Metabotropic glutamate receptors: Allosteric structural modulation and new treatments for anxiety disorders

What is stress?

Anxiety disorders are divided into 5 categories: post-traumatic stress disorder (PTSD), specific and social phobias, generalized anxiety disorder (GAD), panic disorder, and obsessive-compulsive disorder (OCD).^1,2 While stress and anxiety are distinct phenomena, they are closely interconnected in their neurobiological mechanisms and clinical manifestations. Stress represents the psychological and functional response to environmental demands or threats, which, when chronic or severe, can precipitate or exacerbate anxiety disorders.³ Both stress and anxiety involve overlapping neural circuits, particularly in the amygdala, prefrontal cortex, and hippocampus, with glutamatergic neurotransmission playing a critical role in these pathways.³ In this review, stress is referred to as a chronic pathological condition and can be used alternatively with anxiety.

The current interventions for anxiety-related ailments predominantly target serotonergic and gamma-aminobutyric acid (GABA) neurons. They include benzodiazepines, selective serotonin reuptake inhibitors (SSRIs), and partial agonists of the serotonin 5-HT1A receptor.^1,4 However, certain types of anxiety diseases may prove resistant to these treatments.^5,6 Additionally, some medications, such as benzodiazepines and SSRIs, have significant side effects, including sedation, memory loss, dependence, withdrawal symptoms, poor sex drive, and weight gain.^1,7 Therefore, there is an increasing need for alternative treatments.

What is the connection between glutamate and stress?

Glutamate is a neurotransmitter that is crucial to emotional, cognitive, and motor functions. It interacts with 2 categories of receptors include kainate, ionotropic receptors (N-methyl-d-aspartate [NMDA], and alpha-amino-3hydroxy-5-methyl-4-isoxazolepropoinate [AMPA]) and mGluRs.^1,8,9 Dysfunctional glutamatergic transmission can lead to depression.¹ In addition, the imbalance between inhibitory and stimulatory signals in the brain can lead to anxiety due to excessive activity of excitatory glutamatergic neurons.^10,11 Various preclinical studies and, to a lesser extent, clinical explorations have revealed the beneficial effects of using glutamate modulators in the treatment of anxiety.¹²

What are mGluRs?

Metabotropic glutamate receptors can be divided into three groups (Table 1).¹ Group I includes mGluR1 and mGluR5, which are located mainly on postsynaptic neurons and activate phospholipase C.^1,9 Group II includes mGluR2 and mGluR3, which are located on pre- and postsynaptic neurons.^13,14 Finally, group III includes mGluR4, mGluR6, mGluR7, and mGluR8, mainly located in the presynaptic terminals of GABAergic and glutamatergic neurons.^15,16 Both group II and III mGluRs inhibit adenylyl cyclase.^17,18

How are mGluRs involved in stress?

Table 2 provides a summary of the research on the effects of mGluR agonists and antagonists on anxiety and other conditions.

Group I mGluRs are situated on postsynaptic excitatory terminals in brain regions linked to behavior and emotion such as olgactory tubercle, amygdala, limbic cortex, basal ganglia, and hippocampal gyrus.^19,20 mGluR1 modulators increase GABA receptor activity,²³ and many preclinical studies have demonstrated that mGluR1 antagonists cause anxiolytic effects.²⁰ However, mGluR1 antagonists have been shown to trigger amnestic responses in preclinical studies; this suggests that they may have serious side effects if used to treat anxiety disorders.²⁴ mGluR5 shows stimulating effects on the stress-related region of the brain (amygdala).²⁵ Other studies have shown that antagonist treatments increase anxiety in experimental animals and are promising for managing anxiety-related disorders. These medications have demonstrated effectiveness in reducing anxiety in preclinical settings.²⁰ The differences in findings between studies may be due to variations in specific brain activation. According to Tatarczyńska et al.,²⁶ 3-((2-methyl-1,3-thiazol-4-yl)ethynyl) pyridine (MTEP) shows a significant reduction in anxiety-like responses in rodent models. MTEP may be suitable for clinical use based on its pharmacological properties.²⁷ Clinical trials involving fenobam (McN-3377) in animals or humans have shown it to be a safe, potent, and specific antagonist of mGluR5; furthermore, it has been demonstrated to be effective in managing depression.²⁸

