Exploring the activation mechanism of metabotropic glutamate receptor 2.

Homomeric dimerization of metabotropic glutamate receptors (mGlus) is essential for the modulation of their functions and represents a promising avenue for the development of novel therapeutic approaches to address central nervous system diseases. Yet, the scarcity of detailed molecular and energetic data on mGlu2 impedes our in-depth comprehension of their activation process. Here, we employ computational simulation methods to elucidate the activation process and key events associated with the mGlu2, including a detailed analysis of its conformational transitions, the binding of agonists, Gi protein coupling, and the guanosine diphosphate (GDP) release. Our results demonstrate that the activation of mGlu2 is a stepwise process and several energy barriers need to be overcome. Moreover, we also identify the rate-determining step of the mGlu2's transition from the agonist-bound state to its active state. From the perspective of free-energy analysis, we find that the conformational dynamics of mGlu2's subunit follow coupled rather than discrete, independent actions. Asymmetric dimerization is critical for receptor activation. Our calculation results are consistent with the observation of cross-linking and fluorescent-labeled blot experiments, thus illustrating the reliability of our calculations. Besides, we also identify potential key residues in the Gi protein binding position on mGlu2, mGlu2 dimer's TM6-TM6 interface, and Gi α5 helix by the change of energy barriers after mutation. The implications of our findings could lead to a more comprehensive grasp of class C G protein-coupled receptor activation.

Heterodimerization of Chemoreceptors TAS1R3 and mGlu2 in Human Blood Leukocytes.

The expression of canonical chemosensory receptors of the tongue, such as the heteromeric sweet taste (TAS1R2/TAS1R3) and umami taste (TAS1R1/TAS1R3) receptors, has been demonstrated in many extra-oral cells and tissues. Gene expression studies have revealed transcripts for all TAS1 and metabotropic glutamate (mGlu) receptors in different types of immune cells, where they are involved, for example, in the chemotaxis of human neutrophils and the protection of T cells from activation-induced cell death. Like other class-C G protein-coupling receptors (GPCRs), TAS1Rs and mGlu receptors form heteromers within their families. Since mGlu receptors and TAS1R1/TAS1R3 share the same ligand, monosodium glutamate (MSG), we hypothesized their hitherto unknown heteromerization across receptor families in leukocytes. Here we show, by means of immunocytochemistry and co-IP/Western analysis, that across class-C GPCR families, mGlu2 and TAS1R3 co-localize and heterodimerize in blood leukocytes. Expressing the recombinant receptors in HEK-293 cells, we validated their heterodimerization by bioluminescence resonance energy transfer. We demonstrate MSG-induced, mGlu2/TAS1R3 heteromer-dependent gain-of-function and pertussis toxin-sensitive signaling in luminescence assays. Notably, we show that mGlu2/TAS1R3 is necessary and sufficient for MSG-induced facilitation of N-formyl-methionyl-leucyl-phenylalanine-stimulated IL-8 secretion in neutrophils, using receptor-specific antagonists. In summary, our results demonstrate mGlu2/TAS1R3 heterodimerization in leukocytes, suggesting cellular function-tailored chemoreceptor combinations to modulate cellular immune responses.

Asymmetric activation of dimeric GABAB and metabotropic glutamate receptors.

G protein-coupled receptors (GPCRs) represent the largest family of membrane proteins and are important drug targets. GPCRs are allosteric machines that transduce an extracellular signal to the cell by activating heterotrimeric G proteins. Herein, we summarize the recent advancements in the molecular activation mechanism of the γ-aminobutyric acid type B (GABAB) and metabotropic glutamate (mGlu) receptors, the most important class C GPCRs that modulate synaptic transmission in the brain. Both are mandatory dimers, this quaternary structure being needed for their function The structures of these receptors in different conformations and in complexes with G proteins have revealed their asymmetric activation. This asymmetry is further highlighted by the recent discovery of mGlu heterodimers, where the eight mGlu subunits can form specific and functional heterodimers. Finally, the development of allosteric modulators has revealed new possibilities for regulating the function of these receptors by targeting the transmembrane dimer interface. This family of receptors never ceases to astonish and serve as models to better understand the diversity and asymmetric functioning of GPCRs.NEW & NOTEWORTHY γ-aminobutyric acid type B (GABAB) and metabotropic glutamate (mGlu) receptors form constitutive dimers, which are required for their function. They serve as models to better understand the diversity and activation of G protein-coupled receptors (GPCRs). The structures of these receptors in different conformations and in complexes with G proteins have revealed their asymmetric activation. This asymmetry is further highlighted by the recent discovery of specific and functional mGlu heterodimers. Allosteric modulators can be developed to target the transmembrane interface and modulate the asymmetry.

