Efficacy switching SAR of mGluR5 allosteric modulators: highly potent positive and negative modulators from one chemotype

A series of metabotropic glutamate 5 receptor (mGluR5) allosteric ligands with positive, negative or no modulatory efficacy is described. The ability of this series to yield both mGluR5 PAMs and NAMs with single-digit nanomolar potency is unusual, and the underlying SAR is detailed.     

3-(Imidazolyl methyl)-3-aza-bicyclo[3.1.0]hexan-6-yl)methyl ethers: a novel series of mGluR2 positive allosteric modulators

The synthesis and structure-activity relationship (SAR) of a novel series of 3-(imidazolyl methyl)-3-aza-bicyclo[3.1.0]hexan-6-yl)methyl ethers, derived from a high throughput screening (HTS), are described. Subsequent optimization led to identification of potent, metabolically stable and orally available mGluR2 positive allosteric modulators (PAMs).     

N-Aryl pyrrolidinonyl oxadiazoles as potent mGluR5 positive allosteric modulators

A novel series of N-aryl pyrrolidinonyl oxadiazoles were identified as mGluR5 positive allosteric modulators (PAMs). Optimization of the initial lead compound 6a led to the identification of the 12c (-) enantiomer as a potent compound with acceptable in vitro clearance, CYP, hERG and PK properties. Para substituted N-aryl pyrrolidinonyl oxadiazoles are mGluR5 PAMs while the meta and ortho substituted N-aryl pyrrolidinonyl oxadiazoles are negative allosteric modulators (NAMs). Para fluoro substitution on the N-aryl group and meta chloro or methyl substituents on the aryl oxadiazole moiety are optimal for mGluR5 PAM efficacy. The existence of an exquisitely sensitive 'PAM to NAM switch' within this chemotype making it challenging for simultaneous optimization of potency and drug-like properties.     

Discovery and SAR of novel mGluR5 non-competitive antagonists not based on an MPEP chemotype

This Letter describes the discovery and SAR of three novel series of mGluR5 non-competitive antagonists/negative allosteric modulators (NAMs) not based on manipulation of an MPEP/MTEP chemotype. This work demonstrates fundamentally new mGluR5 NAM chemotypes with submicromolar potencies, and the first example of a mode of pharmacology ‘switch’ to provide PAMs with a non-MPEP scaffold.     

Synthesis and SAR of novel,non-MPEP chemotype mGluR5 NAMs identified by functional HTS

This Letter describes the discovery and SAR of three novel series of mGluR5 non-competitive antagonists/negative allosteric modulators (NAMs) not based on manipulation of an MPEP/MTEP chemotype identified by a functional HTS approach. This work demonstrates fundamentally new mGluR5 NAM chemotypes with submicromolar potencies, and further examples of a mode of pharmacology ‘switch’ to provide PAMs with a non-MPEP scaffold.     

Chronic Adolescent CDPPB Treatment Alters Short-Term,but not Long-Term,Glutamatergic Receptor Expression

Dysfunction of the glutamatergic system is believed to underlie many neurodevelopmental disorders including autism, Rett syndrome and schizophrenia. Metabotropic glutamate receptor (mGluR5) positive allosteric modulators (PAM) potentiate glutamatergic signaling, particularly indirectly via the NMDA receptor. Preclinical studies report mGluR5 PAMs can improve schizophrenia-relevant behaviours. Furthermore, adolescent administration has shown to prevent cognitive induced deficits in adult rodents. However, there is limited understanding of the short- and long-term neurochemical effects of mGluR5 PAMs, which may underlie their therapeutic effects. We examined the effect of 7-day adolescent (PN28-34) treatment with the mGluR5 PAM, CDDPB (30 mg/kg), on glutamatergic receptor expression at adolescence (PN35) and adulthood (PN96). Immunoblot analysis revealed that 7-day adolescent CDPPB treatment increased protein expression of glutamatergic receptors including the NMDA receptor subunits, NR1 and NR2A and the AMPA subunits (GluA1 and GluA2) in the adolescent hippocampus, changes that did not extend to adulthood. In contrast, there were no changes in the adolescent frontal cortex, however elevated mGluR5 protein expression was observed at adulthood following adolescent CDPPB treatment. The present study indicates adolescent CDPPB treatment may cause brain region dependent effects on the glutamatergic system, which do not persist into adulthood. These findings may have implications for the preclinical development of mGluR5 PAMs for the treatment of neurodevelopmental disorders.     

