Increased Potency and Selectivity for Group III Metabotropic Glutamate Receptor Agonists Binding at Dual sites.

A group III metabotropic glutamate (mGlu) receptor agonist (PCEP) was identified by virtual HTS. This orthosteric ligand is composed by an l-AP4-derived fragment that mimics glutamate and a chain that binds into a neighboring pocket, offering possibilities to improve affinity and selectivity. Herein we describe a series of derivatives where the distal chain is replaced by an aromatic or heteroaromatic group. Potent agonists were identified, including some with a mGlu4 subtype preference, e.g., 17m (LSP1-2111) and 16g (LSP4-2022). Molecular modeling suggests that aromatic functional groups may bind at either one of the two chloride regulatory sites. These agonists may thus be considered as particular bitopic/dualsteric ligands. 17m was shown to reduce GABAergic synaptic transmission at striatopallidal synapses. We now demonstrate its inhibitory effect at glutamatergic parallel fiber-Purkinje cell synapses in the cerebellar cortex. Although these ligands have physicochemical properties that are markedly different from typical CNS drugs, they hold significant therapeutic potential.

Time-resolved FRET strategy to screen GPCR ligand library.

Screening chemical libraries to find specific drugs for G protein-coupled receptors is still of major interest. Indeed, because of their major roles in all physiological functions, G protein-coupled receptors remain major targets for drug development programs. Currently, interest in GPCRs as drug targets has been boosted by the discovery of biased ligands, thus allowing the development of drugs not only specific for one target but also for the specific signaling cascade expected to have the therapeutic effect. Such molecules are then expected to display fewer side effects. To reach such a goal, there is much interest in novel, efficient, simple, and direct screening assays that may help identify any drugs interacting with the target, these being then analyzed for their biased activity. Here, we present an efficient strategy to screen ligands on their binding properties. The method described is based on time-resolved FRET between a receptor and a ligand. This method has already been used to develop new assays called Tag-lite(®) binding assays for numerous G protein-coupled receptors, proving its broad application and its power.

Time-resolved FRET binding assay to investigate hetero-oligomer binding properties: proof of concept with dopamine D1/D3 heterodimer.

G protein-coupled receptors (GPCRs) have been described to form hetero-oligomers. The importance of these complexes in physiology and pathology is considered crucial, and heterodimers represent promising new targets to discover innovative therapeutics. However, there is a lack of binding assays to allow the evaluation of ligand affinity for GPCR hetero-oligomers. Using dopamine receptors and more specifically the D1 and D3 receptors as GPCR models, we developed a new time-resolved FRET (TR-FRET) based assay to determine ligand affinity for the D1/D3 heteromer. Based on the high-resolution structure of the dopamine D3 receptor (D3R), six fluorescent probes derived from a known D3R partial agonist (BP 897) were designed, synthesized and evaluated as high affinity and selective ligands for the D3/D2 receptors, and for other dopamine receptor subtypes. The highest affinity ligand 21 was then employed in the development of the D1/D3 heteromer assay. The TR-FRET was monitored between a fluorescent tag donor carried by the D1 receptor (D1R) and a fluorescent acceptor D3R ligand 21. The newly reported assay, easy to implement on other G protein-coupled receptors, constitutes an attractive strategy to screen for heteromer ligands.

CTAB turbidimetric method for assaying hyaluronic acid in complex environments and under cross-linked form.

The cetyltrimethylammonium bromide turbidimetric method (CTM) has been developed to quantify the hyaluronic acid (HA) in complex media to overcome the lack of selectivity and specificity of the standard carbazole method. The objective of this work is to assess the potential application of CTM to determine HA concentration. Factors such as duration of incubation, linearity range, HA size and form (natural linear HA or cross linked HA), pH and ionic environment impact were investigated. The incubation time was set to 10 min and the calibration curve was linear up to 0.6 g L(-1). The quantitative method was relevant whatever the HA size and form, and also for a wide range of conditions. The robustness of the CTM added to its high specificity and simplicity demonstrated that the CTM is a valuable method that would be an interesting substitute to the carbazole assay for HA quantification.

