mGluR5 Negative Allosteric Modulators Research Articles

The novel peptide LCGM-10 attenuates metabotropic glutamate receptor 5 activity and demonstrates behavioral effects in animal models.

We employed a structural bioinformatics approach to develop novel peptides with predicted affinity to the binding site for negative allosteric modulators (NAMs) of metabotropic glutamate receptor 5 (mGluR5). Primary screening in zebrafish (Danio rerio) revealed a stimulatory effect of two peptides, LCGM-10 and LCGM-15. Target validation studies using calcium ion flux imaging and a luciferase reporter assay confirmed mGluR5 as the target. LCGM-10 showed greater potency than LCGM-15; it was comparable to that of the mGluR5 NAM 2-methyl-6-(phenylethynyl) pyridine (MPEP). Rodent behavioral screening in the open field and elevated plus maze revealed increased locomotor activity in both tests after acute LCGM-10 treatment, supported by further analysis of home cage spontaneous locomotor activity (SLA). The stimulating effect of a single LCGM-10 administration on SLA was evident up to 60 min after administration and was not accompanied by hypokinetic rebound observed for caffeine. According to our results, LCGM-10 has therapeutic potential to treat hypo- and dyskinesias of various etiologies. Further investigation of LCGM-10 effects in the delay discounting model of impulsive choice in rats revealed reduced trait impulsivity after single and chronic administrations, suggesting potential implication for attention deficit hyperactivity disorder, obsessive compulsive disorder, and addictions.

Inhibiting metabotropic glutamate receptor 5 after stroke restores brain function and connectivity.

Stroke results in local neural disconnection and brain-wide neuronal network dysfunction leading to neurological deficits. Beyond the hyper-acute phase of ischaemic stroke, there is no clinically-approved pharmacological treatment that alleviates sensorimotor impairments. Functional recovery after stroke involves the formation of new or alternative neuronal circuits including existing neural connections. The type-5 metabotropic glutamate receptor (mGluR5) has been shown to modulate brain plasticity and function and is a therapeutic target in neurological diseases outside of stroke. We investigated whether mGluR5 influences functional recovery and network reorganization rodent models of focal ischaemia. Using multiple behavioural tests, we observed that treatment with negative allosteric modulators (NAMs) of mGluR5 (MTEP, fenobam and AFQ056) for 12 days, starting 2 or 10 days after stroke, restored lost sensorimotor functions, without diminishing infarct size. Recovery was evident within hours after initiation of treatment and progressed over the subsequent 12 days. Recovery was prevented by activation of mGluR5 with the positive allosteric modulator VU0360172 and accelerated in mGluR5 knock-out mice compared with wild-type mice. After stroke, multisensory stimulation by enriched environments enhanced recovery, a result prevented by VU0360172, implying a role of mGluR5 in enriched environment-mediated recovery. Additionally, MTEP treatment in conjunction with enriched environment housing provided an additive recovery enhancement compared to either MTEP or enriched environment alone. Using optical intrinsic signal imaging, we observed brain-wide disruptions in resting-state functional connectivity after stroke that were prevented by mGluR5 inhibition in distinct areas of contralesional sensorimotor and bilateral visual cortices. The levels of mGluR5 protein in mice and in tissue samples of stroke patients were unchanged after stroke. We conclude that neuronal circuitry subserving sensorimotor function after stroke is depressed by a mGluR5-dependent maladaptive plasticity mechanism that can be restored by mGluR5 inhibition. Post-acute stroke treatment with mGluR5 NAMs combined with rehabilitative training may represent a novel post-acute stroke therapy.

MPEP Attenuates Intrahepatic Fat Accumulation in Obese Mice.