Regarding group II mGluRs, mGluR3 is widely distributed in many parts of the brain, including emotion-related areas such as the amygdala, prefrontal cortex, hippocampus, and bed nucleus of the stria terminalis.¹³ In contrast, according to Jian and Yongling,²⁰ mGluR2 exhibits a more restricted distribution, with strong expression in the olfactory regions and dentate gyrus and limited expression in the cortex, thalamus, and striatum. This receptor is also considered to be significant in the regulation of stress.^20,29 mGluR2/3 activators reduce glutamate release in the limbic region during the development of anxiety,^20,30 and various mGluR2/3 agonists such as LY544344, LY354740, LY314582, CBiPES, and LY566332 have been studied in animal models for preclinical studies of anxiety. Among these, the agonist LY354740 has anti-anxiety properties.^31,32 In pioneering work, Schoepp et al.³³ demonstrated that LY354740 effectively reduces anxiety-like behaviors across multiple experimental paradigms. Clinical studies have shown that mGluR2/3 antagonists (LY341495 and MSG0039) induce anxiolytic responses^34,35 and potentiate ketamine-like responses.³⁶ Shimazaki et al³³ were the first to characterize MSG0039’s anxiolytic effects; they found that this compound demonstrates rapid and robust antianxiety properties. Clinical studies demonstrating the impact of LY354740 on GAD have shown notable antagonism to the mGluR2/3.^32,37 Additionally, LY544344, which is derived from LY354740, has been shown to have higher bioavailability than LY354740 and to reduce panic symptoms and cholecystokinin tetrapeptide (CCK-4)-induced anxiety.³⁸ LY354740 showed efficacy in managing GAD in a double-blind, placebo-controlled study.³⁷ Although LY354740 and LY544344 are well tolerated in humans, clinical studies have documented a greater likelihood of convulsions with repeated doses.³⁷ No recent studies evaluate the toxicological and clinical effects of LY354740 or LY544344. The signaling pathway of this group involves binding to Gαi/o proteins; when activated, they suppress adenylyl cyclase activity and the synthesis of cyclic adenosine monophosphate (cAMP), thus reducing protein kinase A (PKA) activation.³⁹

Despite these promising findings, several clinical trials with mGluR ligands have produced negative or inconclusive results. For example, Witkin et al⁴⁰ documented that the mGluR2/3 agonist LY2140023 (pomaglumetad methionil) did not show effectiveness in phase III clinical trials for schizophrenia despite showing initial promise. Similarly, basimglurant, a negative allosteric modulator (NAM) of mGluR5, showed limited efficacy in clinical trials for major depressive disorder and fragile X syndrome.⁴⁰ These negative results highlight the challenges in translating preclinical findings to clinical success and underscore the complexity of glutamatergic signaling in psychiatric disorders.⁴⁰

Within the group III mGluRs, mGluR6 is found mainly in the retina; mGluR7 is found mainly in the brain and forebrain; and mGluR8 is found mainly in the cerebellum, hippocampus, and olfactory mass.^21,22 In addition, mGluR6 presents low expression in the hippocampus. mGluR4, mGluR7, and mGluR8 are also present in other key regions, such as the prefrontal cortex, hypothalamus, and amygdala.²⁰ All three receptors are distributed in presynaptic neurons and directly control glutamate and GABA transmission.^21,41 Therefore, they are relevant when developing antianxiety drugs. According to Palazzo et al,⁴² mGluR6 is only found in the retina and may not be related to stress.

