Isabelle Brabet

Time-resolved FRET between GPCR ligands reveals oligomers in native tissues

G protein-coupled receptor (GPCR) oligomers have been proposed to play critical roles in cell signaling, but confirmation of their existence in a native context remains elusive, as no direct interactions between receptors have been reported. To demonstrate their presence in native tissues, we developed a time-resolved FRET strategy that is based on receptor labeling with selective fluorescent ligands. Specific FRET signals were observed with four different receptors expressed in cell lines, consistent with their dimeric or oligomeric nature in these transfected cells. More notably, the comparison between FRET signals measured with sets of fluorescent agonists and antagonists was consistent with an asymmetric relationship of the two protomers in an activated GPCR dimer. Finally, we applied the strategy to native tissues and succeeded in demonstrating the presence of oxytocin receptor dimers and/or oligomers in mammary gland.

Closure of the Venus flytrap module of mGlu8 receptor and the activation process: Insights from mutations converting antagonists into agonists

Ca2+, pheromones, sweet taste compounds, and the main neurotransmitters glutamate and γ-aminobutyric acid activate G protein-coupled receptors (GPCRs) that constitute the GPCR family 3. These receptors are dimers, and each subunit has a large extracellular domain called a Venus flytrap module (VFTM), where agonists bind. This module is connected to a heptahelical domain that activates G proteins. Recently, the structure of the dimer of mGlu1 VFTMs revealed two important conformational changes resulting from glutamate binding. First, agonists can stabilize a closed state of at least one VFTM in the dimer. Second, the relative orientation of the two VFTMs in the dimer is different in the presence of glutamate, such that their C-terminal ends (which are connected to the G protein-activating heptahelical domain) become closer by more than 20 Å. This latter change in orientation has been proposed to play a key role in receptor activation. To elucidate the respective role of VFTM closure and the change in orientation of the VFTMs in family 3 GPCR activation, we analyzed the mechanism of action of the mGlu8 receptor antagonists ACPT-II and MAP4. Molecular modeling studies suggest that these two compounds prevent the closure of the mGlu8 VFTM because of ionic and steric hindrance, respectively. We show here that the replacement of the residues responsible for these hindrances (Asp-309 and Tyr-227, respectively) by Ala allows ACPT-II or MAP4 to fully activate the receptors. These data are consistent with the requirement of the VFTM closure for family 3 GPCR activation.

Molecular determinants involved in the allosteric control of agonist affinity in the GABAB receptor by the GABAB2 subunit.

The γ-aminobutyric acid type B (GABAB) receptor is an allosteric complex made of two subunits, GABAB1 (GB1) and GABAB2 (GB2). Both subunits are composed of an extracellular Venus flytrap domain (VFT) and a heptahelical domain (HD). GB1 binds GABA, and GB2 plays a major role in G-protein activation as well as in the high agonist affinity state of GB1. How agonist affinity in GB1 is regulated in the receptor remains unknown. Here, we demonstrate that GB2 VFT is a major molecular determinant involved in this control. We show that isolated versions of GB1 and GB2 VFTs in the absence of the HD and C-terminal tail can form hetero-oligomers as shown by time-resolved fluorescence resonance energy transfer (based on HTRF® technology). GB2 VFT and its association with GB1 VFT controlled agonist affinity in GB1 in two ways. First, GB2 VFT exerted a direct action on GB1 VFT, as it slightly increased agonist affinity in isolated GB1 VFT. Second and most importantly, GB2 VFT prevented inhibitory interaction between the two main domains (VFT and HD) of GB1. According to this model, we propose that GB1 HD prevents the possible natural closure of GB1 VFT. In contrast, GB2 VFT facilitates this closure. Finally, such inhibitory contacts between HD and VFT in GB1 could be similar to those important to maintain the inactive state of the receptor.

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 [EC50: 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.—Goudet, C., Vilar, B., Courtiol, T., Deltheil, T., Bessiron, T., Brabet, I., Oueslati, N., Rigault, D., Bertrand, H.‐O., McLean, H., Daniel, H., Amalric, M., Acher, F., Pin, J.‐P. A novel selective metabotropic glutamate receptor 4 agonist reveals new possibilities for developing subtype‐selective ligands with therapeutic potential. FASEB J. 26, 1682‐1693 (2012). www.fasebj.org

Aminobicyclo[2.2.1.]heptane dicarboxylic acids (ABHD), rigid analogs of ACPD and glutamic acid: synthesis and pharmacological activity on metabotropic receptors mGluR1 and mGluR2.

Isomeric norbornane-derived rigid analogs mimicking different potential conformations of ACPD (1-aminocyclopentane-1,3-dicarboxylic acid) and glutamic acid have been synthesized, via the hydantoin route, to be used as conformational probes for bioactive conformations at the glutamatergic receptors of the central nervous system. Activities on metabotropic receptors mGluR1 and mGluR2 are reported and discussed.

Allosteric modulation of metabotropic glutamate receptors by chloride ions

Metabotropic glutamate receptors (mGluRs) play key roles in the modulation of many synapses. Chloride (Cl‐) is known to directly bind and regulate the function of different actors of neuronal activity, and several studies have pointed to the possible modulation of mGluRs by Cl‐. Herein, we demonstrate that Cl‐ behaves as a positive allosteric modulator of mGluRs. For example, whereas glutamate potency was 3.08 ± 0.33 μM on metabotropic glutamate (mGlu) 4 receptors in high‐Cl‐ buffer, signaling activity was almost abolished in low Cl_ in cell‐based assays. Cl‐ potency was 78.6 ± 3.5 mM. Cl‐ possesses a high positive cooperativity with glutamate (Hill slope ã6 on mGlu4), meaning that small variations in [Cl‐] lead to large variations in glutamate action. Using molecular modeling and mutagenesis, we have identified 2 well‐conserved Cl‐binding pockets in the extracellular domain of mGluRs. Moreover, modeling of activity‐dependent Cl‐ variations at GABAergic synapses suggests that these variations may be compatible with a dynamic modulation of the most sensitive mGluRs present in these synapses. Taken together, these data reveal a necessary role of Cl‐ for the glutamate activation of manymGluRs. Exploiting Cl‐ binding pockets may yield to the development of innovative regulators of mGluR activity.—Tora, A. S., Rovira, X., Dione, I., Bertrand, H.‐O., Brabet, I., De Koninck, Y., Doyon, N., Pin, J.P., Acher, F., Goudet, C. Allosteric modulation of metabotropic glutamate receptors by chloride ions. FASEB J. 29, 4174‐4188 (2015). www.fasebj.org

Synthesis of conformationally-constrained stereospecific analogs of glutamic acid as antagonists of metabotropic receptors

Abstract Rigid analogs of ACPD have been synthesized to mimick different potential conformations of ACPD in aqueous solution. One of them, (±)-ABHD-I is a competitive antagonist at mGluR1a receptor with a K B value of 300 μM.

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.