Novel Inhibitory Site Revealed by XAP044 Mode of Action on the Metabotropic Glutamate 7 Receptor Venus Flytrap Domain.

Metabotropic glutamate (mGlu) receptors play a key role in modulating most synapses in the brain. The mGlu7 receptors inhibit presynaptic neurotransmitter release and offer therapeutic possibilities for post-traumatic stress disorders or epilepsy. Screening campaigns provided mGlu7-specific allosteric modulators as the inhibitor XAP044 (Gee et al. J. Biol. Chem. 2014). In contrast to other mGlu receptor allosteric modulators, XAP044 does not bind in the transmembrane domain but to the extracellular domain of the mGlu7 receptor and not at the orthosteric site. Here, we identified the mode of action of XAP044, combining synthesis of derivatives, modeling and docking experiments, and mutagenesis. We propose a unique mode of action of these inhibitors, preventing the closure of the Venus flytrap agonist binding domain. While acting as a noncompetitive antagonist of L-AP4, XAP044 and derivatives act as apparent competitive antagonists of LSP4-2022. These data revealed more potent XAP044 analogues and new possibilities to target mGluRs.

Epitope Identification of an mGlu5 Receptor Nanobody Using Physics-Based Molecular Modeling and Deep Learning Techniques.

The world has witnessed a revolution in therapeutics with the development of biological medicines such as antibodies and antibody fragments, notably nanobodies. These nanobodies possess unique characteristics including high specificity and modulatory activity, making them promising candidates for therapeutic applications. Identifying their binding mode is essential for their development. Experimental structural techniques are effective to get such information, but they are expensive and time-consuming. Here, we propose a computational approach, aiming to identify the epitope of a nanobody that acts as an agonist and a positive allosteric modulator at the rat metabotropic glutamate receptor 5. We employed multiple structure modeling tools, including various artificial intelligence algorithms for epitope mapping. The computationally identified epitope was experimentally validated, confirming the success of our approach. Additional dynamics studies provided further insights on the modulatory activity of the nanobody. The employed methodologies and approaches initiate a discussion on the efficacy of diverse techniques for epitope mapping and later nanobody engineering.

Design, Synthesis, Pharmacology, and In Silico Studies of (1S,2S,3S)-2-((S)-Amino(carboxy)methyl)-3-(carboxymethyl)cyclopropane-1-carboxylic Acid (LBG30300): A Picomolar Potency Subtype-Selective mGlu2 Receptor Agonist.

Metabotropic glutamate (Glu) receptors (mGlu receptors) play a key role in modulating excitatory neurotransmission in the central nervous system (CNS). In this study, we report the structure-based design and pharmacological evaluation of densely functionalized, conformationally restricted glutamate analogue (1S,2S,3S)-2-((S)-amino(carboxy)methyl)-3-(carboxymethyl)cyclopropane-1-carboxylic acid (LBG30300). LBG30300 was synthesized in a stereocontrolled fashion in nine steps from a commercially available optically active epoxide. Functional characterization of all eight mGlu receptor subtypes showed that LBG30300 is a picomolar agonist at mGlu2 with excellent selectivity over mGlu3 and the other six mGlu receptor subtypes. Bioavailability studies on mice (IV administration) confirm CNS exposure, and an in silico study predicts a binding mode of LBG30300 which induces a flipping of Tyr144 to allow for a salt bridge interaction of the acetate group with Arg271. The Tyr144 residue now prevents Arg271 from interacting with Asp146, which is a residue of differentiation between mGlu2 and mGlu3 and thus could explain the observed subtype selectivity.

Activation of the Mu-Delta Opioid Receptor Heteromers Blocks Morphine Rewarding Effects.

