Delineating the stepwise millisecond allosteric activation mechanism of the class C GPCR dimer mGlu5.

Two-thirds of signaling hormones and one-third of approved drugs exert their effects by binding and modulating the G protein-coupled receptors (GPCRs) activation. While the activation mechanism for monomeric GPCRs has been well-established, little is known about GPCRs in dimeric form. Here, by combining transition pathway generation, extensive atomistic simulation-based Markov state models, and experimental signaling assays, we reveal an asymmetric, stepwise millisecond allosteric activation mechanism for the metabotropic glutamate receptor subtype 5 receptor (mGlu5), an obligate dimeric class C GPCR. The dynamic picture is presented that agonist binding induces dimeric ectodomains compaction, amplified by the precise association of the cysteine-rich domains, ultimately loosely bringing the intracellular 7-transmembrane (7TM) domains into proximity and establishing an asymmetric TM6-TM6 interface. The active inter-domain interface enhances their intra-domain flexibility, triggering the activation of micro-switches crucial for downstream signal transduction. Furthermore, we show that the positive allosteric modulator stabilizes both the active inter-domain 7TM interface and an open, extended intra-domain ICL2 conformation. This stabilization leads to the formation of a pseudo-cavity composed of the ICL2, ICL3, TM3, and C-terminus, which facilitates G protein coordination. Our strategy may be generalizable for characterizing millisecond events in other allosteric systems.

Stepwise activation of a metabotropic glutamate receptor.

Metabotropic glutamate receptors belong to a family of G protein-coupled receptors that are obligate dimers and possess a large extracellular ligand-binding domain that is linked via a cysteine-rich domain to their 7-transmembrane domain1. Upon activation, these receptors undergo a large conformational change to transmit the ligand binding signal from the extracellular ligand-binding domain to the G protein-coupling 7-transmembrane domain2. In this manuscript, we propose a model for a sequential, multistep activation mechanism of metabotropic glutamate receptor subtype 5. We present a series of structures in lipid nanodiscs, from inactive to fully active, including agonist-bound intermediate states. Further, using bulk and single-molecule fluorescence imaging, we reveal distinct receptor conformations upon allosteric modulator and G protein binding.

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.

ERK-Directed Phosphorylation of mGlu5 Gates Methamphetamine Reward and Reinforcement in Mouse.

Methamphetamine (MA) is a highly addictive psychomotor stimulant drug. In recent years, MA use has increased exponentially on a global scale, with the number of MA-involved deaths reaching epidemic proportions. There is no approved pharmacotherapy for treating MA use disorder, and we know relatively little regarding the neurobiological determinants of vulnerability to this disease. Extracellular signal-regulated kinase (ERK) is an important signaling molecule implicated in the long-lasting neuroadaptations purported to underlie the development of substance use disorders, but the role for this kinase in the propensity to develop addiction, particularly MA use disorder, is uncharacterized. In a previous MA-induced place-conditioning study of C57BL/6J mice, we characterized mice as MA-preferring, -neutral, or -avoiding and collected tissue from the medial prefrontal cortex (mPFC). Using immunoblotting, we determined that elevated phosphorylated ERK expression within the medial prefrontal cortex (mPFC) is a biochemical correlate of the affective valence of MA in a population of C57BL/6J mice. We confirmed the functional relevance for mPFC ERK activation for MA-induced place-preference via site-directed infusion of the MEK inhibitor U0126. By contrast, ERK inhibition did not have any effect upon MA-induced locomotion or its sensitization upon repeated MA treatment. Through studies of transgenic mice with alanine point mutations on T1123/S1126 of mGlu5 that disrupt ERK-dependent phosphorylation of the receptor, we discovered that ERK-dependent mGlu5 phosphorylation normally suppresses MA-induced conditioned place-preference (MA-CPP), but is necessary for this drug's reinforcing properties. If relevant to humans, the present results implicate individual differences in the capacity of MA-associated cues/contexts to hyper-activate ERK signaling within mPFC in MA Use Disorder vulnerability and pose mGlu5 as one ERK-directed target contributing to the propensity to seek out and take MA.

Understanding Exposure-Receptor Occupancy Relationships for Metabotropic Glutamate Receptor 5 Negative Allosteric Modulators across a Range of Preclinical and Clinical Studies.