4-aryl piperazine and piperidine amides as novel mGluR5 positive allosteric modulators

Positive allosteric modulation of metabotropic glutamate receptor 5 (mGluR5) is regarded as a potential novel treatment for schizophrenic patients. Herein we report the synthesis and SAR of 4-aryl piperazine and piperidine amides as potent mGluR5 positive allosteric modulators (PAMs). Several analogs have excellent activity and desired drug-like properties. Compound 2b was further characterized as a PAM using several in vitro experiments, and produced robust activity in several preclinical animal models.     

Challenges in the development of mGluR5 positive allosteric modulators: the discovery of CPPHA

This Letter describes, for the first time, the synthesis and SAR, developed through an iterative analog library approach, that led to the discovery of the positive allosteric modulator (PAM) of the metabotropic glutamate receptor mGluR5 CPPHA. Binding to a unique allosteric binding site distinct from other mGluR5 PAMs, CPPHA has been the focus of numerous pharmacology studies by several laboratories.     

Discovery of a potent and brain penetrant mGluR5 positive allosteric modulator

This Letter describes the discovery of a novel series of mGluR5 positive allosteric modulators (PAMs). The lead compound, 11c, exhibits excellent potency (EC₅₀ = 30 nM) in vitro, and reaches high brain levels in both rats and mice after oral administration.     

Synthesis and characterization of 8-ethynyl-1,3-dihydro-benzo[b][1,4]diazepin-2-one derivatives: part 2. New potent non-competitive metabotropic glutamate receptor 2/3 antagonists

A series of 1,3-dihydro-benzo[b][1,4]diazepin-2-one derivatives was evaluated as non-competitive mGluR2/3 antagonists. Replacement of a cyano group by a five-membered heterocycle produced compounds inhibiting the binding of [(3)H]-LY354740 to rat mGluR2 with low nanomolar affinity and consistent functional effect at both mGluR2 and mGluR3. Further modification to improve the physicochemical properties led eventually to compounds with the ability to reverse LY354740-mediated inhibition of field excitatory postsynaptic potentials in the rat dentate gyrus.     

Synthesis and characterization of 8-ethynyl-1,3-dihydro-benzo[b][1,4]diazepin-2-one derivatives: new potent non-competitive metabotropic glutamate receptor 2/3 antagonists. Part 1

A series of 1,3-dihydrobenzo[b][1,4]diazepin-2-one derivatives was evaluated as non-competitive mGluR2/3 antagonists. Attachment of an 8-(2-aryl)-ethynyl-moiety produced compounds inhibiting the binding of [³H]-LY354740 to rat mGluR2 with low nanomolar affinity and consistent functional effect at both mGluR2 and mGluR3.     

Benzazoles as allosteric potentiators of metabotropic glutamate receptor 2 (mGluR2): efficacy in an animal model for schizophrenia

Metabotropic glutamate receptor 2 (mGluR2) has been implicated in a variety of CNS disorders, including schizophrenia. Disclosed herein is the development of a new series of allosteric potentiators of mGluR2. Structure-activity relationship studies in conjunction with pharmacokinetic data led to the discovery of indole 5, which is active in an animal model for schizophrenia.     

Positive allosteric modulators of the metabotropic glutamate receptor subtype 4 (mGluR4). Part II: Challenges in hit-to-lead

This Letter describes the synthesis and SAR of two mGluR4 positive allosteric modulator leads, 6 and 7. VU001171 (6) represents the most potent (EC₅₀ = 650 nM), efficacious (141% Glu Max) and largest fold shift (36-fold) of any mGluR4 PAM reported to date. However, this work highlights the challenges in hit-to-lead for mGluR4 PAMs, with multiple confirmed HTS hits displaying little or no tractable SAR.     

