Genetic Reduction or Negative Modulation of mGlu₇ Does Not Impact Anxiety and Fear Learning Phenotypes in a Mouse Model of <i>MECP2</i> Duplication Syndrome

Rett syndrome and <i>MECP2</i> Duplication syndrome are neurodevelopmental disorders attributed to loss-of-function mutations in, or duplication of, the gene encoding methyl-CpG-binding protein 2 (MeCP2), respectively. We recently reported decreased expression and function of the metabotropic glutamate receptor 7 (mGlu₇) in a mouse model of Rett syndrome. Positive allosteric modulation of mGlu₇ activity was sufficient to improve several disease phenotypes including cognition. Here, we tested the hypothesis that mGlu₇ expression would be reciprocally regulated in a mouse model of <i>MECP2</i> Duplication syndrome, such that negative modulation of mGlu₇ activity would exert therapeutic benefit. To the contrary, we report that mGlu₇ is not functionally increased in mice overexpressing MeCP2 and that neither genetic nor pharmacological reduction of mGlu₇ activity impacts phenotypes that are antiparallel to those observed in Rett syndrome model mice. These data expand our understanding of how mGlu₇ expression and function is affected by changes in MeCP2 dosage and have important implications for the therapeutic development of mGlu₇ modulators

Clickable Photoaffinity Ligands for Metabotropic Glutamate Receptor 5 Based on Select Acetylenic Negative Allosteric Modulators

G protein-coupled receptors (GPCRs) represent the largest class of current drug targets. In particular, small-molecule allosteric modulators offer substantial potential for selectively “tuning” GPCR activity. However, there remains a critical need for experimental strategies that unambiguously determine direct allosteric ligand–GPCR interactions, to facilitate both chemical biology studies and rational structure-based drug design. We now report the development and use of first-in-class clickable allosteric photoprobes for a GPCR based on metabotropic glutamate receptor 5 (mGlu₅) negative allosteric modulator (NAM) chemotypes. Select acetylenic mGlu₅ NAM lead compounds were rationally modified to contain either a benzophenone or an aryl azide as a photoreactive functional group, enabling irreversible covalent attachment to mGlu₅ via photoactivation. Additionally, a terminal alkyne or an aliphatic azide was incorporated as a click chemistry handle, allowing chemoselective attachment of fluorescent moieties to the irreversibly mGlu₅-bound probe <i>via</i> tandem photoaffinity labeling-bioorthogonal conjugation. These clickable photoprobes retained submicromolar affinity for mGlu₅ and negative cooperativity with glutamate, interacted with the “common allosteric-binding site,” displayed slow binding kinetics, and could irreversibly label mGlu₅ following UV exposure. We depleted the number of functional mGlu₅ receptors using an irreversibly bound NAM to elucidate and delineate orthosteric agonist affinity and efficacy. Finally, successful conjugation of fluorescent dyes <i>via</i> click chemistry was demonstrated for each photoprobe. In the future, these clickable photoprobes are expected to aid our understanding of the structural basis of mGlu₅ allosteric modulation. Furthermore, tandem photoaffinity labeling-bioorthogonal conjugation is expected to be a broadly applicable experimental strategy across the entire GPCR superfamily

Structure–Activity Relationships of Pan-Gα_q/11 Coupled Muscarinic Acetylcholine Receptor Positive Allosteric Modulators

Recent years have seen a large increase in the discovery of allosteric ligands targeting muscarinic acetylcholine receptors (mAChRs). One of the challenges in screening such compounds is to understand their mechanisms of action and define appropriate parameter estimates for affinity, cooperativity and efficacy. Herein we describe the mechanisms of action and structure–activity relationships for a series of “pan-G_q-coupled” muscarinic acetylcholine (ACh) receptor (mAChR) positive allosteric modulators (PAMs). Using a combination of radioligand binding, functional inositol phosphate accumulation assays, receptor alkylation and operational data analysis, we show that most compounds in the series derive their variable potency and selectivity from differential cooperativity at the M₁, M₃ and M₅ mAChRs. None of the PAMs showed greater than 10-fold subtype selectivity for the agonist-free receptor, but VU6007705, VU6007678, and VU6008555 displayed markedly increased cooperativity compared to the parent molecule and M₅ mAChR-preferring PAM, ML380 (αβ > 100), in the presence of ACh. Most of the activity of these PAMs derives from their ability to potentiate ACh binding affinity at mAChRs, though VU6007678 was notable for also potentiating ACh signaling efficacy and robust allosteric agonist activity. These data provide key insights for the future design of more potent and subtype-selective mAChR PAMs

PF-06827443 Displays Robust Allosteric Agonist and Positive Allosteric Modulator Activity in High Receptor Reserve and Native Systems