A novel selective metabotropic glutamate receptor 4 agonist reveals new possibilities for developing subtype selective ligands with therapeutic potential.

Metabotropic glutamate (mGlu) receptors are promising targets to treat numerous brain disorders. So far, allosteric modulators are the only subtype selective ligands, but pure agonists still have strong therapeutic potential. Here, we aimed at investigating the possibility of developing subtype-selective agonists by extending the glutamate-like structure to hit a nonconsensus binding area. We report the properties of the first mGlu4-selective orthosteric agonist, derived from a virtual screening hit, LSP4-2022 using cell-based assays with recombinant mGlu receptors [EC(50): 0.11 ± 0.02, 11.6 ± 1.9, 29.2 ± 4.2 µM (n>19) in calcium assays on mGlu4, mGlu7, and mGlu8 receptors, respectively, with no activity at the group I and -II mGlu receptors at 100 µM]. LSP4-2022 inhibits neurotransmission in cerebellar slices from wild-type but not mGlu4 receptor-knockout mice. In vivo, it possesses antiparkinsonian properties after central or systemic administration in a haloperidol-induced catalepsy test, revealing its ability to cross the blood-brain barrier. Site-directed mutagenesis and molecular modeling was used to identify the LSP4-2022 binding site, revealing interaction with both the glutamate binding site and a variable pocket responsible for selectivity. These data reveal new approaches for developing selective, hydrophilic, and brain-penetrant mGlu receptor agonists, offering new possibilities to design original bioactive compounds with therapeutic potential.

Homogeneous time-resolved fluorescence-based assay to screen for ligands targeting the growth hormone secretagogue receptor type 1a.

The growth hormone secretagogue receptor type 1a (GHS-R1a) belongs to class A G-protein-coupled receptors (GPCR). This receptor mediates pleiotropic effects of ghrelin and represents a promising target for dysfunctions of growth hormone secretion and energy homeostasis including obesity. Identification of new compounds which bind GHS-R1a is traditionally achieved using radioactive binding assays. Here we propose a new fluorescence-based assay, called Tag-lite binding assay, based on a fluorescence resonance energy transfer (FRET) process between a terbium cryptate covalently attached to a SNAP-tag fused GHS-R1a (SNAP-GHS-R1a) and a high-affinity red fluorescent ghrelin ligand. The long fluorescence lifetime of the terbium cryptate allows a time-resolved detection of the FRET signal. The assay was made compatible with high-throughput screening by using prelabeled cells in suspension under a 384-well plate format. K(i) values for a panel of 14 compounds displaying agonist, antagonist, or inverse agonist properties were determined using both the radioactive and the Tag-lite binding assays performed on the same batches of GHS-R1a-expressing cells. Compound potencies obtained in the two assays were nicely correlated. This study is the first description of a sensitive and reliable nonradioactive binding assay for GHS-R1a in a format amenable to high-throughput screening.

A fluorescent ligand-binding alternative using Tag-lite® technology.

G-protein-coupled receptors (GPCRs) are crucial cell surface receptors that transmit signals from a wide range of extracellular ligands. Indeed, 40% to 50% of all marketed drugs are thought to modulate GPCR activity, making them the major class of targets in the drug discovery process. Binding assays are widely used to identify high-affinity, selective, and potent GPCR drugs. In this field, the use of radiolabeled ligands has remained so far the gold-standard method. Here the authors report a less hazardous alternative for high-throughput screening (HTS) applications by the setup of a nonradioactive fluorescence-based technology named Tag-lite(®). Selective binding of various fluorescent ligands, either peptidic or not, covering a large panel of GPCRs from different classes is illustrated, particularly for chemokine (CXCR4), opioid (δ, µ, and κ), and cholecystokinin (CCK1 and CCK2) receptors. Affinity constants of well-known pharmacological agents of numerous GPCRs are in line with values published in the literature. The authors clearly demonstrate that the Tag-lite binding assay format can be successfully and reproducibly applied by using different cellular materials such as transient or stable recombinant cells lines expressing SNAP-tagged GPCR. Such fluorescent-based binding assays can be performed with adherent cells or cells in suspension, in 96- or 384-well plates. Altogether, this new technology offers great advantages in terms of flexibility, rapidity, and user-friendliness; allows easy miniaturization; and makes it completely suitable for HTS applications.