Synthesis and characterization of 1,3-dihydro-benzo[b][1,4]diazepin-2-one derivatives: Part 3. New potent non-competitive metabotropic glutamate receptor 2/3 antagonists

A series of 1,3-dihydro-benzo[b][1,4]diazepin-2-one derivatives was evaluated as non-competitive mGluR2/3 antagonists. Replacement of the (2-aryl)-ethynyl-moiety in 8-position with smaller less lipophilic substituents produced compounds inhibiting the binding of [3H]-LY354740 to rat mGluR2 with low nanomolar affinity and consistent functional effect at both mGluR2 and mGluR3. These compounds were able to reverse LY354740-mediated inhibition of field excitatory postsynaptic potentials in the rat dentate gyrus and in vivo activity could be demonstrated by reversal of the LY354740-induced hypoactivity in mice after oral administration.     

3-(Pyridin-2-yl-ethynyl)benzamide metabotropic glutamate receptor 5 negative allosteric modulators: hit to lead studies

A series of 3-(pyridin-2-yl-ethynyl)benzamide negative allosteric modulators of the metabotropic glutamate receptor 5 (mGluR5 NAMs) have been prepared. Starting from HTS hit 1 (IC₅₀: 926 nM), potent mGluR5 NAMs showing excellent potencies (IC₅₀s <50 nM) and good physicochemical profiles were prepared by monitoring LipE values. One compound 26 showed excellent mGluR5 binding (K_i: 21 nM) and antagonism (IC₅₀: 8 nM), an excellent rat PK profile (CL: 12 mL/min/kg, %F: 85) and showed oral activity in a mouse 4-Plate Behavioral model of anxiety (MED: 30 mpk) and a mouse Stress Induced Hyperthermia model of anxiety (MED 17.8 mpk).     

一种天然植物抗菌液对HEPG-2细胞的杀伤及作用机制研究

目的:采用多种方法探究天然植物抗菌液(PAMs)对人肝癌细胞HEPG-2的杀伤及初步分子作用机理。方法:运用形态学观察、MTT法测定PAMs对HEPG-2的抑制作用,AO/EB细胞双染、Annexin V-FITC/PI流式细胞术检测PAMs作用于HEPG-2细胞后细胞的凋亡情况,再通过qRT-PCR与western blot检测HEPG-2中相关基因在m RNA水平和蛋白水平的表达量变化。最后利用划痕实验和transwell小室检测PAMs对HEPG-2细胞迁移能力的抑制。结果:PAMs可以抑制和杀伤HEPG-2细胞,作用效果呈浓度和时间依赖性,且与细胞的凋亡途径密切相关。PAMs可下调HEPG-2细胞中foxm1、survivin、bcl-2的m RNA表达水平,同时在蛋白水平下调foxm1的表达量,上调p53的表达量。除此之外,PAMs还能够有效地抑制HEPG-2细胞的体外迁移。结论:PAMs杀伤HEPG-2细胞的途径之一是通过下调foxm1、上调p53基因的表达量,从而抑制或减少其下游靶基因survivin和bcl-2的转录表达,促进细胞凋亡。本研究为基于PAMs抗癌创新民族医药的研发提供了重要的前期工作基础。     

Synthesis of N(1)-substituted analogues of (2R,4R)-4-amino-pyrrolidine-2,4-dicarboxylic acid as agonists, partial agonists, and antagonists of group II metabotropic glutamate receptors.

The chemical synthesis of a series of N(1)-substituted derivatives of (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylic acid [(2R,4R)-APDC] as constrained analogues of gamma-substituted glutamic acids is described. Appropriate substitution of the N(1)-position results in agonist, partial agonist, or antagonist activity at mGluR2, mGluR3, and/or mGluR6.     