The psychiatric mechanisms of group III mGluRs are similar to those of group II mGluRs. They signal by activating the G protein subunit Gαi/o, which inhibits adenylyl cyclase and thus decreased cAMP levels.⁴³ Gβγ can also be activated, resulting in the regulation of voltage-gated ion channels, limiting Ca2+ influx and opening G protein-coupled inwardly rectifying K+ channels (GIRK).⁴³ This pathway works to inhibit the release of glutamate and GABA. Other signaling pathways that group III mGluRs can activate include the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt), which promotes cell survival, and the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway.⁴⁴ Compared with group I and II mGluR ligands, group III mGluR ligands have received less attention from researchers concerning their effectiveness in treating anxiety.⁴⁵ However, there is substantial ongoing research involving this receptor group. Importantly, the use of group III mGluR agonists has shown similar results in reducing anxiety and depression in experimental animal models.⁴⁵ For instance, physical application of the mGluR8 agonist (S)-3,4-dicarboxyphenylglycine (DCPG) increases c-fos expression in the stress-related brains of normal mice but not in mice lacking mGluR8; indicating that mGluR8 is involved in stress regulation.⁴⁵ The initial evidence for the anxiolytic potential of N-phenyl-7-(hydroxylamine)cycloprop[b]chromene-1a-methamide (PHCCC), a mGluR4 activation, was reported by Stachowicz et al,⁴⁶ who demonstrated that selective modulation of this receptor subtype produces marked antianxiety effects. It has recently been shown that an agonist specifically targeting mGluR7 can also produce anxiety-reducing effects.⁴⁷ Stachowicz et al⁴⁸ were the first to characterize this mGluR7-mediated anxiolytic action, showing its promise as a new target for anxiety disorders. Despite the progress made in this field over time, the exact role of group III mGluRs in stress is still unclear.²⁰ Hence, further research is required to explicate their precise roles and importance in anxiety-related behaviors.²⁰

What are the properties of mGluRs?

The seven-transmembrane (7TM) domain is a defining structural feature of G protein-coupled receptors (GPCRs), also referred to as 7TM domain receptors (7TMRs).^49,50 A large N-terminal region called the Venus flytrap domain (VFD) contains the linear ligand binding domain.⁴⁹ Examination of the VFD crystal structure of mGluR1, mGluR3, and mGluR7 revealed the presence of 2 lobes in each receptor that contain its glutamate-binding domain.^49,51 mGluRs function as hetero- and homodimers; one receptor binds via disulfide bonds to the VFD of another receptor.⁴⁹ When glutamate binds to an mGluR, it causes both lobes of the VFD to close, and cysteine residues in the 7TM activate the transcription factor, initiating intracellular signaling.^49,51 The cysteine residue is a unique class C 7TMR family member, forming a strong disulfide bond that connects the VFD and the 7TM domain.^49,52 All known allosteric modulators of mGluRs are thought to interact with cysteine residues in the 7TM domain. For more information on the properties of mGluRs, see the review by Ludovic et al.⁴⁹

It is important to identify the binding site of the allosteric modulator of mGluR to cysteine residues in the 7TM domain.⁵³ This method has also been used to determine the selectivity of orthosteric agonists.^53,54 For example, 7(hydroxylamine)cycloprop[b]chromene-1α-carboxylic acid ethyl ester (CPCOEt) is a specific allosteric modulator of mGluR1 that combines the genetic structures of mGluR1 (calcium-sensing receptor) and other mGluR subunits.⁵⁵ This approach is important for distinguishing mGluR NAMs from positive allosteric modulators (PAMs).^55,56 Another technique for characterizing the interaction of allosteric modulators containing the 7TM domain uses truncated receptor constructs. For example, “headless” mGluRs with only the extracellular VFD retain intact and functional 7TM cysteine residues and the C terminus.^57,58 Therefore, they retain intracellular signaling capacity without orthotopic binding sites. Importantly, ligands can contribute positively or negatively to regulating headless mGluRs. For example, PAMs act as agonists of mGluRs, whereas NAMs act as antagonists of mGluRs.⁵⁷ Genetic and headless receptor structures provide insight into the mechanism of allosteric action and generalization of the allosteric binding site.⁵⁷

Although the crystal structure of the 7TM cysteine residue in class C 7TMRs remains unclear, using the class A 7TMR structure in the homology model facilitates the identification of the structure connected to the mGluR product.⁵⁹ Unfortunately, the similarity between class A and C 7TMRs is low (<20%), reducing the accuracy of the structural information obtained from the class C 7TMR homology model.⁵⁹ To overcome this limitation, homology models combined with mutagenesis experiments have proven effective. Early studies of the mGluR1 and mGluR5 allosteric binding regions at the cysteine residues in the 7TM domain were similar to the rhodopsin/biogenic amine orthosteric binding sites.⁵⁹ These studies are consistent with the current knowledge and research on mGluRs and GPCRs. Improving the study of allosteric mechanisms and interaction sites of GPCRs provides opportunities for further research that builds on the available literature.