In silico binding affinity prediction for metabotropic glutamate receptors using both endpoint free energy methods and a machine learning-based scoring function.

Metabotropic glutamate receptors (mGluRs) play an important role in regulating glutamate signal pathways, which are involved in neuropathy and periphery homeostasis. mGluR4, which belongs to Group III mGluRs, is most widely distributed in the periphery among all the mGluRs. It has been proved that the regulation of this receptor is involved in diabetes, colorectal carcinoma and many other diseases. However, the application of structure-based drug design to identify small molecules to regulate the mGluR4 receptor is limited due to the absence of a resolved mGluR4 protein structure. In this work, we first built a homology model of mGluR4 based on a crystal structure of mGluR8, and then conducted hierarchical virtual screening (HVS) to identify possible active ligands for mGluR4. The HVS protocol consists of three hierarchical filters including Glide docking, molecular dynamic (MD) simulation and binding free energy calculation. We successfully prioritized active ligands of mGluR4 from a set of screening compounds using HVS. The predicted active ligands based on binding affinities can almost cover all the experiment-determined active ligands, with only one ligand missed. The correlation between the measured and predicted binding affinities is significantly improved for the MM-PB/GBSA-WSAS methods compared to the Glide docking method. More importantly, we have identified hotspots for ligand binding, and we found that SER157 and GLY158 tend to contribute to the selectivity of mGluR4 ligands, while ALA154 and ALA155 could account for the ligand selectivity to mGluR8. We also recognized other 5 key residues that are critical for ligand potency. The difference of the binding profiles between mGluR4 and mGluR8 can guide us to develop more potent and selective modulators. Moreover, we evaluated the performance of IPSF, a novel type of scoring function trained by a machine learning algorithm on residue-ligand interaction profiles, in guiding drug lead optimization. The cross-validation root-mean-square errors (RMSEs) are much smaller than those by the endpoint methods, and the correlation coefficients are comparable to the best endpoint methods for both mGluRs. Thus, machine learning-based IPSF can be applied to guide lead optimization, albeit the total number of actives/inactives are not big, a typical scenario in drug discovery projects.

Allosteric Modulators of Metabotropic Glutamate Receptors as Novel Therapeutics for Neuropsychiatric Disease.

Metabotropic glutamate (mGlu) receptors, a family of G-protein-coupled receptors, have been identified as novel therapeutic targets based on extensive research supporting their diverse contributions to cell signaling and physiology throughout the nervous system and important roles in regulating complex behaviors, such as cognition, reward, and movement. Thus, targeting mGlu receptors may be a promising strategy for the treatment of several brain disorders. Ongoing advances in the discovery of subtype-selective allosteric modulators for mGlu receptors has provided an unprecedented opportunity for highly specific modulation of signaling by individual mGlu receptor subtypes in the brain by targeting sites distinct from orthosteric or endogenous ligand binding sites on mGlu receptors. These pharmacological agents provide the unparalleled opportunity to selectively regulate neuronal excitability, synaptic transmission, and subsequent behavioral output pertinent to many brain disorders. Here, we review preclinical and clinical evidence supporting the utility of mGlu receptor allosteric modulators as novel therapeutic approaches to treat neuropsychiatric diseases, such as schizophrenia, substance use disorders, and stress-related disorders. SIGNIFICANCE STATEMENT: Allosteric modulation of metabotropic glutamate (mGlu) receptors represents a promising therapeutic strategy to normalize dysregulated cellular physiology associated with neuropsychiatric disease. This review summarizes preclinical and clinical studies using mGlu receptor allosteric modulators as experimental tools and potential therapeutic approaches for the treatment of neuropsychiatric diseases, including schizophrenia, stress, and substance use disorders.

