanced by the presence of LY354740. New residues in the allosteric rat mGlu2 binding pocket are identified to be crucial for the PAMs ligand binding, among these Tyr3.40 and Asn5.46. Also of remark, in the described experimental conditions S731A (Ser5.42) residue is important only for the mGlu2 PAM LY487379 and not for the compound PAM-1: an example of the structural differences among these mGlu2 PAMs. This study provides a summary of the information generated in the past decade on mGlu2 PAMs adding a detailed molecular investigation of PAM binding mode. Differences among mGlu2 PAM compounds are discussed as well as the mGlu2 regions interacting with mGlu2 PAM and NAM agents and residues driving mGlu2 PAM selectivity. This article is part of the Special Issue entitled 'Metabotropic Glutamate Receptors, 5 years on'.

Pharmacological and molecular characterization of the positive allosteric modulators of metabotropic glutamate receptor 2.

The metabotropic glutamate receptor 2 (mGlu2) plays an important role in the presynaptic control of glutamate release and several mGlu2 positive allosteric modulators (PAMs) have been under assessment for their potential as antipsychotics. The binding mode of mGlu2 PAMs is better characterized in functional terms while few data are available on the relationship between allosteric and orthosteric binding sites. Pharmacological studies characterizing binding and effects of two different chemical series of mGlu2 PAMs are therefore carried out here using the radiolabeled mGlu2 agonist 3[H]-LY354740 and mGlu2 PAM 3[H]-2,2,2-TEMPS. A multidimensional approach to the PAM mechanism of action shows that mGlu2 PAMs increase the affinity of 3[H]-LY354740 for the orthosteric site of mGlu2 as well as the number of 3[H]-LY354740 binding sites. 3[H]-2,2,2-TEMPS binding is also enhanced by the presence of LY354740. New residues in the allosteric rat mGlu2 binding pocket are identified to be crucial for the PAMs ligand binding, among these Tyr3.40 and Asn5.46. Also of remark, in the described experimental conditions S731A (Ser5.42) residue is important only for the mGlu2 PAM LY487379 and not for the compound PAM-1: an example of the structural differences among these mGlu2 PAMs. This study provides a summary of the information generated in the past decade on mGlu2 PAMs adding a detailed molecular investigation of PAM binding mode. Differences among mGlu2 PAM compounds are discussed as well as the mGlu2 regions interacting with mGlu2 PAM and NAM agents and residues driving mGlu2 PAM selectivity.

Structural determinants of allosteric antagonism at metabotropic glutamate receptor 2: mechanistic studies with new potent negative allosteric modulators.

BACKGROUND AND PURPOSE: Altered glutamatergic neurotransmission is linked to several neurological and psychiatric disorders. Metabotropic glutamate receptor 2 (mGlu2) plays an important role on the presynaptic control of glutamate release and negative allosteric modulators (NAMs) acting on mGlu2/3 receptors are under assessment for their potential as antidepressants, neurogenics and cognitive enhancers. Two new potent mGlu2/3 NAMs, RO4988546 and RO5488608, are described in this study and the allosteric binding site in the transmembrane (TM) domain of mGlu2 is characterized. EXPERIMENTAL APPROACH: Site directed mutagenesis, functional measurements and ß2-adrenoceptor-based modelling of mGlu2 were employed to identify important molecular determinants of two new potent mGlu2/3 NAMs. KEY RESULTS: RO4988546 and RO5488608 affected both [³H]-LY354740 agonist binding at the orthosteric site and the binding of a tritiated positive allosteric modulator (³H-PAM), indicating that NAMs and PAMs could have overlapping binding sites in the mGlu2 TM domain. We identified eight residues in the allosteric binding pocket that are crucial for non-competitive antagonism of agonist-dependent activation of mGlu2 and directly interact with the NAMs: Arg³·²8, Arg³·²9, Phe³·³6, His(E2.52) , Leu5·4³, Trp6·48, Phe6·55 and Val7·4³. The mGlu2 specific residue His(E2.52) is likely to be involved in selectivity and residues located in the outer part of the binding pocket are more important for [³H]-LY354740 agonist binding inhibition, which is independent of the highly conserved Trp6·48 residue. CONCLUSIONS AND IMPLICATIONS: This is the first complete molecular investigation of the allosteric binding pocket of mGlu2 and Group II mGluRs and provides new information on what determines mGlu2 NAMs selective interactions and effects.