The blockade of metabotropic glutamate receptor type 5 (mGluR5) was previously found to reduce fat accumulation in HEPG2 cells. Here, we evaluated the effects of mGluR5 blockade in a mouse model of steatosis. Male ob/ob mice fed a high-fat diet were treated with MPEP or vehicle. After 7 weeks, liver biopsies were collected, and nuclei were isolated from fresh tissue. Lipid droplet area and collagen deposition were evaluated on tissue slices; total lipids, lipid peroxidation, and ROS were evaluated on tissue homogenates; PPARα, SREBP-1, mTOR, and NF-κB were assayed on isolated nuclei by Western Blot. Target genes of the above-mentioned factors were assayed by RT-PCR. Reduced steatosis and hepatocyte ballooning were observed in the MPEP group with respect to the vehicle group. Concomitantly, increased nuclear PPARα and reduced nuclear SREBP-1 levels were observed in the MPEP group. Similar trends were obtained in target genes of PPARα and SREBP-1, Acox1 and Acc1, respectively. MPEP administration also reduced oxidative stress and NF-κB activation, probably via NF-κB inhibition. Levels of common markers of inflammation (Il-6, Il1β and Tnf-α) and oxidative stress (Nrf2) were significantly reduced. mTOR, as well as collagen deposition, were unchanged. Concluding, MPEP, a selective mGluR5 negative allosteric modulator, reduces both fat accumulation and oxidative stress in a 7-week murine model of steatosis. Although underlying mechanisms need to be further investigated, this is the first in vivo study showing the beneficial effects of MPEP in a murine model of steatosis.

Loss of schizophrenia-related miR-501-3p in mice impairs sociability and memory by enhancing mGluR5-mediated glutamatergic transmission.

Schizophrenia is a polygenetic disease, the heterogeneity of which is likely complicated by epigenetic modifications yet to be elucidated. Here, we performed transcriptomic analysis of peripheral blood RNA from monozygotic twins discordant for schizophrenia and identified a schizophrenia-associated down-regulated microRNA, miR-501-3p. We showed that the loss of miR-501-3p in germline knockout (KO) male mice resulted in dendritic structure defects, glutamatergic transmission enhancement, and sociability, memory, and sensorimotor gating disruptions, which were attenuated when miR-501 expression was conditionally restored in the nervous system. Combining the results of proteomic analyses with the known genes linked to schizophrenia revealed that metabotropic glutamate receptor 5 (mGluR5) was one of the miR-501-3p targets and was elevated in vivo upon loss of miR-501. Treatment with the mGluR5 negative allosteric modulator 3-2((-methyl-4-thiazolyl) ethynyl) pyridine or the N-methyl-d-aspartate receptor antagonist 2-amino-5-phosphonopentanoic acid ameliorated the deficits observed in Mir501-KO mice. The epigenetic and pathophysiological mechanism that links miR-501-3p to the modulation of glutamatergic transmission provides etiological implications for schizophrenia.

The mGlu5 Receptor Protomer-Mediated Dopamine D2 Receptor Trans-Inhibition Is Dependent on the Adenosine A2A Receptor Protomer: Implications for Parkinson’s Disease

The adenosine A2A receptor (A2AR), dopamine D2 receptor (D2R) and metabotropic glutamate receptor type 5 (mGluR5) form A2AR-D2R-mGluR5 heteroreceptor complexes in living cells and in rat striatal neurons. In the current study, we present experimental data supporting the view that the A2AR protomer plays a major role in the inhibitory modulation of the density and the allosteric receptor-receptor interaction within the D2R-mGluR5 heteromeric component of the A2AR-D2R-mGluR5 complex in vitro and in vivo. The A2AR and mGluR5 protomers interact and modulate D2R protomer recognition and signalling upon forming a trimeric complex from these receptors. Expression of A2AR in HEK293T cells co-expressing D2R and mGluR5 resulted in a significant and marked increase in the formation of the D2R-mGluR5 heteromeric component in both bioluminescence resonance energy transfer and proximity ligation assays. A highly significant increase of the the high-affinity component of D2R (D2RKi High) values was found upon cotreatment with the mGluR5 and A2AR agonists in the cells expressing A2AR, D2R and mGluR5 with a significant effect observed also with the mGluR5 agonist alone compared to cells expressing only D2R and mGluR5. In cells co-expressing A2AR, D2R and mGluR5, stimulation of the cells with an mGluR5 agonist like or D2R antagonist fully counteracted the D2R agonist-induced inhibition of the cAMP levels which was not true in cells only expressing mGluR5 and D2R. In agreement, the mGluR5-negative allosteric modulator raseglurant significantly reduced the haloperidol-induced catalepsy in mice, and in A2AR knockout mice, the haloperidol action had almost disappeared, supporting a functional role for mGluR5 and A2AR in enhancing D2R blockade resulting in catalepsy. The results represent a relevant example of integrative activity within higher-order heteroreceptor complexes.