BACKGROUND: Evidence has accumulated demonstrating the existence of opioid receptor heteromers, and recent data suggest that targeting these heteromers could reduce opioid side effects while retaining therapeutic effects. Indeed, CYM51010 characterized as a MOR (mu opioid receptor)/DOR (delta opioid receptor) heteromer-preferring agonist promoted antinociception comparable with morphine but with less tolerance. In the perspective of developing these new classes of pharmacological agents, data on their putative side effects are mandatory. METHODS: Therefore, in this study, we investigated the effects of CYM51010 in different models related to drug addiction in mice, including behavioral sensitization, conditioned place preference and withdrawal. RESULTS: We found that, like morphine, CYM51010 promoted acute locomotor activity as well as psychomotor sensitization and rewarding effect. However, it induced less physical dependence than morphine. We also investigated the ability of CYM51010 to modulate some morphine-induced behavior. Whereas CYM51010 was unable to block morphine-induced physical dependence, it blocked reinstatement of an extinguished morphine induced-conditioned place preference. CONCLUSIONS: Altogether, our results reveal that targeting MOR-DOR heteromers could represent a promising strategy to block morphine reward.

Metabotropic glutamate receptor orthosteric ligands and their binding sites.

Metabotropic glutamate receptors (mGluRs) have been discovered almost four decades ago. Since then, their pharmacology has been largely developed as well as their structural organization. Indeed mGluRs are attractive therapeutic targets for numerous psychiatric and neurological disorders because of their modulating role of synaptic transmission. The more recent drug discovery programs have mostly concentrated on allosteric modulators. However, orthosteric agonists and antagonists have remained unavoidable pharmacological tools as, although not expected, many of them can reach the brain, or can be modified to reach the brain. This review focuses on the most common orthosteric ligands as well as on the few allosteric modulators interacting with the glutamate binding domain. The 3D-structures of these ligands at their binding sites are reported. For most of them, X-Ray structures or docked homology models are available. Because of the high conservation of the binding site, subtype selective agonists were not easy to find. Yet, some were discovered when extending their chemical structures in order to reach selective sites of the receptors.

A nanobody activating metabotropic glutamate receptor 4 discriminates between homo- and heterodimers.

There is growing interest in developing biologics due to their high target selectivity. The G protein-coupled homo- and heterodimeric metabotropic glutamate (mGlu) receptors regulate many synapses and are promising targets for the treatment of numerous brain diseases. Although subtype-selective allosteric small molecules have been reported, their effects on the recently discovered heterodimeric receptors are often not known. Here, we describe a nanobody that specifically and fully activates homodimeric human mGlu4 receptors. Molecular modeling and mutagenesis studies revealed that the nanobody acts by stabilizing the closed active state of the glutamate binding domain by interacting with both lobes. In contrast, this nanobody does not activate the heterodimeric mGlu2-4 but acts as a pure positive allosteric modulator. These data further reveal how an antibody can fully activate a class C receptor and bring further evidence that nanobodies represent an alternative way to specifically control mGlu receptor subtypes.

LSP2-9166, an orthosteric mGlu4 and mGlu7 receptor agonist, reduces cocaine self-administration under a progressive ratio schedule in rats.

Cocaine addiction is a serious health issue in Western countries. Despite the regular increase in cocaine consumption across the population, there is no specific treatment for cocaine addiction. Critical roles for glutamate neurotransmission in the rewarding effects of psychostimulants as well as relapse have been suggested and accumulating evidence indicates that targeting mGlu group III receptors could represent a promising strategy to develop therapeutic compounds to treat addiction. In this context, the aim of our study was to examine the effect of LSP2-9166, a mGlu4/mGlu7 receptor orthosteric agonist, on the motivation for cocaine intake. We used an intravenous self-administration paradigm in male Wistar rats as a reliable model of voluntary drug intake. We first evaluated the direct impact of cocaine on Grm4 and Grm7 gene expression. Voluntary cocaine intake under a fixed ratio schedule of injections induced an increase of both mGlu4 and mGlu7 receptor transcripts in nucleus accumbens and hippocampus. We then evaluated the ability of LSP2-9166 to affect cocaine self-administration under a progressive ratio schedule of reinforcement. We found that this compound inhibits the motivation to obtain the drug, although it induced a hypolocomotor effect which could biais motivation index. Our findings demonstrate that mGlu group III receptors represent new targets for decreasing motivation to self-administer cocaine.

Illuminating the allosteric modulation of the calcium-sensing receptor.