Unusual manifestation of posttransplant lymphoproliferative disorder in the esophagus.

Early manifestations of posttransplant lymphoproliferative disorders (PTLD) are mainly associated with a primary Epstein-Barr virus (EBV) infection. Rapid increases in peripheral blood EBV DNA load are supposed to reliably predict PTLD. We report a boy who 6 months after living-related kidney transplantation presented with an extranodal esophageal manifestation of PTLD. Despite a primary EBV infection with tonsillitis, the peripheral blood EBV DNA remained low, hiding the progression to PTLD.

Pharmacological properties of native metabotropic glutamate receptors in freshly dissociated Golgi cells of the rat cerebellum.

We have examined the pharmacological properties of native metabotropic glutamate (mGlu) receptors in freshly isolated rat cerebellar Golgi cells using the whole-cell configuration of the patch-clamp technique. Group II mGlu receptor agonists inhibited voltage-gated Ca(2+) channels (VGCC) currents in a reversible and concentration-dependent manner with a rank order of potency being LY354740> DCG-IV > L-CCG-I > glutamate >>1S,3R-ACPD > NAAG. The maximum degree of inhibition obtained was similar for all drugs tested, saturating at about 33-41%, except for NAAG that had a non saturating effect of 50% at 1mM. Two novel group II mGlu receptor antagonists, LY341495 and Ro 65-3479, reversed VGCC current inhibition by LY354740 with pK(B) values of 7.0 and 6.3, respectively. In a subpopulation of Golgi cells, the antagonistic effect of LY341495 was only partial, suggesting a remaining effect of group I mGlu receptors. This was confirmed by experiments with S-DHPG, a selective group I mGlu receptor agonist. These experiments suggest that Golgi cells of the cerebellum express group II mGlu receptors that couple to the inhibition of VGCCs. Therefore, inhibition of VGCCs in cerebellar Golgi cells is a useful model system to evaluate novel group II mGlu receptor ligands.

Characterization of [(3)H]-LY354740 binding to rat mGlu2 and mGlu3 receptors expressed in CHO cells using semliki forest virus vectors.

The binding properties of [(3)H]-LY354740 were characterized on rat metabotropic glutamate receptors mGlu2 and mGlu3 expressed in Chinese hamster ovary (CHO) cells using Semliki Forest virus vectors. The saturation isotherm gave K(D) values of 20+/-5 and 53+/-8 nM and B(max) values of 474+/-161 and 667+/-89 fmol/mg protein for mGlu2 and mGlu3 receptors, respectively. NMDA, CaCl(2), DHPG and kainate were inactive up to 1 mM, whereas LY341495, DCG IV and ibotenate inhibited [(3)H]-LY354740 binding with similar potencies on both receptors. L-CCG I, L-AP4, L-AP5, LY354740 and 1S,3R-ACPD were 2- to 4-fold more potent inhibitors of [(3)H]-LY354740 binding to mGlu2 than mGlu3 receptors. However, MPPG and L-AP3 had a 6-fold and DTT a 28-fold preference for mGlu2 over mGlu3. ZnCl(2), at 10 mM, inhibited more than 70% of [(3)H]-LY354740 binding to mGlu2 receptors. At the same concentration it did not affect significantly [(3)H]-LY354740 binding to mGlu3 receptors. On the contrary, glutamate, quisqualate, EGLU and NAAG showed a 3-, 5-, 7- and 12-fold preference for mGlu3 over mGlu2. Finally, GTPgammaS, which partially inhibited the binding on mGlu2 receptors, was inactive to inhibit [(3)H]-LY354740 binding on mGlu3 receptors.