The metabotropic glutamate receptor 5 (mGlu5) is a recognized central nervous system therapeutic target for which several negative allosteric modulator (NAM) drug candidates have or are continuing to be investigated for various disease indications in clinical development. Direct measurement of target receptor occupancy (RO) is extremely useful to help design and interpret efficacy and safety in nonclinical and clinical studies. In the mGlu5 field, this has been successfully achieved by monitoring displacement of radiolabeled ligands, specifically binding to the mGlu5 receptor, in the presence of an mGlu5 NAM using in vivo and ex vivo binding in rodents and positron emission tomography imaging in cynomolgus monkeys and humans. The aim of this study was to measure the RO of the mGlu5 NAM HTL0014242 in rodents and cynomolgus monkeys and to compare its plasma and brain exposure-RO relationships with those of clinically tested mGlu5 NAMs dipraglurant, mavoglurant, and basimglurant. Potential sources of variability that may contribute to these relationships were explored. Distinct plasma exposure-response relationships were found for each mGlu5 NAM, with >100-fold difference in plasma exposure for a given level of RO. However, a unified exposure-response relationship was observed when both unbound brain concentration and mGlu5 affinity were considered. This relationship showed <10-fold overall difference, was fitted with a Hill slope that was not significantly different from 1, and appeared consistent with a simple Emax model. This is the first time this type of comparison has been conducted, demonstrating a unified brain exposure-RO relationship across several species and mGlu5 NAMs with diverse properties. SIGNIFICANCE STATEMENT: Despite the long history of mGlu5 as a therapeutic target and progression of multiple compounds to the clinic, no formal comparison of exposure-receptor occupancy relationships has been conducted. The data from this study indicate for the first time that a consistent, unified relationship can be observed between exposure and mGlu5 receptor occupancy when unbound brain concentration and receptor affinity are taken into account across a range of species for a diverse set of mGlu5 negative allosteric modulators, including a new drug candidate, HTL0014242.

[Recent advances in the structural biology of the class C G protein-coupled receptors: The metabotropic Glutamate receptor 5]. / Les avancées récentes dans le domaine de la biologie structurale des récepteurs couplés aux protéines G de la classe C : Le récepteur métabotropique du glutamate 5.

Class C GPCRs, that include metabotropic glutamate receptors (mGlu), taste receptors, GABAB receptor and Calcium-sensing receptor, are unusual in terms of their molecular architecture and allosteric regulation. They all form obligatory dimers, dimerization being fundamental for their function. More specifically, the mGlu are activated by the main excitatory neurotransmitter, L-glutamate. mGlu activation by glutamate binding in the venus flytrap domain (VFT) triggers conformational changes that are transmitted, through the Cystein-Rich Domain (CRD), to the conserved fold of 7 transmembrane helices (7TM), that couples to intracellular G protein. mGlu activity can also be allosterically modulated by positive (PAM) or negative (NAM) allosteric modulators binding to the 7TM. Recent progress in cryo-electron microscopy (cryoEM) has allowed unprecedented advances in deciphering the structural and molecular basis of their activation mechanism. The agonist induces a large movement between the subunits, bringing the 7TMs together and stabilizing a 7TM conformation structurally similar to the inactive state. The diversity of inactive conformations for the class C was unexpected but allows PAM stabilising a 7TM active conformation independent of the conformational changes induced by agonists, representing an alternative mode of mGlu activation. Here we present and discuss recent structural characterisation of mGlu receptors, highlighting findings that make the class C of GPCR unique. Understanding the structural basis of mGlu dimer signaling represents a landmark achievement and paves the way for structural investigation of GPCR dimer signaling in general. Structural information will open new avenues for structure-based drug design.