Metabotropic Glutamate Receptor 5 Antagonism Reduces Pathology and Differentially Improves Symptoms in Male and Female Heterozygous zQ175 Huntington's Mice.

Huntington’s disease (HD) is an inherited autosomal dominant neurodegenerative disorder that leads to progressive motor and cognitive impairment. There are currently no available disease modifying treatments for HD patients. We have previously shown that pharmacological blockade of metabotropic glutamate receptor 5 (mGluR5) signaling rescues motor deficits, improves cognitive impairments and mitigates HD neuropathology in male zQ175 HD mice. Mounting evidence indicates that sex may influence HD progression and we have recently reported a sex-specific pathological mGluR5 signaling in Alzheimer’s disease (AD) mice. Here, we compared the outcomes of treatment with the mGluR5 negative allosteric modulator CTEP (2-chloro-4-[2-[2,5-dimethyl-1-[4-(trifluoromethoxy)phenyl]imidazol-4-yl]ethynyl]pyridine) in both male and female symptomatic zQ175 mice. We found that female zQ175 mice required a longer treatment duration with CTEP than male mice to show improvement in their rotarod performance. Unlike males, chronic CTEP treatment did not improve the grip strength nor reverse the cognitive decline of female zQ175 mice. However, CTEP reduced the number of huntingtin aggregates, improved neuronal survival and decreased microglia activation in the striatum of both male and female zQ175 mice. Together, our results indicate that mGluR5 antagonism can reduce HD neuropathology in both male and female zQ175 HD mice, but sex has a clear impact on the efficacy of the treatment and must be taken into consideration for future HD drug development.

Upregulated mGluR5 induces ER stress and DNA damage by regulating the NMDA receptor subunit NR2B.

Dysfunction caused by mGluR5 expression or activation is an important mechanism in the development of Parkinson's disease (PD). Early clinical studies on mGluR5 negative allosteric modulators have shown some limitations. It is therefore necessary to find a more specific approach to block mGluR5-mediated neurotoxicity. Here, we determined the role of N-methyl-D-aspartate (NMDA) receptor subunit NR2B in mGluR5-mediated ER stress and DNA damage. In vitro study, rotenone-induced ER stress and DNA damage were accompanied by an increase in mGluR5 expression and overexpressed or activated mGluR5 with agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) induced ER stress and DNA damage, while blocking mGluR5 with antagonist 2-methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP) alleviated the effect. Furthermore, the damage caused by CHPG was blocked by NMDA receptor antagonist MK-801. Additionally, rotenone or CHPG increased the p-Src and p-NR2B, which was inhibited by MPEP. Blocking p-Src or NR2B with PP2 or CP101,606 alleviated CHPG-induced ER stress and DNA damage. Overactivation of mGluR5 accompanied with the increase of p-Src and p-NR2B in the ER stress and DNA damage was found in rotenone-induced PD rat model. These findings suggest a new mechanism wherein mGluR5 induces ER stress and DNA damage through the NMDA receptor and propose NR2B as the molecular target for therapeutic strategy for PD.

MGluR5 Negative Modulators for Fragile X: Treatment Resistance and Persistence.