Synthesis of heterocyclic enol ethers and their use as group 2 metabotropic glutamate receptor antagonists.

Heterocyclic enol ethers of type 1 were studied with respect to the inhibition of 1S,3R-ACPD (10 microM)-stimulated GTP gamma35S binding on rat mGluR2 transfected cell membranes. The structure activity relationship with regard to the substitution pattern of the phenyl ring, the oxygen substituent and the nature of the heterocycle is discussed.

Structure-activity relationships of substituted 5H-thiazolo[3,2-a]pyrimidines as group 2 metabotropic glutamate receptor antagonists.

A series of 5H-thiazolo[3,2-a]pyrimidine derivatives 1 was studied with respect to the inhibition of 1S,3R-ACPD (10 microM)-stimulated GTP gamma35S binding on rat mGlu2 receptor transfected cell membranes. The influence of substituents at position 6 and 7 as well as the substitution pattern of the two phenyl-rings in position 2 and 5 on the activity is discussed.

Design, synthesis and biological evaluation of aryl-substituted sialyl Lewis X mimetics prepared via cross-metathesis of C-fucopeptides.

Several aryl substituted C-fucopeptides have been developed as sialyl Lewis X mimetics. Although the compounds have a much simpler structure compared to SLe(x), up to 3-times higher binding affinity toward E-selectin and > 1000 times toward P-selectin was observed. Furthermore, a convenient strategy for generating a number of analogues from a SLe(x) mimetic template at a very late stage of the synthesis was introduced, using a ruthenium catalyzed cross olefin metathesis under benchtop conditions.

Characterization of (2S,2'R,3'R)-2-(2',3'-[3H]-dicarboxycyclopropyl)glycine binding in rat brain.

[(2S,2'R,3'R)-2-(2',3'-[3H]Dicarboxycyclopropyl)glycine ([3H]DCG IV) binding was characterized in vitro in rat brain cortex homogenates and rat brain sections. In cortex homogenates, the binding was saturable and the saturation isotherm indicated the presence of a single binding site with a K(D) value of 180 +/- 33 nM and a Bmax of 780 +/- 70 fmol/mg of protein. The nonspecific binding, measured using 100 microM LY354740, was <30%. NMDA, AMPA, kainate, L(-)-threo-3-hydroxyaspartic acid, and (S)-3,5-dihydroxyphenylglycine were all inactive in [3H]DCG IV binding up to 1 mM. However, several compounds inhibited [3H]DCG IV binding in a concentration-dependent manner with the following rank order of potency: LY341495 = LY354740 > DCG IV = (2S,1'S,2'S)-2-(2-carboxycyclopropyl)glycine > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid > (2S,1'S,2'S)-2-methyl-2-(2-carboxycyclopropyl)glycine > L-glutamate = ibotenate > quisqualate > (RS)-alpha-methyl-4-phosphonophenylglycine = L(+)-2-amino-3-phosphonopropionic acid > (S)-alpha-methyl-4-carboxyphenylglycine > (2S)-alpha-ethylglutamic acid > L(+)-2-amino-4-phosphonobutyric acid. N-Acetyl-L-aspartyl-L-glutamic acid inhibited the binding in a biphasic manner with an IC50 of 0.2 microM for the high-affinity component. The binding was also affected by GTPgammaS, reducing agents, and CdCl2. In parasagittal sections of rat brain, a high density of specific binding was observed in the accessory olfactory bulb, cortical regions (layers 1, 3, and 4 > 2, 5, and 6), caudate putamen, molecular layers of the hippocampus and dentate gyrus, subiculum, presubiculum, retrosplenial cortex, anteroventral thalamic nuclei, and cerebellar granular layer, reflecting its preferential (perhaps not exclusive) affinity for pre- and postsynaptic metabotropic glutamate mGlu2 receptors. Thus, the pharmacology, tissue distribution, and sensitivity to GTPgammaS show that [3H]DCG IV binding is probably to group II metabotropic glutamate receptors in rat brain.