Title: Les avancées récentes dans le domaine de la biologie structurale des récepteurs couplés aux protéines G de la classe C : Le récepteur métabotropique du glutamate 5. Abstract: La classe C des Récepteurs Couplés aux Protéines G (RCPG) comprend plusieurs membres aux fonctions physiologiques importantes comme par exemple les récepteurs des principaux neurotransmetteurs excitateurs (glutamate) et inhibiteurs (GABA) du système nerveux, les récepteurs des goûts umami et sucré et les récepteurs sensibles au calcium. Ces récepteurs possèdent une architecture moléculaire particulière, caractérisée par la présence d'un large domaine extracellulaire (ECD) relié à un domaine membranaire composé de 7 hélices transmembranaires (7TM). De plus, ils forment tous des dimères obligatoires, la dimérisation étant fondamentale pour leur fonction. La fixation d'agoniste dans l'ECD induit l'activation du récepteur. L'activité des agonistes peut être modulée de manière allostérique par des modulateurs positifs (PAM) ou négatifs (NAM), se liant au domaine 7TM. Il est important de comprendre comment les changements de conformation induits par la liaison des agonistes au sein du domaine extracellulaire sont transmis au domaine transmembranaire mais aussi de comprendre les bases structurales et moléculaires de la régulation allostérique des récepteurs de la classe C. Les progrès récents de la microscopie électronique en conditions cryogéniques (cryoEM) ont permis des avancées sans précédent dans le décryptage des bases structurelles et moléculaires des mécanismes d'activation des RCPG de classe C, et notamment du récepteur métabotropique du glutamate de type 5 (mGlu5). Le glutamate entraîne une fermeture et un changement d'orientation des domaines extracellulaires qui induit un mouvement important entre les sous-unités, rapprochant les 7TM et stabilisant la conformation active du récepteur. La diversité de conformations inactives pour les récepteurs de la classe C était inattendue mais propice à une activation possible par des PAM. Ces derniers stabilisent une conformation active des 7TM, indépendante des changements conformationnels induits par les agonistes, représentant un mode alternatif d'activation des récepteurs mGlu. Nous présentons et discutons ici les caractérisations structurales récentes des récepteurs de classe C, en soulignant les résultats qui rendent cette famille de récepteurs unique. La compréhension de la base structurelle de la signalisation des dimères de mGlu représente une réalisation historique et ouvre la voie à l'analyse de la signalisation des dimères de RCPG en général. Ces analyses structurales devraient également ouvrir de nouvelles voies pour la conception de médicaments ciblant cette famille de récepteurs qui sont aussi des cibles thérapeutiques.

Lead optimization of the VU0486321 series of mGlu1 PAMs. Part 4: SAR reveals positive cooperativity across multiple mGlu receptor subtypes leading to subtype unselective PAMs.

Further optimization of the VU0486321 series of highly selective and CNS-penetrant mGlu1 PAMs identified unique 'molecular switches' on the central aromatic ring that engendered positive cooperativity with multiple mGlu subtypes across the receptor family, resulting in compounds with comparable activity at Group I (mGlu1/5) and Group III (mGlu4/6/7/8) mGlu receptors, receptors. These exciting data suggests this PAM chemotype appears to bind to multiple mGlu receptors, and that subtype selectivity is dictated by the degree of cooperativity, not a subtype selective, unique allosteric binding site. Moreover, there is interesting therapeutic potential for mGlu1/4/7/8 PAMs, as well as the first report of a GPCR allosteric 'privileged structure'.

Subtype-selective mechanisms of negative allosteric modulators binding to group I metabotropic glutamate receptors.

Group I metabotropic glutamate receptors (mGlu1 and mGlu5) are promising targets for multiple psychiatric and neurodegenerative disorders. Understanding the subtype selectivity of mGlu1 and mGlu5 allosteric sites is essential for the rational design of novel modulators with single- or dual-target mechanism of action. In this study, starting from the deposited mGlu1 and mGlu5 crystal structures, we utilized computational modeling approaches integrating docking, molecular dynamics simulation, and efficient post-trajectory analysis to reveal the subtype-selective mechanism of mGlu1 and mGlu5 to 10 diverse drug scaffolds representing known negative allosteric modulators (NAMs) in the literature. The results of modeling identified six pairs of non-conserved residues and four pairs of conserved ones as critical features to distinguish the selective NAMs binding to the corresponding receptors. In addition, nine pairs of residues are beneficial to the development of novel dual-target NAMs of group I metabotropic glutamate receptors. Furthermore, the binding modes of a reported dual-target NAM (VU0467558) in mGlu1 and mGlu5 were predicted to verify the identified residues that play key roles in the receptor selectivity and the dual-target binding. The results of this study can guide rational structure-based design of novel NAMs, and the approach can be generally applicable to characterize the features of selectivity for other G-protein-coupled receptors.