Positive allosteric modulators (PAMs) of the M₁ subtype of muscarinic acetylcholine receptor have attracted intense interest as an exciting new approach for improving the cognitive deficits in schizophrenia and Alzheimer’s disease. Recent evidence suggests that the presence of intrinsic agonist activity of some M₁ PAMs may reduce efficacy and contribute to adverse effect liability. However, the M₁ PAM PF-06827443 was reported to have only weak agonist activity at human M₁ receptors but produced M₁-dependent adverse effects. We now report that PF-06827443 is an allosteric agonist in cell lines expressing rat, dog, and human M₁ and use of inducible cell lines shows that agonist activity of PF-06827443 is dependent on receptor reserve. Furthermore, PF-06827443 is an agonist in native tissue preparations and induces behavioral convulsions in mice similar to other ago-PAMs. These findings suggest that PF-06827443 is a robust ago-PAM, independent of species, in cell lines and native systems

A Novel Class of Succinimide-Derived Negative Allosteric Modulators of Metabotropic Glutamate Receptor Subtype 1 Provides Insight into a Disconnect in Activity between the Rat and Human Receptors

Recent progress in the discovery of mGlu₁ allosteric modulators has suggested the modulation of mGlu₁ could offer possible treatment for a number of central nervous system disorders; however, the available chemotypes are inadequate to fully investigate the therapeutic potential of mGlu₁ modulation. To address this issue, we used a fluorescence-based high-throughput screening assay to screen an allosteric modulator-biased library of compounds to generate structurally diverse mGlu₁ negative allosteric modulator hits for chemical optimization. Herein, we describe the discovery and characterization of a novel mGlu₁ chemotype. This series of succinimide negative allosteric modulators, exemplified by VU0410425, exhibited potent inhibitory activity at rat mGlu₁ but was, surprisingly, inactive at human mGlu₁. VU0410425 and a set of chemically diverse mGlu₁ negative allosteric modulators previously reported in the literature were utilized to examine this species disconnect between rat and human mGlu₁ activity. Mutation of the key transmembrane domain residue 757 and functional screening of VU0410425 and the literature compounds suggests that amino acid 757 plays a role in the activity of these compounds, but the contribution of the residue is scaffold specific, ranging from critical to minor. The operational model of allosterism was used to estimate the binding affinities of each compound to compare to functional data. This novel series of mGlu₁ negative allosteric modulators provides valuable insight into the pharmacology underlying the disconnect between rat and human mGlu₁ activity, an issue that must be understood to progress the therapeutic potential of allosteric modulators of mGlu₁

Development of Novel, CNS Penetrant Positive Allosteric Modulators for the Metabotropic Glutamate Receptor Subtype 1 (mGlu₁), Based on an <i>N</i>‑(3-Chloro-4-(1,3-dioxoisoindolin-2-yl)phenyl)-3-methylfuran-2-carboxamide Scaffold, That Potentiate Wild Type and Mutant mGlu₁ Receptors Found in Schizophrenics

The therapeutic potential of selective mGlu₁ activation is vastly unexplored relative to the other group I mGlu receptor, mGlu₅; therefore, our lab has focused considerable effort toward developing mGlu₁ positive allosteric modulators (PAMs) suitable as in vivo proof of concept tool compounds. Optimization of a series of mGlu₁ PAMs based on an <i>N</i>-(3-chloro-4-(1,3-dioxoisoindolin-2-yl)­phenyl)-3-methylfuran-2-carboxamide scaffold provided <b>17e</b>, a potent (mGlu₁ EC₅₀ = 31.8 nM) and highly CNS penetrant (brain to plasma ratio (<i>K</i>_p) of 1.02) mGlu₁ PAM tool compound, that potentiated not only wild-type human mGlu₁ but also mutant mGlu₁ receptors derived from deleterious <i>GRM1</i> mutations found in schizophrenic patients. Moreover, both electrophysiological and in vivo studies indicate the mGlu₁ ago-PAMs/PAMs do not possess the same epileptiform adverse effect liability as mGlu₅ ago-PAMs/PAMs and maintain temporal activity suggesting a broader therapeutic window

Discovery of (<i>R</i>)‑(2-Fluoro-4-((-4-methoxyphenyl)ethynyl)phenyl) (3-Hydroxypiperidin-1-yl)methanone (ML337), An mGlu₃ Selective and CNS Penetrant Negative Allosteric Modulator (NAM)

A multidimensional, iterative parallel synthesis effort identified a series of highly selective mGlu₃ NAMs with submicromolar potency and good CNS penetration. Of these, ML337 resulted (mGlu₃ IC₅₀ = 593 nM, mGlu₂ IC₅₀ >30 μM) with B:P ratios of 0.92 (mouse) to 0.3 (rat). DMPK profiling and shallow SAR led to the incorporation of deuterium atoms to address a metabolic soft spot, which subsequently lowered both in vitro and in vivo clearance by >50%

