Technology combination to address GPCR allosteric modulator drug-discovery pitfalls.

Allosteric modulators (AMs) represent a novel paradigm to therapeutically target G-protein-coupled receptors (GPCRs). However, their identification and characterization using standard functional assays remain elusive due to the 'context-dependent phenomena'. Novel technological approaches such as combining a Fluorescence Resonance Energy Transfer (FRET)-based library filtering with a Bioluminescence Resonance Energy Transfer (BRET)-based multiparametric compound profiling can circumvent the limitations of current GPCR screening processes and simplify the discovery of biased AMs.

Chemical switch of a metabotropic glutamate receptor 2 silent allosteric modulator into dual metabotropic glutamate receptor 2/3 negative/positive allosteric modulators.

Using an mGluR2 FRET-based binding assay, binders of the transmembrane region devoid of functional activity were identified. It is reported that slight chemical modifications of these SAMs can dramatically change activity of the resulting analogues without altering their affinities. Starting from compound 1, three mGluR2 NAMs showing also mGluR3 PAM activities were obtained. SAMs therefore represent a useful approach to explore the chemical space for GPCR allosteric modulator identification.

Functional interaction between adenosine A2A and group III metabotropic glutamate receptors to reduce parkinsonian symptoms in rats.

Non-dopaminergic drugs acting either on adenosine A2A or metabotropic glutamate (mGlu) receptors reduce motor impairment in animal models of Parkinson's disease (PD), suggesting a possible functional interaction between these receptors to regulate basal ganglia function. The present study therefore tested the behavioural effects of compounds acting selectively on A2A or on specific mGlu receptor subtypes, alone or in combination, in rodent models of PD. Acute administration of the adenosine A2A receptor antagonists CSC or MSX-3 at the highest doses tested (5 and 1.25mg/kg, respectively) significantly reduces haloperidol-induced catalepsy. Furthermore, the anticataleptic effect of MSX-3 was enhanced by a 3-week treatment. Acute administration of the selective group III mGlu agonist ACPT-I produces potent anticataleptic effects and prolongs time on rotarod of 6-OHDA-lesioned rats. In contrast, acute or chronic administration of MPEP (mGlu5 receptor antagonist) has no anticataleptic action. Furthermore, the acute co-administration of ACPT-I 1mg/kg, but not 5mg/kg, with CSC markedly reduces catalepsy. Opposite effects are observed after a 3-week co-administration. The co-administration of ACPT-I with MSX-3 has anticataleptic effects both after acute or chronic treatment. In contrast, acute combination of subthreshold doses of CSC and MPEP has no effect. After a 3-week treatment, however, the combination of CSC and MPEP was found to reduce haloperidol-induced catalepsy. Altogether, these results show for the first time that systemic activation of group III mGlu receptors with ACPT-I provides benefits in parkinsonian rats and underlie a possible interaction with A2A receptors to regulate basal ganglia motor function.

Amino-pyrrolidine tricarboxylic acids give new insight into group III metabotropic glutamate receptor activation mechanism.

Like most class C G-protein-coupled receptors, metabotropic glutamate (mGlu) receptors possess a large extracellular domain where orthosteric ligands bind. Crystal structures revealed that this domain, called Venus FlyTrap (VFT), adopts a closed or open conformation upon agonist or antagonist binding, respectively. We have described amino-pyrrolidine tricarboxylic acids (APTCs), including (2S,4S)-4-amino-1-[(E)-3-carboxyacryloyl]pyrrolidine-2,4-dicarboxylic acid (FP0429), as new selective group III mGlu agonists. Whereas FP0429 is an almost full mGlu4 agonist, it is a weak and partial agonist of the closely related mGlu8 subtype. To get more insight into the activation mechanism of mGlu receptors, we aimed to elucidate why FP0429 behaves differently at these two highly homologous receptors by focusing on two residues within the binding site that differ between mGlu4 and mGlu8. Site-directed mutagenesis of Ser157 and Gly158 of mGlu4 into their mGlu8 homologs (Ala) turned FP0429 into a weak partial agonist. Conversely, introduction of Ser and Gly residues into mGlu8 increased FP0429 efficacy. Docking of FP0429 in mGlu4 VFT 3D model helped us characterize the role of each residue. Indeed, mGlu4 Ser157 seems to have an important role in FP0429 binding, whereas Gly158 may allow a deeper positioning of this agonist in the cavity of lobe I, thereby ensuring optimal interactions with lobe II residues in the fully closed state of the VFT. In contrast, the presence of a methyl group in mGlu8 (Ala instead of Gly) weakens the interactions with the lobe II residues. This probably results in a less stable or a partially closed form of the mGlu8 VFT, leading to partial receptor activation.

Design and synthesis of APTCs (aminopyrrolidinetricarboxylic acids): identification of a new group III metabotropic glutamate receptor selective agonist.