Probe dependence and biased potentiation of metabotropic glutamate receptor 5 is mediated by differential ligand interactions in the common allosteric binding site.

The metabotropic glutamate receptor 5 (mGlu5) is a promising therapeutic target for multiple CNS disorders. Recent mGlu5 drug discovery has focused on targeting binding sites within the mGlu5 7-transmembrane domain (7TM) that are topographically distinct from that of the endogenous ligand. mGlu5 primarily couples to Gq/11 proteins leading to mobilization of intracellular Ca2+ (iCa2+), but also activates iCa2+ independent signaling pathways, with biased agonism/modulation operative for multiple positive allosteric modulator (PAM) and PAM-agonist chemotypes. Although several residues within the common allosteric binding pocket are key determinants of PAM activity, how these residues affect biased modulation is unknown. The current study probed the molecular basis of mGlu5 PAM biased modulation. Modulation of mGlu5 activity by four chemically distinct mGlu5 PAMs (VU0424465, DPFE, VU29 and VU0409551) was assessed across two distinct receptor endpoints (iCa2+ mobilization and ERK1/2 phosphorylation) at mGlu5 receptors containing single-point mutations of allosteric binding pocket residues informed by computational modeling. Many mutations had differential effects on PAM affinity and cooperativity across signaling endpoints, resulting in gain or reversal of bias at the level of both affinity and functional cooperativity. Additionally, mutants had differential effects on functional cooperativity between the orthosteric ligands, DHPG and glutamate, and the PAMs, DPFE and VU29, but not VU0409551, indicating that probe dependence is linked to orthosteric agonists conferring activation states that differentially influence allosteric ligand-receptor interactions in a chemotype dependent fashion. Collectively, these data provide crucial insight into the residues that govern different activation states adopted by mGlu5 in order to signal via distinct intracellular pathways when co-bound by orthosteric agonists and PAMs.

Structure-based discovery and development of metabotropic glutamate receptor 5 negative allosteric modulators.

The metabotropic glutamate (mGlu) receptors are a family of eight class C G protein-coupled receptors (GPCRs) which modulate cell signaling and synaptic transmission to the major excitatory neurotransmitter l-glutamate (l-glutamic acid). Due to their role in modulating glutamate response, their widespread distribution in the central nervous system (CNS) and some evidence of dysregulation in disease, the mGlu receptors have become attractive pharmacological targets. As the orthosteric (glutamate) binding site is highly conserved across the eight mGlu receptors, it is difficult not only to generate ligands with subtype selectivity but, due to the nature of the binding site, with suitable drug-like properties to allow oral bioavailability and CNS penetration. Selective pharmacological targeting of a single receptor subtype can be achieved by targeting alternative (allosteric) binding sites. The nature of the allosteric binding pockets allows ligands to be developed that have good physical chemical properties as evidenced by several allosteric modulators of mGlu receptors entering clinical trials. The first negative allosteric modulators of the metabotropic glutamate 5 (mGlu5) receptor were discovered from high throughput screening activities. An alternative approach to drug discovery is to use structural knowledge to enable structure-based drug design (SBDD), which allows the design of molecules in a more rational, rather than empirical, fashion. Here we will describe the process of SBDD in the discovery of the mGlu5 negative allosteric modulator HTL0014242 and describe how knowledge of receptor structure can also be used to gain insights into the receptor activation mechanisms.

Design, Synthesis and Characterization of a New Series of Fluorescent Metabotropic Glutamate Receptor Type 5 Negative Allosteric Modulators.

In recent years, new drug discovery approaches based on novel pharmacological concepts have emerged. Allosteric modulators, for example, target receptors at sites other than the orthosteric binding sites and can modulate agonist-mediated activation. Interestingly, allosteric regulation may allow a fine-tuned regulation of unbalanced neurotransmitter' systems, thus providing safe and effective treatments for a number of central nervous system diseases. The metabotropic glutamate type 5 receptor (mGlu5R) has been shown to possess a druggable allosteric binding domain. Accordingly, novel allosteric ligands are being explored in order to finely regulate glutamate neurotransmission, especially in the brain. However, before testing the activity of these new ligands in the clinic or even in animal disease models, it is common to characterize their ability to bind mGlu5Rs in vitro. Here, we have developed a new series of fluorescent ligands that, when used in a new NanoBRET-based binding assay, will facilitate screening for novel mGlu5R allosteric modulators.