Group I Metabotropic Glutamate Receptors and Interacting Partners: An Update.

Group I metabotropic glutamate (mGlu) receptors (mGlu1/5 subtypes) are G protein-coupled receptors and are broadly expressed in the mammalian brain. These receptors play key roles in the modulation of normal glutamatergic transmission and synaptic plasticity, and abnormal mGlu1/5 signaling is linked to the pathogenesis and symptomatology of various mental and neurological disorders. Group I mGlu receptors are noticeably regulated via a mechanism involving dynamic protein-protein interactions. Several synaptic protein kinases were recently found to directly bind to the intracellular domains of mGlu1/5 receptors and phosphorylate the receptors at distinct amino acid residues. A variety of scaffolding and adaptor proteins also interact with mGlu1/5. Constitutive or activity-dependent interactions between mGlu1/5 and their interacting partners modulate trafficking, anchoring, and expression of the receptors. The mGlu1/5-associated proteins also finetune the efficacy of mGlu1/5 postreceptor signaling and mGlu1/5-mediated synaptic plasticity. This review analyzes the data from recent studies and provides an update on the biochemical and physiological properties of a set of proteins or molecules that interact with and thus regulate mGlu1/5 receptors.

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.

Receptor mapping using methoxy phenyl piperazine derivative: Preclinical PET imaging.

This study aimed at assessing 2-methoxyphenyl piperazine derivative for its binding specificity and suitability in mapping metabotropic glutamate receptor subtype 1, which is implicated in several neuropsychiatric disorders. N-(2-(4-(2-Methoxyphenyl)piperazin-1-yl)ethyl)-N-methylpyridin-2-amine was synthesised and evaluated for brain imaging subsequent to radiolabelling with [11C] radioisotope via methylation process in 98.9% purity and 52 ± 6% yield (decay corrected). The specific activity was in the range of 72-93 GBq/µmol. The haemolysis of blood was 2-5% for initial 4 hr and remained < 10% after 24 h of incubation indicating low toxicity. In vitro autoradiograms after coincubation with unlabelled ligand confirmed the high uptake of the PET radioligand in the mGluR1 receptor rich regions. The PET as well as biodistribution studies also showed high activity in the brain with a direct correlation between receptor abundance distribution pattern and tracer activity. The biodistribution analyses revealed initial high brain uptake (4.18 ± 0.48). The highest uptake was found in cerebellum (SUV 4.7 ± 0.2), followed by thalamus (SUV 3.5 ± 0.1), and striatum (SUV 3 ± 0.1). In contrast, pons had negligible tracer activity. The high uptake observed in all the regions with known mGluR1 activity indicates suitability of the ligand for mGluR1 imaging.

Exploring the binding mechanism of positive allosteric modulators in human metabotropic glutamate receptor 2 using molecular dynamics simulations.

Positive allosteric modulators (PAMs) of human metabotropic glutamate receptor 2 (hmGlu2) are well-known in the treatment of psychiatric disorders for their higher selectivity and lower tolerance risk. A variety of PAMs have been reported over the last decade and two compounds were in Phase II clinical trials for schizophrenia and anxiety. These trials were discontinued on account of the unsatisfactory therapeutic efficacy, but PAMs were explored as novel treatments for addiction and epilepsy. Thus, it is still important to explore novel hmGlu2 PAMs in the near future. Nowadays, the challenges in optimizing drug potency and improving scaffold diversity for PAMs are the noncomprehensive character analyses of multiple scaffolds; the exploration of the binding modes of PAMs in the allosteric binding site have been proposed to reduce this difficulty. However, there has been no comprehensive research about the binding profiles of PAMs in the hmGlu2 receptor. To address this issue, this work explores the binding characters of eight PAMs representing five chemical series by multiple computational methods. As a result, the shared binding modes of the eight studied PAMs interacting with 15 residues in the allosteric binding site were defined. In addition, the reduced hydrophobicity with low electronegativity of R1, increased hydrophobicity with low negative electron density of R2 and the electronegativity of the linker were identified as indicators that regulate the affinity of PAMs. This finding agrees well with the physicochemical properties of reported multiple series PAMs. This comprehensive work sheds additional light on the binding mechanism and physicochemical regularity underlining PAMs affinity and could be further utilized as a structural and energetic blueprint for discovering and assessing novel PAMs for hmGlu2.