Fragile X syndrome (FXS) is caused by silencing of the human FMR1 gene and is the leading monogenic cause of intellectual disability and autism. Abundant preclinical data indicated that negative allosteric modulators (NAMs) of metabotropic glutamate receptor 5 (mGluR5) might be efficacious in treating FXS in humans. Initial attempts to translate these findings in clinical trials have failed, but these failures provide the opportunity for new discoveries that will improve future trials. The emergence of acquired treatment resistance (“tolerance”) after chronic administration of mGluR5 NAMs is a potential factor in the lack of success. Here we confirm that FXS model mice display acquired treatment resistance after chronic treatment with the mGluR5 NAM CTEP in three assays commonly examined in the mouse model of FXS: (1) audiogenic seizure susceptibility, (2) sensory cortex hyperexcitability, and (3) hippocampal protein synthesis. Cross-tolerance experiments suggest that the mechanism of treatment resistance likely occurs at signaling nodes downstream of glycogen synthase kinase 3α (GSK3α), but upstream of protein synthesis. The rapid emergence of tolerance to CTEP begs the question of how previous studies showed an improvement in inhibitory avoidance (IA) cognitive performance after chronic treatment. We show here that this observation was likely explained by timely inhibition of mGluR5 during a critical period, as brief CTEP treatment in juvenile mice is sufficient to provide a persistent improvement of IA behavior measured many weeks later. These data will be important to consider when designing future fragile X clinical trials using compounds that target the mGluR5-to-protein synthesis signaling cascade.

Cold ischemic injury is reduced by the mGluR5 negative allosteric modulator MPEP in rat livers from cardiac death donors

Background: we previously demonstrated that the blockade of mGluR5 reduces inflammation and necrosis in both cold and warm ischemia/reperfusion injury models; in fact, the administration of 2-methyl-6(phenylethylnyl) pyridine (MPEP) reduced Bax/Bcl-2, TNF-α and iNOS protein levels after the normothermic reperfusion of rat and mouse livers. In this study, we evaluated whether MPEP reduces the hepatic preservation injury in rat livers from cardiac death donors (DCD).

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.

MGluR5 Allosteric Modulation Promotes Neurorecovery in a 6-OHDA-Toxicant Model of Parkinson's Disease.

Parkinson's disease is a neurodegenerative disease characterized by a loss of dopaminergic substantia nigra neurons and depletion of dopamine. To date, current therapeutic approaches focus on managing motor symptoms and trying to slow neurodegeneration, with minimal capacity to promote neurorecovery. mGluR5 plays a key role in neuroplasticity, and altered mGluR5 signaling contributes to synucleinopathy and dyskinesia in patients with Parkinson's disease. Here, we tested whether the mGluR5-negative allosteric modulator, (2-chloro-4-[2[2,5-dimethyl-1-[4-(trifluoromethoxy) phenyl] imidazol-4-yl] ethynyl] pyridine (CTEP), would be effective in improving motor deficits and promoting neural recovery in a 6-hydroxydopamine (6-OHDA) mouse model. Lesions were induced by 6-ODHA striatal infusion, and 30 days later treatment with CTEP (2 mg/kg) or vehicle commenced for either 1 or 12 weeks. Animals were subjected to behavioral, pathological, and molecular analyses. We also assessed how long the effects of CTEP persisted, and finally, using rapamycin, determined the role of the mTOR pathway. CTEP treatment induced a duration-dependent improvement in apomorphine-induced rotation and performance on rotarod in lesioned mice. Moreover, CTEP promoted a recovery of striatal tyrosine hydroxylase-positive fibers and normalized FosB levels in lesioned mice. The beneficial effects of CTEP were paralleled by an activation of mammalian target of rapamycin (mTOR) pathway and elevated brain-derived neurotrophic factor levels in the striatum of lesioned mice. The mTOR inhibitor, rapamycin (sirolimus), abolished CTEP-induced neurorecovery and rescue of motor deficits. Our findings indicate that mTOR pathway is a useful target to promote recovery and that mGluR5 allosteric regulators may potentially be repurposed to selectively target this pathway to enhance neuroplasticity in patients with Parkinson's disease.

PDZ Scaffold Protein CAL Couples with Metabotropic Glutamate Receptor 5 to Protect Against Cell Apoptosis and Is a Potential Target in the Treatment of Parkinson's Disease.