A virtual screening hit reveals new possibilities for developing group III metabotropic glutamate receptor agonists.

(R)-PCEP (3-amino-3-carboxypropyl-2'-carboxyethyl phosphinic acid, 1), a new metabotropic glutamate receptor 4 (mGlu4R) agonist, was discovered in a previously reported virtual screening. The (S)-enantiomer and a series of derivatives were synthesized and tested on recombinant mGlu4 receptors. A large number of derivatives activated this receptor but was not able to discriminate between mGlu4 and mGlu8 receptors. The most potent ones 6 and 12 displayed an EC(50) of 1.0 +/- 0.2 microM at mGlu4R. Interestingly these agonists with longer alkyl chains revealed a new binding pocket adjacent to the glutamate binding site, which is lined with residues that differ among the mGluR subtypes and that will allow the design of more selective compounds. Additionally 6 was able to activate mGlu7 receptor with an EC(50) of 43 +/- 16 microM and is thus significantly more potent than L-AP4 (EC(50) of 249 +/- 106 microM).

Metabotropic glutamate receptor subtype 4 selectively modulates both glutamate and GABA transmission in the striatum: implications for Parkinson's disease treatment.

Alterations of striatal synaptic transmission have been associated with several motor disorders involving the basal ganglia, such as Parkinson's disease. For this reason, we investigated the role of group-III metabotropic glutamate (mGlu) receptors in regulating synaptic transmission in the striatum by electrophysiological recordings and by using our novel orthosteric agonist (3S)-3-[(3-amino-3-carboxypropyl(hydroxy)phosphinyl)-hydroxymethyl]-5-nitrothiophene (LSP1-3081) and l-2-amino-4-phosphonobutanoate (L-AP4). Here, we show that both drugs dose-dependently reduced glutamate- and GABA-mediated post-synaptic potentials, and increased the paired-pulse ratio. Moreover, they decreased the frequency, but not the amplitude, of glutamate and GABA spontaneous and miniature post-synaptic currents. Their inhibitory effect was abolished by (RS)-alpha-cyclopropyl-4-phosphonophenylglycine and was lost in slices from mGlu4 knock-out mice. Furthermore, (S)-3,4-dicarboxyphenylglycine did not affect glutamate and GABA transmission. Finally, intrastriatal LSP1-3081 or L-AP4 injection improved akinesia measured by the cylinder test. These results demonstrate that mGlu4 receptor selectively modulates striatal glutamate and GABA synaptic transmission, suggesting that it could represent an interesting target for selective pharmacological intervention in movement disorders involving basal ganglia circuitry.

Functioning of the dimeric GABA(B) receptor extracellular domain revealed by glycan wedge scanning.

The G-protein-coupled receptor (GPCR) activated by the neurotransmitter GABA is made up of two subunits, GABA(B1) and GABA(B2). GABA(B1) binds agonists, whereas GABA(B2) is required for trafficking GABA(B1) to the cell surface, increasing agonist affinity to GABA(B1), and activating associated G proteins. These subunits each comprise two domains, a Venus flytrap domain (VFT) and a heptahelical transmembrane domain (7TM). How agonist binding to the GABA(B1) VFT leads to GABA(B2) 7TM activation remains unknown. Here, we used a glycan wedge scanning approach to investigate how the GABA(B) VFT dimer controls receptor activity. We first identified the dimerization interface using a bioinformatics approach and then showed that introducing an N-glycan at this interface prevents the association of the two subunits and abolishes all activities of GABA(B2), including agonist activation of the G protein. We also identified a second region in the VFT where insertion of an N-glycan does not prevent dimerization, but blocks agonist activation of the receptor. These data provide new insight into the function of this prototypical GPCR and demonstrate that a change in the dimerization interface is required for receptor activation.