Allosteric modulators enhance agonist efficacy by increasing the residence time of a GPCR in the active state.

Much hope in drug development comes from the discovery of positive allosteric modulators (PAM) that display target subtype selectivity and act by increasing agonist potency and efficacy. How such compounds can allosterically influence agonist action remains unclear. Metabotropic glutamate receptors (mGlu) are G protein-coupled receptors that represent promising targets for brain diseases, and for which PAMs acting in the transmembrane domain have been developed. Here, we explore the effect of a PAM on the structural dynamics of mGlu2 in optimized detergent micelles using single molecule FRET at submillisecond timescales. We show that glutamate only partially stabilizes the extracellular domains in the active state. Full activation is only observed in the presence of a PAM or the Gi protein. Our results provide important insights on the role of allosteric modulators in mGlu activation, by stabilizing the active state of a receptor that is otherwise rapidly oscillating between active and inactive states.

G-protein activation by a metabotropic glutamate receptor.

Family C G-protein-coupled receptors (GPCRs) operate as obligate dimers with extracellular domains that recognize small ligands, leading to G-protein activation on the transmembrane (TM) domains of these receptors by an unknown mechanism1. Here we show structures of homodimers of the family C metabotropic glutamate receptor 2 (mGlu2) in distinct functional states and in complex with heterotrimeric Gi. Upon activation of the extracellular domain, the two transmembrane domains undergo extensive rearrangement in relative orientation to establish an asymmetric TM6-TM6 interface that promotes conformational changes in the cytoplasmic domain of one protomer. Nucleotide-bound Gi can be observed pre-coupled to inactive mGlu2, but its transition to the nucleotide-free form seems to depend on establishing the active-state TM6-TM6 interface. In contrast to family A and B GPCRs, G-protein coupling does not involve the cytoplasmic opening of TM6 but is facilitated through the coordination of intracellular loops 2 and 3, as well as a critical contribution from the C terminus of the receptor. The findings highlight the synergy of global and local conformational transitions to facilitate a new mode of G-protein activation.

Regulation and functional consequences of mGlu4 RNA editing.

Metabotropic glutamate receptor 4 (mGlu4) is one of eight mGlu receptors within the Class C G protein-coupled receptor superfamily. mGlu4 is primarily localized to the presynaptic membrane of neurons where it functions as an auto and heteroreceptor controlling synaptic release of neurotransmitter. mGlu4 is implicated in numerous disorders and is a promising drug target; however, more remains to be understood about its regulation and pharmacology. Using high-throughput sequencing, we have validated and quantified an adenosine-to-inosine (A-to-I) RNA editing event that converts glutamine 124 to arginine in mGlu4; additionally, we have identified a rare but novel K129R site. Using an in vitro editing assay, we then validated the pre-mRNA duplex that allows for editing by ADAR enzymes and predicted its conservation across the mammalian species. Structural modeling of the mGlu4 protein predicts the Q124R substitution to occur in the B helix of the receptor that is critical for receptor dimerization and activation. Interestingly, editing of a receptor homodimer does not disrupt G protein activation in response to the endogenous agonist, glutamate. Using an assay designed to specifically measure heterodimer populations at the surface, however, we found that Q124R substitution decreased the propensity of mGlu4 to heterodimerize with mGlu2 and mGlu7 Our study is the first to extensively describe the extent and regulatory factors of RNA editing of mGlu4 mRNA transcripts. In addition, we have proposed a novel functional consequence of this editing event that provides insights regarding its effects in vivo and expands the regulatory capacity for mGlu receptors.

Structures of human mGlu2 and mGlu7 homo- and heterodimers.