Targeting mGluR5 has been an attractive strategy to modulate glutamate excitotoxicity for neuroprotection. Although human clinical trials using mGluR5 negative allosteric modulators (NAMs) have included some disappointments, recent investigations have added several more attractive small molecules to this field, providing a promise that the identification of more additional strategies to modulate mGluR5 activity might be potentially beneficial for the advancement of PD treatment. Here, we determined the role of the interacting partner CAL (cystic fibrosis transmembrane conductance regulator-associated ligand) in mGluR5-mediated protection in vitro and in vivo. In astroglial C6 cells, CAL deficiency blocked (S)-3, 5-dihydroxyphenylglycine (DHPG)-elicited p-AKT and p-ERK1/2, subsequently prevented group I mGluRs-mediated anti-apoptotic protection, which was blocked by receptor antagonist 1-aminoindan-1, 5-dicarboxylic acid (AIDA), and PI3K or MEK inhibitor LY294002 or U0126. In rotenone-treated MN9D cells, both CAL and mGluR5 expressions were decreased in a time- and dose-dependent manner, and the correlation between these 2 proteins was confirmed by lentivirus-delivered CAL overexpression and knockdown. Moreover, CAL coupled with mGluR5 upregulated mGluR5 protein expression by inhibition of ubiquitin-proteasome-dependent degradation to suppress mGluR5-mediated p-JNK and to protect against cell apoptosis. Additionally, CAL also inhibited rotenone-induced glutamate release to modulate mGluR5 activity. Furthermore, in the rotenone-induced rat model of PD, AAV-delivered CAL overexpression attenuated behavioral deficits and dopaminergic neuronal death, while CAL deficiency aggravated rotenone toxicity. On the other hand, the protective effect of the mGluR5 antagonist MPEP was weakened by knocking down CAL. In vivo experiments also confirmed that CAL inhibited ubiquitination-proteasome-dependent degradation to modulate mGluR5 expression and JNK phosphorylation. Our findings show that CAL protects against cell apoptosis via modulating mGluR5 activity, and may be a new molecular target for an effective therapeutic strategy for PD.

E-pharmacophore-based screening of mGluR5 negative allosteric modulators for central nervous system disorder

Glutamate, a major neurotransmitter in the central nervous system of human, plays a crucial role in various neurological pathways by activating the ligand-gated ion channels such as mGluR and iGluR. Dysfunction of mGluR 5 can cause Alzheimer’s disease, Parkinson’s disease, epilepsy, depression, anxiety, etc. In the current study, we have developed the energetically optimized pharmacophore model to screen the eMolecules database having more than 6 million compounds with the help of reported cocrystal structure with 3-chloro-5-[6-(5-fluoropyridin-2 yl)pyrimidin-4-yl]benzonitrile (PDB ID: 5CGD). The obtained hits were docked into the allosteric site of the target and further validation of E-pharmacophore was done by enrichment calculations followed by the molecular dynamics simulations to analyze the specific amino acid interactions with the compound present in the allosteric site of the receptor.

In Vitro Functional Characterization of GET73 as Possible Negative Allosteric Modulator of Metabotropic Glutamate Receptor 5.

The present study was aimed to further characterize the pharmacological profile of N-[4-(trifluoromethyl) benzyl]-4-methoxybutyramide (GET73), a putative negative allosteric modulator (NAM) of metabotropic glutamate subtype 5 receptor (mGluR5) under development as a novel medication for the treatment of alcohol dependence. This aim has been accomplished by means of a series of in vitro functional assays. These assays include the measure of several down-stream signaling [intracellular Ca++ levels, inositol phosphate (IP) formation and CREB phosphorylation (pCREB)] which are generally affected by mGluR5 ligands. In particular, GET73 (0.1 nM–10 μM) was explored for its ability to displace the concentration-response curve of some mGluR5 agonists/probes (glutamate, L-quisqualate, CHPG) in different native preparations. GET73 produced a rightward shift of concentration-response curves of glutamate- and CHPG-induced intracellular Ca++ levels in primary cultures of rat cortical astrocytes. The compound also induced a rightward shift of concentration response curve of glutamate- and L-quisqualate-induced increase in IP turnover in rat hippocampus slices, along with a reduction of CHPG (10 mM)-induced increase in IP formation. Moreover, GET73 produced a rightward shift of concentration-response curve of glutamate-, CHPG- and L-quisqualate-induced pCREB levels in rat cerebral cortex neurons. Although the engagement of other targets cannot be definitively ruled out, these data support the view that GET73 acts as an mGluR5 NAM and support the significance of further investigating the possible mechanism of action of the compound.

