3-((2-methyl-1-3-thiazol-4-yl)ethynyl)piperidine and fenobam

2-Methyl-6-(phenylethynyl)pyridine (MPEP), a negative allosteric modulator of the metabotropic glutamate receptor (mGluR) 5, protects hepatocytes from ischemic injury. In astrocytes and microglia, MPEP depletes ATP. These findings seem to be self-contradictory, since ATP depletion is a fundamental stressor in ischemia. This study attempted to reconstruct the mechanism of MPEP-mediated ATP depletion and the consequences of ATP depletion on protection against ischemic injury. We compared the effects of MPEP and other mGluR5 negative modulators on ATP concentration when measured in rat hepatocytes and acellular solutions. We also evaluated the effects of mGluR5 blockade on viability in rat hepatocytes exposed to hypoxia. Furthermore, we studied the effects of MPEP treatment on mouse livers subjected to cold ischemia and warm ischemia reperfusion. We found that MPEP and 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) deplete ATP in hepatocytes and acellular solutions, unlike fenobam. This finding suggests that mGluR5s may not be involved, contrary to previous reports. MPEP, as well as MTEP and fenobam, improved hypoxic hepatocyte viability, suggesting that protection against ischemic injury is independent of ATP depletion. Significantly, MPEP protected mouse livers in two different ex vivo models of ischemia reperfusion injury, suggesting its possible protective deployment in the treatment of hepatic inflammatory conditions.

Topics: Adenosine Triphosphate; Animals; Cell Hypoxia; Disease Models, Animal; Hepatocytes; Imidazoles; Liver; Mice; Mitochondria, Liver; Piperidines; Pyridines; Rats; Receptor, Metabotropic Glutamate 5; Reperfusion Injury; Thiazoles; Tumor Necrosis Factor-alpha

Metabotropic glutamate receptor 5 (mGlu5) is a biological target implicated in major neurological and psychiatric disorders. In the present study, we have investigated structural determinants of the interaction of negative allosteric modulators (NAMs) with the seven-transmembrane (7TM) domain of mGlu5. A homology model of the 7TM receptor domain built on the crystal structure of the mGlu1 template was obtained, and the binding modes of known NAMs, namely MPEP and fenobam, were investigated by docking and molecular dynamics simulations. The results were validated by comparison with mutagenesis data available in the literature for these two ligands, and subsequently corroborated by the recently described mGlu5 crystal structure. Moreover, a new series of NAMs was synthesized and tested, providing compounds with nanomolar affinity. Several structural modifications were sequentially introduced with the aim of identifying structural features important for receptor binding. The synthesized NAMs were docked in the validated homology model and binding modes were used to interpret and discuss structure-activity relationships within this new series of compounds. Finally, the models of the interaction of NAMs with mGlu5 were extended to include important non-aryl alkyne mGlu5 NAMs taken from the literature. Overall, the results provide useful insights into the molecular interaction of negative allosteric modulators with mGlu5 and may facilitate the design of new modulators for this class of receptors.

Topics: Allosteric Regulation; Allosteric Site; Antipsychotic Agents; Drug Discovery; Humans; Imidazoles; Kinetics; Ligands; Molecular Docking Simulation; Molecular Dynamics Simulation; Protein Binding; Protein Structure, Tertiary; Pyridines; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Structural Homology, Protein; Structure-Activity Relationship

Pre-clinical studies suggest that negative allosteric modulators (NAMs) of the metabotropic glutamate receptor subtype 5 (mGluR5), including 2-methyl-6-(phenylethynyl)pyridine (MPEP), 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) and fenobam are highly effective in attenuating drug-taking and drug-seeking behaviors. However, both MPEP and MTEP have no translational potential for use in humans because of their off-target effects and short half-lives. Here, we report that 3-fluoro-5-[(6-methylpyridin-2-yl)ethynyl]benzonitrile (MFZ 10-7), a novel mGluR5 NAM, is more potent and selective than MPEP, MTEP and fenobam in both in vitro binding and functional assays. Similar to MTEP, intraperitoneal administration of MFZ 10-7 inhibited intravenous cocaine self-administration, cocaine-induced reinstatement of drug-seeking behavior and cocaine-associated cue-induced cocaine-seeking behavior in rats. Although MFZ 10-7 and MTEP lowered the rate of oral sucrose self-administration, they did not alter total sucrose intake. Further, MFZ 10-7 appeared to be more potent than MTEP in inducing downward shifts in the cocaine dose-response curve, but less effective than MTEP in attenuating sucrose-induced reinstatement of sucrose-seeking behavior. MFZ 10-7 and MTEP had no effect on basal locomotor behavior. These findings not only provide additional evidence supporting an important role for mGluR5 in cocaine reward and addiction, but also introduce a new tool for both in vitro and in vivo investigations with which to further characterize this role.

Topics: Allosteric Regulation; Analysis of Variance; Animals; Binding, Competitive; Cocaine; Cues; Dopamine Uptake Inhibitors; Dose-Response Relationship, Drug; Drug-Seeking Behavior; Enzyme-Linked Immunosorbent Assay; Excitatory Amino Acid Antagonists; HEK293 Cells; Humans; Imidazoles; In Vitro Techniques; Infusions, Intravenous; Inhibitory Concentration 50; Male; Motor Activity; Nitriles; Piperidines; Pyridines; Random Allocation; Rats; Receptor, Metabotropic Glutamate 5; Reinforcement Schedule; Reward; Secondary Prevention; Self Administration; Sucrose; Thiazoles