How are similarities in allosteric sites within and between mGluRs represented in modulators?

Allosteric modulators share similar binding sites between different mGluRs, allowing the identification of modulators without the need for them to be specific for certain isoforms. For example, the mGluR5 NAMs 2-methyl-6-(2-phenylvinyl)pyridine (SIB-1893) and 2-methyl-6-(phenylethynyl)pyridine (MPEP) are mGlu4 PAMs. L-2-amino-4-phosphobutyrate (L-AP4) is an orthosteric agonist of group III mGluRs (60,61). DFB is an mGluR5 PAM with weak NAM activity for mGluR4.^62,63 Moreover, PHCCC is an mGluR4 PAM that acts as a mGluR1 NAM.⁶⁰

Radiolabeled mGluR allosteric modulators have been developed to allow the dissection of allosteric binding sites.⁶³ Three specific radiolabeled allosteric ligands for mGluR1 have been developed: [3H]-1-(3,4-dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-2-phenyl-1-ethanone (R214127),^63,64 [3H]-1-ethyl-2-methyl-6-oxo-4-(1,2,4,5-tetrahydrofuran)hydro-benzo[d]azepin-3-yl)-1,6-dihydro-pyrimidine-5-carbonitrile (EM-TBPC),^63,65 and [3H]-6-amino-N-cyclohexyl-N,3-dimethylthiazolo[3,2-a]benzimidazole-2-carboxamide (YM298198).^63,66 Additionally, radiolabeled and positron tomography (PET) ligands for mGluR5^63,67 have been developed to facilitate the study of allosteric small-molecule binding to mGluR5. The first reported radiolabeled mGluR2 PAM was [3H]2,2,2-TEMPS.^63,68 Competitive binding assays using allosteric radiolabeled ligands can help to distinguish between competitive and allosteric binding sites for novel allosteric modulators of established allosteric sites.^63,64 In addition, the discovery of neutral allosteric ligands called silent allosteric modulators has increased our understanding of the connection between allosteric modulators and enabled the classification of these modulators into competitive and non-competitive drugs.^63,69 The mGluR allosteric modulator binding site has been mutated to identify a region similar to the site bound by the antagonist 11-cis-retinal in bovine rhodopsin.^63,70

General conclusions and future directions

mGluRs play important roles in anxiety: They are located in areas of the brain that are associated with anxiety and are potentially associated with decreased glutamatergic transmission. Hence, mGluRs have potential in the development of drugs for the treatment of stress. Although there have been few clinical studies evaluating the effectiveness of mGluR ligands in disorders such as GAD and panic attacks, their efficacy in other anxiety disorders such as OCD and PTSD has been evaluated. Future research should explore this potential. The relationship between stress and anxiety disorders is complex but critical to understand the therapeutic potential of mGluR modulators. While not all anxiety disorders are directly linked to stress, chronic stress can precipitate or exacerbate anxiety symptoms through dysregulation of glutamatergic transmission in key brain regions. This relationship explains why compounds targeting glutamatergic signaling may have broad applications across various anxiety disorders. Several types of mGluR allosteric modulators have been identified for each mGluR, including isoform-selective modulators, PAMs, NAMs, and neutral modulators. Targeting allosteric sites or exploring other sites holds promise for developing effective therapies, particularly for group III mGluRs; this endeavor requires pharmacological agents to identify modulators that bind to other allosteric sites. Several mGluR5 NAMs have entered clinical trials to examine their potential in the treatment of anxiety disorders. Additionally, 7TMRs are capable therapeutic targets, and some agents targeting them are being evaluated in preclinical and clinical trials for treating depression.

Despite promising preclinical findings, clinical trials with mGluR ligands have shown mixed results. The challenge may lie in the complexity of glutamatergic signaling and the need for more selective compounds that can modulate specific aspects of the glutamate system without disrupting its usual physiological roles. Future research should aim at more selective allosteric modulators with improved pharmacokinetic properties, fewer side effects, and personalized approaches that account for individual differences in glutamatergic signaling.