Many membrane receptors are regulated by nutrients. However, how these nutrients control a single receptor remains unknown, even in the case of the well-studied calcium-sensing receptor CaSR, which is regulated by multiple factors, including ions and amino acids. Here, we developed an innovative cell-free Förster resonance energy transfer (FRET)-based conformational CaSR biosensor to clarify the main conformational changes associated with activation. By allowing a perfect control of ambient nutrients, this assay revealed that Ca2+ alone fully stabilizes the active conformation, while amino acids behave as pure positive allosteric modulators. Based on the identification of Ca2+ activation sites, we propose a molecular basis for how these different ligands cooperate to control CaSR activation. Our results provide important information on CaSR function and improve our understanding of the effects of genetic mutations responsible for human diseases. They also provide insights into how a receptor can integrate signals from various nutrients to better adapt to the cell response.

Amino Acids Bearing Aromatic or Heteroaromatic Substituents as a New Class of Ligands for the Lysosomal Sialic Acid Transporter Sialin.

Sialin, encoded by the SLC17A5 gene, is a lysosomal sialic acid transporter defective in Salla disease, a rare inherited leukodystrophy. It also enables metabolic incorporation of exogenous sialic acids, leading to autoantibodies against N-glycolylneuraminic acid in humans. Here, we identified a novel class of human sialin ligands by virtual screening and structure-activity relationship studies. The ligand scaffold is characterized by an amino acid backbone with a free carboxylate, an N-linked aromatic or heteroaromatic substituent, and a hydrophobic side chain. The most potent compound, 45 (LSP12-3129), inhibited N-acetylneuraminic acid 1 (Neu5Ac) transport in a non-competitive manner with IC50 ≈ 2.5 µM, a value 400-fold lower than the KM for Neu5Ac. In vitro and molecular docking studies attributed the non-competitive character to selective inhibitor binding to the Neu5Ac site in a cytosol-facing conformation. Moreover, compound 45 rescued the trafficking defect of the pathogenic mutant (R39C) causing Salla disease. This new class of cell-permeant inhibitors provides tools to investigate the physiological roles of sialin and help develop pharmacological chaperones for Salla disease.

Asc-1 Transporter (SLC7A10): Homology Models And Molecular Dynamics Insights Into The First Steps Of The Transport Mechanism.

The alanine-serine-cysteine transporter Asc-1 regulates the synaptic availability of D-serine and glycine (the two co-agonists of the NMDA receptor) and is regarded as an important drug target. To shuttle the substrate from the extracellular space to the cytoplasm, this transporter undergoes multiple distinct conformational states. In this work, homology modeling, substrate docking and molecular dynamics simulations were carried out to learn more about the transition between the "outward-open" and "outward-open occluded" states. We identified a transition state involving the highly-conserved unwound TM6 region in which the Phe243 flips close to the D-serine substrate without major movements of TM6. This feature and those of other key residues are proposed to control the binding site and substrate translocation. Competitive inhibitors ACPP, LuAE00527 and SMLC were docked and their binding modes at the substrate binding site corroborated the key role played by Phe243 of TM6. For ACPP and LuAE00527, strong hydrophobic interactions with this residue hinder its mobility and prevent the uptake and the efflux of substrates. As for SMLC, the weaker interactions maintain the flexibility of Phe243 and the efflux process. Overall, we propose a molecular basis for the inhibition of substrate translocation of the Asc-1 transporter that should be valuable for rational drug design.

Antidepressant efficacy of a selective organic cation transporter blocker in a mouse model of depression.