A new family of mGlu receptor orthosteric ligands called APTCs was designed and synthesized using a parallel chemistry approach. Amongst 65 molecules tested on mGlu4, mGlu6 and mGlu8 subtypes, (2S,4S)-4-amino-1-[(E)-3-carboxyacryloyl]pyrrolidine-2,4-dicarboxylic acid (8a06-FP0429) has been shown to be a full mGlu4 agonist and a partial mGlu8 agonist. In addition, 8a06 was shown to be selective versus group I and II mGlu subtypes. A possible explanation for this efficacy difference is proposed by docking experiment performed with molecular model of the receptor.

Among the twenty classical L-amino acids, only glutamate directly activates metabotropic glutamate receptors.

Under pathophysiological conditions, cellular amino acids can be profusely released from cells into the cerebral interstitial space. Because several class-C G protein coupled receptors (GPCRs) display a broad natural ligand spectrum, being sensitive to more than one endogenous ligand, we wondered whether the related metabotropic glutamate (mGlu) receptors could be modulated by various types of L-amino acids, allowing them to sense large increase in extracellular amino acid concentration. Here, the agonist, antagonist and allosteric effects of the twenty classical L-amino acids were evaluated on the eight mGlu receptor subtypes. We show that, in addition to glutamate (Glu), cysteine, aspartate and asparagine also lead to the activation of mGlu3, 4 and 5. Interestingly, our data demonstrate that the effect of these three amino acids did not result from a direct activation of the receptors, but from an indirect action involving Glu-transporters/exchangers. These data first demonstrate that mGlu receptors, unlike other class-C GPCRs, display an extremely high selectivity towards one ligand. Moreover, our results also show that Glu transport systems allow mGlu receptors to sense large increase in the extracellular concentration of some amino acids. Such a system will certainly lead to a large increase in some mGlu receptor activity under pathological conditions, such as seizure, ischemia or other brain injuries.

Adenoviral-mediated skeletal muscle transcriptional targeting using chimeric tissue-specific promoters.

BACKGROUND: Adenoviral vectors are promising tools to achieve skeletal muscle gene transfer for the treatment of peripheral ischemia. However, the use of ubiquitous viral promoters represents a major safety issue that could limit their use. Cellular regulatory sequences that allow strong and tissue-specific expression could circumvent this problem. MATERIAL/METHODS: Adenoviral vectors encoding the firefly luciferase under the control of the human skeletal a-actin promoter, alone or combined with the b-enolase or creatine kinase enhancer, were studied in vitro in murine C2C12 cells and in vivo in C57BL/6 mice. The expression of the reporter gene was measured in cell lysates and animal tissue homogenates. Adenoviral distribution was evaluated by PCR on DNA extracted from liver, spleen, heart and lungs. RESULTS: Skeletal a-actin promoter-based expression cassettes follow the regulation of the endogenous skeletal a-actin gene in vitro as luciferase expression strongly increases with myoblast differentiation into myotubes. The addition of the cellular b enolase or the creatine kinase enhancer improves the specificity of the skeletal a-actin promoter in vitro as well as in vivo. When adenoviral vectors are locally injected into skeletal muscles, the chimeric promoters drive a relatively strong gene expression, ranging from 16 to 28% of the Rous sarcoma virus promoter-related expression. CONCLUSIONS: Chimeric regulatory sequences based on the skeletal a-actin promoter are highly specific and allow transgene expression in vivo at high levels. These results indicate that expression cassettes designed for the treatment of peripheral ischemia by gene therapy can efficiently target gene expression to skeletal muscle.

Stimulation of angiogenesis by Cyr61 gene: a new therapeutic candidate.

Cyr61 is a secreted, cysteine-rich heparin-binding protein that is associated with extracellular matrix and cell surface, and has been demonstrated to be proangiogenic in vitro. In the present study we evaluated the angiogenic effect of human Cyr61 in an adenoviral context in the rabbit ischemic hindlimb model. For this purpose, three randomized groups of New Zealand White rabbits received intramuscular injections of 5 x 10(8) infectious units of an adenovirus carrying either the Cyr61 gene (Ad-Cyr61), the vascular endothelial growth factor gene (Ad-VEGF(165)) used as the angiogenic gene of reference, or no transgene (Ad-Null), 10 days after femoral artery excision in one limb. Perfusion of the ischemic limb was evaluated before adenoviral treatment (day 10) and 30 days postinjection (day 40). Angiographic, hemodynamic, and histologic parameters indicated that animals in the Ad-Cyr61 group had significantly better perfusion than in the Ad-Null group. Interestingly, this improvement exceeded that achieved with Ad-VEGF(165). In conclusion, Cyr61 gene transfer appears potent in stimulating limb revascularization, thereby promoting great improvement in tissue perfusion in the ischemic limb. These findings indicate that Cyr61 could be a promising therapeutic candidate for treating severe peripheral ischemic diseases.