In vitro binding characteristics of a new selective group II metabotropic glutamate receptor radioligand, [3H]LY354740, in rat brain.

The in vitro binding of [3H]LY354740, the first high affinity group II-selective metabotropic glutamate (mGlu) receptor radioligand, was characterized in rat cortical, hippocampal, and thalamic membranes as well as in rat brain sections. [3H]LY354740 binding was saturable in all regions investigated. Nonspecific binding (in the presence of 10 microM DCG-IV) was approximately 8% of the total. Ionotropic glutamate receptor agonists, N-methyl-D-aspartate, (R,S)-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid/kainate, a Na+-dependent glutamate uptake blocker as well as a group I-selective mGlu receptor agonist (all up to 1 mM) did not inhibit [3H]LY354740 binding to cortical membranes. However, several known metabotropic receptor ligands inhibited the binding with the following rank order of potency: LY354740 = LY341495 > (2S,2'R, 3'R)-2-(2',3'-dicarboxycyclopropyl)glycine = (2S,1'S, 2'S)-2-(2-carboxycyclopropyl)glycine > glutamate = (1S, 3R)-1-aminocyclopentane-1,3-dicarboxylic acid > (2S,1'S, 2'S)-2-methyl-2-(2-carboxycyclopropyl)-glycine > quisqualate > ibotenate > L-2-amino-3-phosphonopropionic acid = (S)-alpha-methyl-4-carboxyphenylglycine > L-(+)-2-amino-4-phosphonobutyric acid. N-Acetyl-aspartyl-glutamate, (2S)-alpha-ethylglutamic acid, and (R, S)-alpha-methyl-4-phosphonophenylglycine inhibited [3H]LY354740 binding in a biphasic manner. Guanosine-5'-O-(3-thiotriphosphate concentration-dependently and almost completely inhibited the binding. Finally, in parasagittal sections of rat brain, a high density of specific binding was observed in the accessory olfactory bulb, cortical regions (layers 1-3 > 4-6), caudate putamen, molecular layers of the hippocampus and dentate gyrus, presubiculum, retrosplenial cortex, anteroventral thalamic nuclei, and cerebellar granular layer, reflecting its preferential (perhaps not exclusive) affinity for presynaptic and postsynaptic mGlu2 receptors. Thus, the pharmacology, tissue distribution, and sensitivity to guanosine-5'-O-(3-thiotriphosphate show that [3H]LY354740 binding probably occurs to group II mGlu receptors in rat brain.

Design and synthesis of C-linked fucosides as inhibitors of E-selectin.

Two series of C-linked fucosides as mimetics for the tetrasaccharide sialyl Lewis X have been synthesized and tested as inhibitors of E-Selectin. The fucopeptides have been prepared from three key intermediates, including alpha-C-allyl fucose, natural and unnatural amino acids bearing hydroxyl groups and an alpha, omega-diacid moiety for the imitation of the essential three parts of SLex, i.e., the Fuc, Gal, and NeuAc. The nature and distance of the linkage of the fucose moiety to the amino acids as well as the distance between the amino acids and the terminal carboxylic acid group turned out to be crucial for the biological activity. In addition the necessity of both OH groups (4- and 6-OH) in the Gal part could be confirmed. Conformational NMR study of the most active mimetic supports the structure-activity relationship. A second series of mimetics was prepared, where Fuc and Gal moieties were purely C-linked. In the synthesis of beta-C-allyl galactose an intramolecular 1,2-hydride shift led to an interesting side product. However, the substituted glycosidic oxygens led to a substantial loss of conformational constrain, which could not be compensated and resulted in low activity.