MGluR5 Modulation of Behavioral and Epileptic Phenotypes in a Mouse Model of Tuberous Sclerosis Complex.

Drugs targeting metabotropic glutamate receptor 5 (mGluR5) have therapeutic potential in autism spectrum disorders (ASD), including tuberous sclerosis complex (TSC). The question whether inhibition or potentiation of mGluR5 could be beneficial depends, among other factors, on the specific indication. To facilitate the development of mGluR5 treatment strategies, we tested the therapeutic utility of mGluR5 negative and positive allosteric modulators (an mGluR5 NAM and PAM) for TSC, using a mutant mouse model with neuronal loss of Tsc2 that demonstrates disease-related phenotypes, including behavioral symptoms of ASD and epilepsy. This model uniquely enables the in vivo characterization and rescue of the electrographic seizures associated with TSC. We demonstrate that inhibition of mGluR5 corrects hyperactivity, seizures, and elevated de novo synaptic protein synthesis. Conversely, positive allosteric modulation of mGluR5 results in the exacerbation of hyperactivity and epileptic phenotypes. The data suggest a meaningful therapeutic potential for mGluR5 NAMs in TSC, which warrants clinical exploration and the continued development of mGluR5 therapies.

Effect of the mGluR5-NAM Basimglurant on Behavior in Adolescents and Adults with Fragile X Syndrome in a Randomized, Double-Blind, Placebo-Controlled Trial: FragXis Phase 2 Results

Preclinical data suggest that inhibition of the metabotropic glutamate receptor 5 (mGluR5) receptor might hold therapeutic benefits in Fragile X syndrome (FXS). Treatment of Fmr1 knockout mice with mGluR5-negative allosteric modulators (NAMs) has been reported to correct a broad range of phenotypes related to FXS. The early short-term clinical trials with mGluR5 NAMs, including basimglurant, assessing the effects in individuals with FXS, were supportive of further exploration in larger, well-controlled trials. We evaluated basimglurant, a potent and selective mGluR5 NAM, in a 12-week, double-blind, parallel-group study of 183 adults and adolescents (aged 14–50, mean 23.4 years) with FXS. Individuals with an FMR1 full mutation were randomized to placebo or one of two doses of basimglurant. The primary efficacy endpoint was the change from baseline in behavioral symptoms using the Anxiety Depression and Mood Scale (ADAMS) total score. All treatment arms showed marked behavioral improvements from baseline to week 12 with less improvement in the basimglurant 1.5 mg arm than placebo; however, basimglurant 0.5 mg was inferior to placebo in the ADAMs total score. Treatment with basimglurant was overall well-tolerated. A higher incidence of adverse events classified as psychiatric disorders were reported in patients treated with basimglurant, including three patients with hallucinations or psychosis. In this phase 2 clinical trial, basimglurant did not demonstrate improvement over placebo. Evaluation of the overall risk-benefit in younger patient populations is an important consideration for the design of potential further investigations of efficacy with this class of medications.

A Phase I randomized clinical trial testing the safety, tolerability and preliminary pharmacokinetics of the mGluR5 negative allosteric modulator GET 73 following single and repeated doses in healthy volunteers