Revisiting the Radiosynthesis of [18F]FPEB and Preliminary PET Imaging in a Mouse Model of Alzheimer's Disease.

[18F]FPEB is a positron emission tomography (PET) radiopharmaceutical used for imaging the abundance and distribution of mGluR5 in the central nervous system (CNS). Efficient radiolabeling of the aromatic ring of [18F]FPEB has been an ongoing challenge. Herein, five metal-free precursors for the radiofluorination of [18F]FPEB were compared, namely, a chloro-, nitro-, sulfonium salt, and two spirocyclic iodonium ylide (SCIDY) precursors bearing a cyclopentyl (SPI5) and a new adamantyl (SPIAd) auxiliary. The chloro- and nitro-precursors resulted in a low radiochemical yield (<10% RCY), whereas both SCIDY precursors and the sulfonium salt precursor produced [18F]FPEB in the highest RCYs of 25% and 36%, respectively. Preliminary PET/CT imaging studies with [18F]FPEB were conducted in a transgenic model of Alzheimer's Disease (AD) using B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J (APP/PS1) mice, and data were compared with age-matched wild-type (WT) B6C3F1/J control mice. In APP/PS1 mice, whole brain distribution at 5 min post-injection showed a slightly higher uptake (SUV = 4.8 ± 0.4) than in age-matched controls (SUV = 4.0 ± 0.2). Further studies to explore mGluR5 as an early biomarker for AD are underway.

Profiling the Structural Determinants of Aryl Benzamide Derivatives as Negative Allosteric Modulators of mGluR5 by In Silico Study.

Glutamate plays a crucial role in the treatment of depression by interacting with the metabotropic glutamate receptor subtype 5 (mGluR5), whose negative allosteric modulators (NAMs) are thus promising antidepressants. At present, to explore the structural features of 106 newly synthesized aryl benzamide series molecules as mGluR5 NAMs, a set of ligand-based three-dimensional quantitative structure-activity relationship (3D-QSAR) analyses were firstly carried out applying comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) methods. In addition, receptor-based analysis, namely molecular docking and molecular dynamics (MD) simulations, were performed to further elucidate the binding modes of mGluR5 NAMs. As a result, the optimal CoMSIA model obtained shows that cross-validated correlation coefficient Q2 = 0.70, non-cross-validated correlation coefficient R2ncv = 0.89, predicted correlation coefficient R2pre = 0.87. Moreover, we found that aryl benzamide series molecules bind as mGluR5 NAMs at Site 1, which consists of amino acids Pro655, Tyr659, Ile625, Ile651, Ile944, Ser658, Ser654, Ser969, Ser965, Ala970, Ala973, Trp945, Phe948, Pro903, Asn907, Val966, Leu904, and Met962. This site is the same as that of other types of NAMs; mGluR5 NAMs are stabilized in the "linear" and "arc" configurations mainly through the H-bonds interactions, π-π stacking interaction with Trp945, and hydrophobic contacts. We hope that the models and information obtained will help understand the interaction mechanism of NAMs and design and optimize NAMs as new types of antidepressants.

Evaluation of Operational Models of Agonism and Allosterism at Receptors with Multiple Orthosteric Binding Sites.