The metabotropic glutamate receptors (mGlus) are involved in the modulation of synaptic transmission and neuronal excitability in the central nervous system1. These receptors probably exist as both homo- and heterodimers that have unique pharmacological and functional properties2-4. Here we report four cryo-electron microscopy structures of the human mGlu subtypes mGlu2 and mGlu7, including inactive mGlu2 and mGlu7 homodimers; mGlu2 homodimer bound to an agonist and a positive allosteric modulator; and inactive mGlu2-mGlu7 heterodimer. We observed a subtype-dependent dimerization mode for these mGlus, as a unique dimer interface that is mediated by helix IV (and that is important for limiting receptor activity) exists only in the inactive mGlu2 structure. The structures provide molecular details of the inter- and intra-subunit conformational changes that are required for receptor activation, which distinguish class C G-protein-coupled receptors from those in classes A and B. Furthermore, our structure and functional studies of the mGlu2-mGlu7 heterodimer suggest that the mGlu7 subunit has a dominant role in controlling dimeric association and G-protein activation in the heterodimer. These insights into mGlu homo- and heterodimers highlight the complex landscape of mGlu dimerization and activation.

Structures of Gi-bound metabotropic glutamate receptors mGlu2 and mGlu4.

The metabotropic glutamate receptors (mGlus) have key roles in modulating cell excitability and synaptic transmission in response to glutamate (the main excitatory neurotransmitter in the central nervous system)1. It has previously been suggested that only one receptor subunit within an mGlu homodimer is responsible for coupling to G protein during receptor activation2. However, the molecular mechanism that underlies the asymmetric signalling of mGlus remains unknown. Here we report two cryo-electron microscopy structures of human mGlu2 and mGlu4 bound to heterotrimeric Gi protein. The structures reveal a G-protein-binding site formed by three intracellular loops and helices III and IV that is distinct from the corresponding binding site in all of the other G-protein-coupled receptor (GPCR) structures. Furthermore, we observed an asymmetric dimer interface of the transmembrane domain of the receptor in the two mGlu-Gi structures. We confirmed that the asymmetric dimerization is crucial for receptor activation, which was supported by functional data; this dimerization may provide a molecular basis for the asymmetric signal transduction of mGlus. These findings offer insights into receptor signalling of class C GPCRs.

Differences in interactions between transmembrane domains tune the activation of metabotropic glutamate receptors.

The metabotropic glutamate receptors (mGluRs) form a family of neuromodulatory G-protein-coupled receptors that contain both a seven-helix transmembrane domain (TMD) and a large extracellular ligand-binding domain (LBD) which enables stable dimerization. Although numerous studies have revealed variability across subtypes in the initial activation steps at the level of LBD dimers, an understanding of inter-TMD interaction and rearrangement remains limited. Here, we use a combination of single molecule fluorescence, molecular dynamics, functional assays, and conformational sensors to reveal that distinct TMD assembly properties drive differences between mGluR subtypes. We uncover a variable region within transmembrane helix 4 (TM4) that contributes to homo- and heterodimerization in a subtype-specific manner and tunes orthosteric, allosteric, and basal activation. We also confirm a critical role for a conserved inter-TM6 interface in stabilizing the active state during orthosteric or allosteric activation. Together this study shows that inter-TMD assembly and dynamic rearrangement drive mGluR function with distinct properties between subtypes.

Neddylation is required for presynaptic clustering of mGlu7 and maturation of presynaptic terminals.

Neddylation is a posttranslational modification in which NEDD8 is conjugated to a target substrate by cellular processes similar to those involved in ubiquitination. Recent studies have identified PSD-95 and cofilin as substrates for neddylation in the brain and have shown that neddylation modulates the maturation and stability of dendritic spines in developing neurons. However, the precise substrates and functional consequences of neddylation at presynaptic terminals remain elusive. Here, we provide evidence that the mGlu7 receptor is a target of neddylation in heterologous cells and rat primary cultured neurons. We found that mGlu7 neddylation is reduced by agonist treatment and is required for the clustering of mGlu7 in the presynaptic active zone. In addition, we observed that neddylation is not required for the endocytosis of mGlu7, but it facilitates the ubiquitination of mGlu7 and stabilizes mGlu7 protein expression. Finally, we demonstrate that neddylation is necessary for the maturation of excitatory presynaptic terminals, providing a key role for neddylation in synaptic function.