Identification of Metabotropic Glutamate Receptor Subtype 5 Potentiators Using Virtual High-Throughput Screening

Selective potentiators of glutamate response at metabotropic glutamate receptor subtype 5 (mGluR5) have exciting potential for the development of novel treatment strategies for schizophrenia. A total of 1,382 compounds with positive allosteric modulation (PAM) of the mGluR5 glutamate response were identified through high-throughput screening (HTS) of a diverse library of 144,475 substances utilizing a functional assay measuring receptor-induced intracellular release of calcium. Primary hits were tested for concentration-dependent activity, and potency data (EC₅₀ values) were used for training artificial neural network (ANN) quantitative structure−activity relationship (QSAR) models that predict biological potency from the chemical structure. While all models were trained to predict EC₅₀, the quality of the models was assessed by using both continuous measures and binary classification. Numerical descriptors of chemical structure were used as input for the machine learning procedure and optimized in an iterative protocol. The ANN models achieved theoretical enrichment ratios of up to 38 for an independent data set not used in training the model. A database of ∼450,000 commercially available drug-like compounds was targeted in a virtual screen. A set of 824 compounds was obtained for testing based on the highest predicted potency values. Biological testing found 28.2% (232/824) of these compounds with various activities at mGluR5 including 177 pure potentiators and 55 partial agonists. These results represent an enrichment factor of 23 for pure potentiation of the mGluR5 glutamate response and 30 for overall mGluR5 modulation activity when compared with those of the original mGluR5 experimental screening data (0.94% hit rate). The active compounds identified contained 72% close derivatives of previously identified PAMs as well as 28% nontrivial derivatives of known active compounds

Design and Synthesis of Systemically Active Metabotropic Glutamate Subtype‑2 and -3 (mGlu_2/3) Receptor Positive Allosteric Modulators (PAMs): Pharmacological Characterization and Assessment in a Rat Model of Cocaine Dependence

As part of our ongoing small-molecule metabotropic glutamate (mGlu) receptor positive allosteric modulator (PAM) research, we performed structure–activity relationship (SAR) studies around a series of group II mGlu PAMs. Initial analogues exhibited weak activity as mGlu₂ receptor PAMs and no activity at mGlu₃. Compound optimization led to the identification of potent mGlu_2/3 selective PAMs with no in vitro activity at mGlu_1,4–8 or 45 other CNS receptors. In vitro pharmacological characterization of representative compound <b>44</b> indicated agonist-PAM activity toward mGlu₂ and PAM activity at mGlu₃. The most potent mGlu_2/3 PAMs were characterized in assays predictive of ADME/T and pharmacokinetic (PK) properties, allowing the discovery of systemically active mGlu_2/3 PAMs. On the basis of its overall profile, compound <b>74</b> was selected for behavioral studies and was shown to dose-dependently decrease cocaine self-administration in rats after intraperitoneal administration. These mGlu_2/3 receptor PAMs have significant potential as small molecule tools for investigating group II mGlu pharmacology

Identification of Specific Ligand–Receptor Interactions That Govern Binding and Cooperativity of Diverse Modulators to a Common Metabotropic Glutamate Receptor 5 Allosteric Site

A common metabotropic glutamate receptor 5 (mGlu₅) allosteric site is known to accommodate diverse chemotypes. However, the structural relationship between compounds from different scaffolds and mGlu₅ is not well understood. In an effort to better understand the molecular determinants that govern allosteric modulator interactions with mGlu₅, we employed a combination of site-directed mutagenesis and computational modeling. With few exceptions, six residues (P654, Y658, T780, W784, S808, and A809) were identified as key affinity determinants across all seven allosteric modulator scaffolds. To improve our interpretation of how diverse allosteric modulators occupy the common allosteric site, we sampled the wealth of mGlu₅ structure–activity relationship (SAR) data available by docking 60 ligands (actives and inactives) representing seven chemical scaffolds into our mGlu₅ comparative model. To spatially and chemically compare binding modes of ligands from diverse scaffolds, the ChargeRMSD measure was developed. We found a common binding mode for the modulators that placed the long axes of the ligands parallel to the transmembrane helices 3 and 7. W784 in TM6 not only was identified as a key NAM cooperativity determinant across multiple scaffolds, but also caused a NAM to PAM switch for two different scaffolds. Moreover, a single point mutation in TM5, G747V, altered the architecture of the common allosteric site such that 4-nitro-<i>N</i>-(1,3-diphenyl-1<i>H</i>-pyrazol-5-yl)­benzamide (VU29) was noncompetitive with the common allosteric site. Our findings highlight the subtleties of allosteric modulator binding to mGlu₅ and demonstrate the utility in incorporating SAR information to strengthen the interpretation and analyses of docking and mutational data