Current operational models of agonism and allosterism quantify ligand actions at receptors where agonist concentration-response relationships are nonhyperbolic by introduction of a transducer slope that relates receptor occupancy to response. However, for some receptors nonhyperbolic concentration-response relationships arise from multiple endogenous agonist molecules binding to a receptor in a cooperative manner. Thus, we developed operational models of agonism in systems with cooperative agonist binding and evaluated the models by simulating data describing agonist effects. The models were validated by analyzing experimental data demonstrating the effects of agonists and allosteric modulators at receptors where agonist binding follows hyperbolic (M4 muscarinic acetylcholine receptors) or nonhyperbolic relationships (metabotropic glutamate receptor 5 and calcium-sensing receptor). For hyperbolic agonist concentration-response relationships, no differences in estimates of ligand affinity, efficacy, or cooperativity were observed when the slope was assigned to either a transducer slope or agonist binding slope. In contrast, for receptors with nonhyperbolic agonist concentration-response relationships, estimates of ligand affinity, efficacy, or cooperativity varied depending on the assignment of the slope. The extent of this variation depended on the magnitude of the slope value and agonist efficacy, and for allosteric modulators on the magnitude of cooperativity. The modified operational models described herein are well suited to analyzing agonist and modulator interactions at receptors that bind multiple orthosteric agonists in a cooperative manner. Accounting for cooperative agonist binding is essential to accurately quantify agonist and drug actions. SIGNIFICANCE STATEMENT: Some orthosteric agonists bind to multiple sites on a receptor, but current analytical methods to characterize such interactions are limited. Herein, we develop and validate operational models of agonism and allosterism for receptors with multiple orthosteric binding sites, and demonstrate that such models are essential to accurately quantify agonist and drug actions. These findings have important implications for the discovery and development of drugs targeting receptors such as the calcium-sensing receptor, which binds at least five calcium ions.

The role of water and protein flexibility in the structure-based virtual screening of allosteric GPCR modulators: an mGlu5 receptor case study.

Stabilizing unique receptor conformations, allosteric modulators of G-protein coupled receptors (GPCRs) might open novel treatment options due to their new pharmacological action, their enhanced specificity and selectivity in both binding and signaling. Ligand binding occurs at intrahelical allosteric sites and involves significant induced fit effects that include conformational changes in the local protein environment and water networks. Based on the analysis of available crystal structures of metabotropic glutamate receptor 5 (mGlu5) we investigated these effects in the binding of mGlu5 receptor negative allosteric modulators. A large set of retrospective virtual screens revealed that the use of multiple protein structures and the inclusion of selected water molecules improves virtual screening performance compared to conventional docking strategies. The role of water molecules and protein flexibility in ligand binding can be taken into account efficiently by the proposed docking protocol that provided reasonable enrichment of true positives. This protocol is expected to be useful also for identifying intrahelical allosteric modulators for other GPCR targets.

Identification of Novel Allosteric Modulators of Metabotropic Glutamate Receptor Subtype 5 Acting at Site Distinct from 2-Methyl-6-(phenylethynyl)-pyridine Binding.

As part of the G-protein coupled receptor (GPCR) family, metabotropic glutamate (mGlu) receptors play an important role as drug targets of cognitive diseases. Selective allosteric modulators of mGlu subtype 5 (mGlu5) have the potential to alleviate symptoms of numerous central nervous system disorders such as schizophrenia in a more targeted fashion. Multiple mGlu5 positive allosteric modulators (PAMs), such as 1-(3-fluorophenyl)-N-((3-fluorophenyl)-methylideneamino)-methanimine (DFB), 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)-benzamide (CDPPB), and 4-nitro-N-(1,3-diphenyl-1H-pyrazol-5-yl)-benzamide (VU-29), exert their actions by binding to a defined allosteric site on mGlu5 located in the seven-transmembrane domain (7TM) and shared by mGlu5 negative allosteric modulator (NAM) 2-methyl-6-(phenylethynyl)-pyridine (MPEP). Actions of the PAM N-{4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl]phenyl}-2-hydroxybenzamide (CPPHA) are mediated by a distinct allosteric site in the 7TM domain different from the MPEP binding site. Experimental evidence confirms these findings through mutagenesis experiments involving residues F585 (TM1) and A809 (TM7). In an effort to investigate mGlu5 PAM selectivity for this alternative allosteric site distinct from MPEP binding, we employed in silico quantitative structure-activity relationship (QSAR) modeling. Subsequent ligand-based virtual screening prioritized a set of 63 candidate compounds predicted from a library of over 4 million commercially available compounds to bind exclusively to this novel site. Experimental validation verified the biological activity for seven of 63 selected candidates. Further, medicinal chemistry optimizations based on these molecules revealed compound VU6003586 with an experimentally validated potency of 174 nM. Radioligand binding experiments showed only partial inhibition at very high concentrations, most likely indicative of binding at a non-MPEP site. Selective positive allosteric modulators for mGlu5 have the potential for tremendous impact concerning devastating neurological disorders such as schizophrenia and Huntington's disease. These identified and validated novel selective compounds can serve as starting points for more specifically tailored lead and probe molecules and thus help the development of potential therapeutic agents with reduced adverse effects.