Current antidepressants act principally by blocking monoamine reuptake by high-affinity transporters in the brain. However, these antidepressants show important shortcomings such as slow action onset and limited efficacy in nearly a third of patients with major depression disorder. Here, we report the development of a prodrug targeting organic cation transporters (OCT), atypical monoamine transporters recently implicated in the regulation of mood. Using molecular modeling, we designed a selective OCT2 blocker, which was modified to increase brain penetration. This compound, H2-cyanome, was tested in a rodent model of chronic depression induced by 7-week corticosterone exposure. In male mice, prolonged administration of H2-cyanome induced positive effects on several behaviors mimicking symptoms of depression, including anhedonia, anxiety, social withdrawal, and memory impairment. Importantly, in this validated model, H2-cyanome compared favorably with the classical antidepressant fluoxetine, with a faster action on anhedonia and better anxiolytic effects. Integrated Z-scoring across these depression-like variables revealed a lower depression score for mice treated with H2-cyanome than for mice treated with fluoxetine for 3 weeks. Repeated H2-cyanome administration increased ventral tegmental area dopaminergic neuron firing, which may underlie its rapid action on anhedonia. H2-cyanome, like fluoxetine, also modulated several intracellular signaling pathways previously involved in antidepressant response. Our findings provide proof-of-concept of antidepressant efficacy of an OCT blocker, and a mechanistic framework for the development of new classes of antidepressants and therapeutic alternatives for resistant depression and other psychiatric disturbances such as anxiety.

LSP5-2157 a new inhibitor of vesicular glutamate transporters.

Vesicular glutamate transporters (VGLUT1-3) mediate the uptake of glutamate into synaptic vesicles. VGLUTs are pivotal actors of excitatory transmission and of almost all brain functions. Their implication in various pathologies has been clearly documented. Despite their functional importance, the pharmacology of VGLUTs is limited to a few dyes such as Trypan Blue, Rose Bengal or Brilliant Yellow type. Here, we report the design and evaluation of new potent analogs based on Trypan Blue scaffold. Our best compound, named LSP5-2157, has an EC50 of 50 nM on glutamate vesicular uptake. Using a 3D homology model of VGLUT1 and docking experiments, we determined its putative binding subdomains within vesicular glutamate transporters and validated the structural requirement for VGLUT inhibition. To better estimate the specificity and potency of LSP5-2157, we also investigated its ability to block glutamatergic transmission in autaptic hippocampal cells. Neither glutamate receptors nor GABAergic transmission or transmission machinery were affected by LSP5-2157. Low doses of compound reversibly reduce glutamatergic neurotransmission in hippocampal autpases. LSP5-2157 had a low and depressing effect on synaptic efficacy in hippocampal slice. Furthermore, LSP5-2157 had no effect on NMDA-R- mediated fEPSP but reduce synaptic plasticity induced by 3 trains of 100 Hz. Finally, LSP5-2157 had the capacity to inhibit VGLUT3-dependent auditory synaptic transmission in the guinea pig cochlea. In this model, it abolished the compound action potential of auditory nerve at high concentration showing the limited permeation of LSP5-2157 in an in-vivo model. In summary, the new ligand LSP5-2157, has a high affinity and specificity for VGLUTs and shows some permeability in isolated neuron, tissue preparations or in vivo in the auditory system. These findings broaden the field of VGLUTs inhibitors and open the way to their use to assess glutamatergic functions in vitro and in vivo.

Conformational pathway provides unique sensitivity to a synaptic mGluR.

Metabotropic glutamate receptors (mGluRs) are dimeric G-protein-coupled receptors that operate at synapses. Macroscopic and single molecule FRET to monitor structural rearrangements in the ligand binding domain (LBD) of the mGluR7/7 homodimer revealed it to have an apparent affinity ~4000-fold lower than other mGluRs and a maximal activation of only ~10%, seemingly too low for activation at synapses. However, mGluR7 heterodimerizes, and we find it to associate with mGluR2 in the hippocampus. Strikingly, the mGluR2/7 heterodimer has high affinity and efficacy. mGluR2/7 shows cooperativity in which an unliganded subunit greatly enhances activation by agonist bound to its heteromeric partner, and a unique conformational pathway to activation, in which mGluR2/7 partially activates in the Apo state, even when its LBDs are held open by antagonist. High sensitivity and an unusually broad dynamic range should enable mGluR2/7 to respond to both glutamate transients from nearby release and spillover from distant synapses.

Infiltrating Myeloid Cells Drive Osteosarcoma Progression via GRM4 Regulation of IL23.