Activation of a dimeric metabotropic glutamate receptor by intersubunit rearrangement.

Although many G protein-coupled receptors (GPCRs) can form dimers, a possible role of this phenomenon in their activation remains elusive. A recent and exciting proposal is that a dynamic intersubunit interplay may contribute to GPCR activation. Here, we examined this possibility using dimeric metabotropic glutamate receptors (mGluRs). We first developed a system to perfectly control their subunit composition and show that mGluR dimers do not form larger oligomers. We then examined an mGluR dimer containing one subunit in which the extracellular agonist-binding domain was uncoupled from the G protein-activating transmembrane domain. Despite this uncoupling in one protomer, agonist stimulation resulted in symmetric activation of either transmembrane domain in the dimer with the same efficiency. This, plus other data, can only be explained by an intersubunit rearrangement as the activation mechanism. Although well established for other types of receptors such as tyrosine kinase and guanylate cyclase receptors, this is the first clear demonstration that such a mechanism may also apply to GPCRs.

L-(+)-2-Amino-4-thiophosphonobutyric acid (L-thioAP4), a new potent agonist of group III metabotropic glutamate receptors: increased distal acidity affords enhanced potency.

L-2-Amino-4-phosphonobutyric acid (l-AP4), l-2-amino-4-thiophosphonobutyric acid (l-thioAP4), and l-2-amino-4-(hydroxy)phosphinylbutyric acid (desmethylphosphinothricin, DMPT) were synthesized from protected vinylglycine. They were tested as agonists at group III metabotropic glutamate receptors (mGluR) along with phosphinothricin (PT). DMPT and PT display a much lower potency at mGlu4 receptor (EC50 = 4.0 and 1100 microM, respectively) in comparison to l-AP4 (EC50 = 0.08 microM), whereas l-thioAP4 has a 2-fold higher potency (EC50 = 0.039 microM). Similar rank orders of potency were observed at mGlu6,7 and mGlu8 receptors. The higher potency of l-thioAP4 is due to its stronger second acidity compared to l-AP4. These pKa values of 5.56 and 6.88, respectively, were determined using 31P NMR chemical shift variations. The second distal negative charge of l-AP4/l-thioAP4 probably provides stronger binding to specific basic residues of the binding sites of group III mGluRs, which stabilizes the active conformation of the receptor.

Synthesis and biological evaluation of 1-amino-2-phosphonomethylcyclopropanecarboxylic acids, new group III metabotropic glutamate receptor agonists.

Stereoisomers of 1-amino-2-phosphonomethylcyclopropanecarboxylic acid (APCPr), conformationally restricted analogues of L-AP4 (2-amino-4-phosphonobutyric acid), have been prepared and evaluated at recombinant group III metabotropic glutamate receptors. They activate these receptors over a broad range of potencies. The most potent isomer (1S,2R)-APCPr displays a similar pharmacological profile as that of L-AP4 (EC50 0.72, 1.95, >500, 0.34 microM at mGlu4, 6, 7, 8 receptors, respectively, and no effect at group I/II mGluRs). It was characterized on native receptors located in the basal ganglia (BG) where it induced a robust and reversible inhibition of synaptic transmission. It was tested in vivo in haloperidol-induced catalepsy, a model of Parkinsonian akinesia, by direct infusion in the globus pallidus of the BG. At a dose of 0.5 nmol/microL, catalepsy was significantly antagonized. This study reveals that (1S,2R)-APCPr is a potent group III mGluR agonist and confirms that these receptors may be considered as a therapeutic target in the Parkinson's disease.