Preclinical work suggests that the metabotropic glutamate receptor subtype 5 (mGlu5) may represent a novel target to treat neuropsychiatric disorders, including alcohol use disorder and obesity. The goal of this first-in-man study was to evaluate the safety, tolerability and pharmacokinetics (PK) of GET 73 (PubChem SID: 329974174), a novel mGluR5 negative allosteric modulator. This was a double-blind, placebo-controlled, ascending dose, Phase I study conducted in healthy male volunteers in two experiments. GET 73 was administered as single ascending doses (N=48; Experiment 1; 10, 30, 100, 300, 450, 600-mg) or multiple ascending doses (N=32; Experiment 2; 100, 300, 450, 450-mg twice a day). Primary endpoints were the incidence of adverse events (AEs) among drug conditions and drug tolerability. The secondary endpoints were the PK parameters of GET 73 and its metabolite MET 2. Single GET 73 doses of up to 600-mg and repeated ascending doses of up to 450-mg twice/day were safe and well-tolerated. There were no serious or severe AEs. All AEs were mild or moderate in severity. Total GET 73 exposure increased with each increased GET 73 dose. A dose-related increase in mean maximum plasma drug concentration was observed after repeated dosing. Maximum plasma drug concentrations occurred between 0.5 and 2.05h after administration in all groups for both single and repeated doses. This first-in-human study indicates that GET 73, as single or multiple ascending doses, is safe and well-tolerated when administered to healthy male volunteers.

Mechanisms of Skin Toxicity Associated with Metabotropic Glutamate Receptor 5 Negative Allosteric Modulators

Cutaneous reactions represent one of the most common adverse drug effects observed in clinical trials leading to substantial compound attrition. Three negative allosteric modulators (NAMs) of metabotropic glutamate receptors (mGluRs), which represent an important target for neurological diseases, developed by Pfizer, were recently failed in preclinical development due to delayed type IV skin hypersensitivity observed in non-human primates (NHPs). Here we employed large-scale phenotypic profiling in standardized panels of human primary cell/co-culture systems to characterize the skin toxicity mechanism(s) of mGluR5 NAMs from two different series. Investigation of a database of chemicals tested in these systems and transcriptional profiling suggested that the mechanism of toxicity may involve modulation of nuclear receptor targets RAR/RXR, and/or VDR with AhR antagonism. The studies reported here demonstrate how phenotypic profiling of preclinical drug candidates using human primary cells can provide insights into the mechanisms of toxicity and inform early drug discovery and development campaigns.

Discovery and Preclinical Characterization of 3-((4-(4-Chlorophenyl)-7-fluoroquinoline-3-yl)sulfonyl)benzonitrile, a Novel Non-acetylenic Metabotropic Glutamate Receptor 5 (mGluR5) Negative Allosteric Modulator for Psychiatric Indications.

Negative allosteric modulators (NAM) of metabotropic glutamate receptor 5 (mGluR5) have been implicated as a potential pharmacotherapy for a number of psychiatric diseases, including anxiety and depression. Most of the mGluR5 NAM clinical candidates can be characterized by the central acetylenic moiety that connects the terminal pharmacophores. Identification of a sulfoquinoline hit via high throughput screening (HTS) followed by optimization provided a 4-phenyl-3-aryl-sulfoquinoline lead compound with the minimal pharmacophore. Optimization of the core and aryl appendages was performed by scanning and matrix libraries synthesized by the multiple parallel synthesis approach. Biological evaluation of matrix libraries provided a number of potent, metabolically stable, and in vivo active compounds. One of these compounds, 25 showed high efficacy and safety in preclinical in vivo models; this allowed its nomination as a novel, nonacetylenic mGluR5 NAM clinical candidate. Compound 25 was advanced to first-in-man trials for the treatment of psychiatric conditions.

Negative Allosteric Modulation of mGluR5 Partially Corrects Pathophysiology in a Mouse Model of Rett Syndrome.

Altered regulation of synaptic protein synthesis has been hypothesized to contribute to the pathophysiology that underlies multiple forms of intellectual disability and autism spectrum disorder. Here, we show in a mouse model of Rett syndrome (Mecp2 KO) that metabotropic glutamate receptor 5 (mGluR5)- and protein-synthesis-dependent synaptic plasticity are abnormal in the hippocampus. We found that a subset of ribosome-bound mRNAs was aberrantly upregulated in hippocampal CA1 neurons of Mecp2 KO mice, that these significantly overlapped with FMRP direct targets and/or SFARI human autism genes, and that chronic treatment of Mecp2 KO mice with an mGluR5-negative allosteric modulator tunes down upregulated ribosome-bound mRNAs and partially improves mutant mice phenotypes.