The glutamate metabotropic receptor 4 (GRM4) locus is linked to susceptibility to human osteosarcoma, through unknown mechanisms. We show that Grm4-/- gene-targeted mice demonstrate accelerated radiation-induced tumor development to an extent comparable with Rb1+/- mice. GRM4 is expressed in myeloid cells, selectively regulating expression of IL23 and the related cytokine IL12. Osteosarcoma-conditioned media induce myeloid cell Il23 expression in a GRM4-dependent fashion, while suppressing the related cytokine Il12. Both human and mouse osteosarcomas express an increased IL23:IL12 ratio, whereas higher IL23 expression is associated with worse survival in humans. Consistent with an oncogenic role, Il23 -/- mice are strikingly resistant to osteosarcoma development. Agonists of GRM4 or a neutralizing antibody to IL23 suppressed osteosarcoma growth in mice. These findings identify a novel, druggable myeloid suppressor pathway linking GRM4 to the proinflammatory IL23/IL12 axis. SIGNIFICANCE: Few novel systemic therapies targeting osteosarcoma have emerged in the last four decades. Using insights gained from a genome-wide association study and mouse modeling, we show that GRM4 plays a role in driving osteosarcoma via a non-cell-autonomous mechanism regulating IL23, opening new avenues for therapeutic intervention.See related commentary by Jones, p. 1484.This article is highlighted in the In This Issue feature, p. 1469.

The mGlu7 receptor provides protective effects against epileptogenesis and epileptic seizures.

Finding new targets to control or reduce seizure activity is essential to improve the management of epileptic patients. We hypothesized that activation of the pre-synaptic and inhibitory metabotropic glutamate receptor type 7 (mGlu7) reduces spontaneous seizures. We tested LSP2-9166, a recently developed mGlu7/4 agonist with unprecedented potency on mGlu7 receptors, in two paradigms of epileptogenesis. In a model of chemically induced epileptogenesis (pentylenetetrazole systemic injection), LSP2-9166 induces an anti-epileptogenic effect rarely observed in preclinical studies. In particular, we found a bidirectional modulation of seizure progression by mGlu4 and mGlu7 receptors, the latter preventing kindling. In the intra-hippocampal injection of kainic acid mouse model that mimics the human mesial temporal lobe epilepsy, we found that LSP2-9166 reduces seizure frequency and hippocampal sclerosis. LSP2-9166 also acts as an anti-seizure drug on established seizures in both models tested. Specific modulation of the mGlu7 receptor could represent a novel approach to reduce pathological network remodeling.

Chloride ions stabilize the glutamate-induced active state of the metabotropic glutamate receptor 3.

Due to the essential roles of glutamate, detection and response to a large range of extracellular concentrations of this excitatory amino acid are necessary for the fine-tuning of brain functions. Metabotropic glutamate receptors (mGluRs) are implicated in shaping the activity of many synapses in the central nervous system. Among the eight mGluR subtypes, there is increasing interest in studying the mGlu3 receptor which has recently been linked to various diseases, including psychiatric disorders. This receptor displays striking functional properties, with a high and, often, full basal activity, making its study elusive in heterologous systems. Here, we demonstrate that Cl- ions exert strong positive allosteric modulation of glutamate on the mGlu3 receptor. We have also identified the molecular and structural determinants lying behind this allostery: a unique interactive "chloride-lock" network. Indeed, Cl- ions dramatically stabilize the glutamate-induced active state of the extracellular domain of the mGlu3 receptor. Thus, the mGlu3 receptors' large basal activity does not correspond to a constitutive activity in absence of agonist. Instead, it results mostly from a Cl-mediated amplified response to low ambient glutamate concentrations, such as those measured in cell media. This strong interaction between glutamate and Cl- ions allows the mGlu3 receptor to sense and efficiently react to sub-micromolar concentrations of glutamate, making it the most sensitive member of mGluR family.

Profiling of orthosteric and allosteric group-III metabotropic glutamate receptor ligands on various G protein-coupled receptors with Tag-lite® assays.