Conformational rearrangement during activation of a metabotropic glutamate receptor.

G protein-coupled receptors (GPCRs) relay information across cell membranes through conformational coupling between the ligand-binding domain and cytoplasmic signaling domain. In dimeric class C GPCRs, the mechanism of this process, which involves propagation of local ligand-induced conformational changes over 12 nm through three distinct structural domains, is unknown. Here, we used single-molecule FRET and live-cell imaging and found that metabotropic glutamate receptor 2 (mGluR2) interconverts between four conformational states, two of which were previously unknown, and activation proceeds through the conformational selection mechanism. Furthermore, the conformation of the ligand-binding domains and downstream domains are weakly coupled. We show that the intermediate states act as conformational checkpoints for activation and control allosteric modulation of signaling. Our results demonstrate a mechanism for activation of mGluRs where ligand binding controls the proximity of signaling domains, analogous to some receptor kinases. This design principle may be generalizable to other biological allosteric sensors.

Absence of metabotropic glutamate receptor homolog(s) accelerates acetylcholine neurotransmission in Caenorhabditis elegans.

Glutamate (Glu) and Acetylcholine (ACh), are excitatory neurotransmitters, acting through ionotropic (iR) and metabotropic receptors (mR). Importantly, both neurotransmitters and their signalling are impaired in the prevalent neurodegenerative disease-Alzheimer disease (AD). Glu and its signalling cascade's influence on ACh-neurotransmission (NT) are sparsely understood. The mGluRs coupled to G-protein signalling acting through PI3K cascade (GrpI) or inhibition of adenylate cyclase-cAMP cascade (GrpII and GrpIII) brings about long-lasting structural/functional changes. These complexities are challenging to decipher. Here, we report that human/mouse mGluRs when compared with their Caenorhabditis elegans homologs, MGL-1-3 showed overall of homology of ∼31-39 %. Phylogeneitc analysis revealed homology of MGL-2 to GrpI, MGL-3 with Grp1 &II and GRM6 of GrpIII and MGL-1, a low homology that falls between GrpI & GrpII. Then, alteration of ACh-NT in C. elegans loss-of-function mutants of mgl-1, mgl-2, mgl-3, PI3K (age-1) and iGluR (NMDA)(nmr-1) was estimated by well-established acute aldicarb (Ald), that increases ACh at synapse, and levamisole (Lev) (postsynaptic activation of levamisole sensitive iAChR) induced time-dependent paralysis assays. Surprisingly, all of them were hypersensitive to Ald and Lev compared to wildtype (in percentage), namely, mgl-1 -17, 54; mgl-2 - 7.2, 24; mgl-3 -52, 64; age-1 - 27, 32; nmr-1- 24, 48; respectively. Of the three, mgl-3 contributes to maximal overall acceleration of ACh-NT. Adenylate cyclase, acy-1 gain-of-function mutant showed less hypersensitivity, Ald - 7% and Lev- 25 %. Together, Glu receptors and signalling cascades are altering ACh-NT permanently, thus establishing the interplay between them thereby provide potential drug targets to be considered for AD.

Allosteric Molecular Switches in Metabotropic Glutamate Receptors.

Metabotropic glutamate receptors (mGlu) are class C G protein-coupled receptors of eight subtypes that are omnipresently expressed in the central nervous system. mGlus have relevance in several psychiatric and neurological disorders, therefore they raise considerable interest as drug targets. Allosteric modulators of mGlus offer advantages over orthosteric ligands owing to their increased potential to achieve subtype selectivity, and this has prompted discovery programs that have produced a large number of reported allosteric mGlu ligands. However, the optimization of allosteric ligands into drug candidates has proved to be challenging owing to induced-fit effects, flat or steep structure-activity relationships and unexpected changes in theirpharmacology. Subtle structural changes identified as molecular switches might modulate the functional activity of allosteric ligands. Here we review these switches discovered in the metabotropic glutamate receptor family..

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.