The identification of structurally novel,selective, orally bioavailable positive modulators of mGluR2

The optimisation of an HTS hit series (1) leading to the identification of structurally novel, selective, orally bioavailable mGluR2 positive modulators GSK1331258 and GSK1331268 is described. Structure–activity relationships, attenuation of dopaminergic activity, and potentiation of mGluR2 responses in rat hippocampal MPP-DG synapses are also reported.     

Phosphorylation-independent regulation of metabotropic glutamate receptor signaling by G protein-coupled receptor kinase 2

The accepted paradigm for G protein-coupled receptor kinase (GRK)-mediated desensitization of G protein-coupled receptors involves GRK-mediated receptor phosphorylation followed by the binding of arrestin proteins. Although GRKs contribute to metabotropic glutamate receptor 1 (mGluR1) inactivation, beta-arrestins do not appear to be required for mGluR1 G protein uncoupling. Therefore, we investigated whether the phosphorylation of serine and threonine residues localized within the C terminus of mGluR1a is sufficient to allow GRK2-mediated attenuation of mGluR1a signaling. We find that the truncation of the mGluR1a C-terminal tail prevents mGluR1a phosphorylation and that GRK2 does not contribute to the phosphorylation of an mGluR1 splice variant (mGluR1b). However, mGluR1a-866Delta- and mGluR1b-stimulated inositol phosphate formation is attenuated following GRK2 expression. The expression of the GRK2 C-terminal domain to block membrane translocation of endogenous GRK2 increases mGluR1a-866Delta- and mGluR1b-stimulated inositol phosphate formation, presumably by blocking membrane translocation of GRK2. In contrast, expression of the kinase-deficient GRK2-K220R mutant inhibits inositol phosphate formation by these unphosphorylated receptors. Expression of the GRK2 N-terminal domain (residues 45-185) also attenuates both constitutive and agonist-stimulated mGluR1a, mGluR1a-866Delta, and mGluR1b signaling, and the GRK2 N terminus co-precipitates with mGluR1a. Taken together, our observations indicate that attenuation of mGluR1 signaling by GRK2 is phosphorylation-independent and that the interaction of the N-terminal domain of GRK2 with mGluR1 contributes to the regulation of mGluR1 G protein coupling.     

The PDZ scaffold NHERF-2 interacts with mGluR5 and regulates receptor activity

The two members of the group I metabotropic glutamate receptor family, mGluR1 and mGluR5, both couple to G(q) to mediate rises in intracellular calcium. The alternatively spliced C termini (CT) of mGluRs1 and 5are known to be critical for regulating receptor activity and to terminate in motifs suggestive of potential interactions with PDZ domains. We therefore screened the CTs of both mGluR1a and mGluR5 against a PDZ domain proteomic array. Out of 96 PDZ domains examined, the domain that bound most strongly to mGluR5-CT was the second PDZ domain of the Na(+)/H(+) exchanger regulatory factor 2 (NHERF-2). This interaction was confirmed by reverse overlay, and a single point mutation to the mGluR5-CT was found to completely disrupt the interaction. Full-length mGluR5 robustly associated with full-length NHERF-2 in cells, as assessed by co-immunoprecipitation and confocal microscopy experiments. In contrast, mGluR1a was found to bind NHERF-2 in vitro with a weaker affinity than mGluR5, and furthermore mGluR1a did not detectably associate with NHERF-2 in a cellular context. Immunohistochemical experiments revealed that NHERF-2 and mGluR5 exhibit overlapping patterns of expression in mouse brain, being found most abundantly in astrocytic processes and postsynaptic neuronal elements. In functional experiments, the interaction of NHERF-2 with mGluR5 in cells was found to prolong mGluR5-mediated calcium mobilization and to also potentiate mGluR5-mediated cell death, whereas coexpression of mGluR1a with NHERF-2 had no evident effects on mGluR1a functional activity. These observations reveal that NHERF-2 can selectively modulate mGluR5 signaling, which may contribute to cell-specific regulation of mGluR5 activity.