Allosteric Modulation Mechanism of the mGluR5 Transmembrane Domain.

Positive allosteric modulators (PAMs) of metabotropic glutamate receptor type 5 (mGluR5), a prototypical class C G protein-coupled receptor (GPCR), have shown therapeutic potential for various neurological disorders. Understanding the allosteric activation mechanism is essential for the rational design of mGluR5 PAMs. We studied the actions of positive and negative allosteric modulators within the transmembrane domain of mGluR5, using enhance-sampling all-atom molecular dynamics simulations. We found dual binding modes of the PAM, associated with distinct shapes of the allosteric pocket. The negative allosteric modulators, in contrast, showed only one binding mode. The simulations revealed the mechanism by which the PAM activated the receptor, in the absence of the orthosteric agonist (the so-called allosteric agonism). The mechanism relied on dynamic communications between amino-acid motifs that are highly conserved across class C GPCRs. The findings may guide structure-based design and virtual screening of allosteric modulators for mGluR5 as well as for other class C GPCRs.

Mechanisms Underlying Allosteric Molecular Switches of Metabotropic Glutamate Receptor 5.

The metabotropic glutamate 5 (mGlu5) receptor is a class C G protein-coupled receptor (GPCR) that is implicated in several CNS disorders making it a popular drug discovery target. Years of research have revealed allosteric mGlu5 ligands showing an unexpected complete switch in functional activity despite only small changes in their chemical structure, resulting in positive allosteric modulators (PAM) or negative allosteric modulators (NAM) for the same scaffold. Up to now, the origins of this effect are not understood, causing difficulties in a drug discovery context. In this work, experimental data was gathered and analyzed alongside docking and Molecular Dynamics (MD) calculations for three sets of PAM and NAM pairs. The results consistently show the role of specific interactions formed between ligand substituents and amino acid side chains that block or promote local movements associated with receptor activation. The work provides an explanation for how such small structural changes lead to remarkable differences in functional activity. While this work can greatly help drug discovery programs avoid these switches, it also provides valuable insight into the mechanisms of class C GPCR allosteric activation. Furthermore, the approach shows the value of applying MD to understand functional activity in drug design programs, even for such close structural analogues.

Structural insights into the activation of metabotropic glutamate receptors.

Metabotropic glutamate receptors are family C G-protein-coupled receptors. They form obligate dimers and possess extracellular ligand-binding Venus flytrap domains, which are linked by cysteine-rich domains to their 7-transmembrane domains. Spectroscopic studies show that signalling is a dynamic process, in which large-scale conformational changes underlie the transmission of signals from the extracellular Venus flytraps to the G protein-coupling domains-the 7-transmembrane domains-in the membrane. Here, using a combination of X-ray crystallography, cryo-electron microscopy and signalling studies, we present a structural framework for the activation mechanism of metabotropic glutamate receptor subtype 5. Our results show that agonist binding at the Venus flytraps leads to a compaction of the intersubunit dimer interface, thereby bringing the cysteine-rich domains into close proximity. Interactions between the cysteine-rich domains and the second extracellular loops of the receptor enable the rigid-body repositioning of the 7-transmembrane domains, which come into contact with each other to initiate signalling.

Structure-Based Optimization Strategies for G Protein-Coupled Receptor (GPCR) Allosteric Modulators: A Case Study from Analyses of New Metabotropic Glutamate Receptor 5 (mGlu5) X-ray Structures.

Two interesting new X-ray structures of negative allosteric modulator (NAM) ligands for the mGlu5 receptor, M-MPEP (3) and fenobam (4), are reported. The new structures show how the binding of the ligands induces different receptor water channel conformations to previously published structures. The structure of fenobam, where a urea replaces the acetylenic linker in M-MPEP and mavoglurant, reveals a binding mode where the ligand is rotated by 180° compared to a previously proposed docking model. The need for multiple ligand structures for accurate GPCR structure-based drug design is demonstrated by the different growing vectors identified for the head groups of M-MPEP and mavoglurant and by the unexpected water-mediated receptor interactions of a new chemotype represented by fenobam. The implications of the new structures for ligand design are discussed, with extensive analysis of the energetics of the water networks of both pseudoapo and bound structures providing a new design strategy for allosteric modulators.