Group-III metabotropic glutamate (mGlu) receptors are important synaptic regulators and are potential druggable targets for Parkinson disease, autism and pain. Potential drugs include orthosteric agonists in the glutamate binding extracellular domain and positive allosteric modulators interacting with seven-pass transmembrane domains. Orthosteric agonists are rarely completely specific for an individual group-III mGlu subtype. Furthermore they often fail to pass the blood-brain barrier and they constitutively activate their target receptor. These properties limit the potential therapeutic use of orthosteric agonists. Allosteric modulators are more specific and maintain the biological activity of the targeted receptor. However, they bind in a hydrophobic pocket and this limits their bio-availability and increases possible off-target action. It is therefore important to characterize the action of potential drug targets with a multifaceted and deeply informative assay. Here we aimed at multifaceted deep profiling of the effect of seven different agonists, and seven positive allosteric modulators on 34 different G protein-coupled receptors by a Tag-lite® assay. Our results did not reveal off-target activity of mGlu orthosteric agonists. However, five allosteric modulators had either positive or negative effects on non-cognate G protein-coupled receptors. In conclusion, we demonstrate the power of the Tag-lite® assay for potential drug ligand profiling on G protein-coupled receptors and its potential to identify positive allosteric compounds.

Mutual activation of glutamatergic mGlu4 and muscarinic M4 receptors reverses schizophrenia-related changes in rodents.

RATIONALE: Metabotropic glutamate receptors and muscarinic M4 receptors have been proposed as novel targets for various brain disorders, including schizophrenia. Both receptors are coupled to Go/i proteins and are expressed in brain circuits that are important in schizophrenia. Therefore, their mutual activation may be an effective treatment and allow minimizing the doses of ligands required for optimal activity. OBJECTIVES: In the present studies, subactive doses of mGlu4 and M4 activators (LSP4-2022 and VU152100, respectively) were administered to investigate the mutual interaction between mGlu4 and M4 receptors in animal models of schizophrenia. METHODS: The behavioral tests used were MK-801-induced hyperactivity, (±)-2.5-dimethoxy-4-iodoamphetamine hydrochloride (DOI)-induced head twitches, the modified forced swim test, and MK-801-induced disruptions of social interactions and novel object recognition. DOI-induced spontaneous excitatory postsynaptic currents (sEPSCs) in brain slices and positron emission tomography (PET) in were used to establish the ability of these compounds to modulate the glutamatergic and dopaminergic systems. Rotarod was used to assess putative adverse effects. RESULTS: The mutual administration of subactive doses of LSP4-2022 and VU152100 exerted similar antipsychotic-like efficacy in animals as observed for active doses of both compounds, indicating their additive actions. VU152100 inhibited the DOI-induced frequency (but not amplitude) of sEPSCs in the frontal cortex, confirming presynaptic regulation of glutamate release. Both compounds reversed amphetamine-induced decrease in D2 receptor levels in the striatum, as measured with [18F]fallypride. The compounds did not induce any motor impartments when measured in rotarod test. CONCLUSIONS: Based on our results, the simultaneous activation of M4 and mGlu4 receptors is beneficial in reversing MK-801- and amphetamine-induced schizophrenia-related changes in animals.

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.

Role of mGlu7 receptor in morphine rewarding effects is uncovered by a novel orthosteric agonist.

Opiate dependence is a major health issue and despite the existence of opioid substitution treatment, relapse frequently occurs. Group III metabotropic glutamate (mGlu) receptors has received much attention as a putative target in ethanol and cocaine addiction, but no data on opiate addiction exist. So we investigated the role of group III mGlu receptors in morphine rewarding effects through the expression and the reinstatement of conditioned place preference (CPP) using a newly synthesized mGlu4/mGlu7 receptor orthosteric agonist, LSP2-9166. We found that LSP2-9166 blocked morphine CPP expression and reinstatement after extinction. Blockade of CPP expression with LSP2-9166 was abolished when using XAP044, a mGlu7 antagonist. We also found that LSP2-9166 at the dose active for blocking morphine reward was devoid of any effect on locomotion, hedonic state, spatial memory, anxiety or depression. Altogether our data demonstrated that group III mGlu receptors, and more specifically mGlu7, might be a valuable target in opiate addiction.