Background Metabotropic glutamate receptor subtype 5 (mGlu5) activators have emerged as a novel approach to the treatment of schizophrenia. Positive allosteric modulators (PAMs) of mGlu5 have generated tremendous excitement and fueled major drug discovery efforts. Although mGlu5 PAMs have robust efficacy in preclinical models of schizophrenia, preliminary reports suggest that these compounds may induce seizure activity. Prototypical mGlu5 PAMs do not activate mGlu5 directly but selectively potentiate activation of mGlu5 by glutamate. This mechanism may be critical to maintaining normal activity-dependence of mGlu5 activation and achieving optimal in vivo effects. Methods Using specially engineered mGlu5 cell lines incorporating point mutations within the allosteric and orthosteric binding sites, as well as brain slice electrophysiology and in vivo electroencephalography and behavioral pharmacology, we found that some mGlu5 PAMs have intrinsic allosteric agonist activity in the absence of glutamate. Results Both in vitro mutagenesis and in vivo pharmacology studies demonstrate that VU0422465 is an agonist PAM that induces epileptiform activity and behavioral convulsions in rodents. In contrast, VU0361747, an mGlu5 PAMs optimized to eliminate allosteric agonist activity, has robust in vivo efficacy and does not induce adverse effects at doses that yield high brain concentrations. Conclusions Loss of the absolute dependence of mGlu5 PAMs on glutamate release for their activity can lead to severe adverse effects. The finding that closely related mGlu5 PAMs can differ in their intrinsic agonist activity provides critical new insights that is essential for advancing these molecules through clinical development for treatment of schizophrenia.

Metabotropic glutamate receptor 5 (mGlu 5 ) is a target for the treatment of central nervous system (CNS) disorders, such as schizophrenia and Alzheimer's disease. Furthermore, mGlu 5 has been shown to play an important role in hippocampal synaptic plasticity, specifically in long-term depression (LTD) and longterm potentiation (LTP), which is thought to be involved in cognition. Multiple mGlu 5 -positive allosteric modulators (PAMs) have been developed from a variety of different scaffolds. Previous work has extensively characterized a common allosteric site on mGlu 5 , termed the MPEP (2-Methyl-6-(phenylethynyl)pyridine) binding site. However, one mGlu 5 PAM, CPPHA (N-(4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl]phenyl)-2-hydroxybenzamide), interacts with a separate allosteric site on mGlu 5. Using cell-based assays and brain slice preparations, we characterized the interaction of a potent and efficacious mGlu 5 PAM from the CPPHA series termed NCFP (N-(4-chloro-2-((4-fluoro-1,3-dioxoisoindolin-2-yl)methyl)phenyl)picolinamide). NCFP binds to the CPPHA site on mGlu 5 and potentiates mGlu 5 -mediated responses in both recombinant and native systems. However, NCFP provides greater mGlu 5 subtype selectivity than does CPPHA, making it more suitable for studies of effects on mGlu 5 in CNS preparations. Of interest, NCFP does not potentiate responses involved in hippocampal synaptic plasticity (LTD/ LTP), setting it apart from other previously characterized MPEP site PAMs. This suggests that although mGlu 5 PAMs may have similar responses in some systems, they can induce differential effects on mGlu 5 -mediated physiologic responses in the CNS. Such stimulus bias by mGlu 5 PAMs may complicate drug discovery efforts but would also allow for specifically tailored therapies, if pharmacological biases can be attributed to different therapeutic outcomes.

Impaired transmission through glutamatergic circuits has been postulated to play a role in the underlying pathophysiology of schizophrenia. Furthermore, inhibition of the N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptors (NMDAR) induces a syndrome that recapitulates many of the symptoms observed in patients with schizophrenia. Selective activation of metabotropic glutamate receptor subtype 5 (mGlu 5 ) may provide a novel therapeutic approach for treatment of symptoms associated with schizophrenia through facilitation of transmission through central glutamatergic circuits. Here, we describe the characterization of two novel N-aryl piperazine mGlu 5 positive allosteric modulators (PAMs): 2-(4-(2-(benzyloxy)acetyl)piperazin-1-yl)benzonitrile (VU0364289) and 1-(4-(2,4-difluorophenyl)piperazin-1-yl)-2-((4-fluorobenzyl)oxy)ethanone (DPFE). VU0364289 and DPFE induced robust leftward shifts in the glutamate concentration-response curves for Ca 21 mobilization and extracellular signal-regulated kinases 1 and 2 phosphorylation. Both PAMs displayed micromolar affinity for the common mGlu 5 allosteric binding site and high selectivity for mGlu 5 . VU0364289 and DPFE possessed suitable pharmacokinetic properties for dosing in vivo and produced robust dose-related effects in reversing amphetamine-induced hyperlocomotion, a preclinical model predictive of antipsychotic-like activity. In addition, DPFE enhanced acquisition of contextual fear conditioning in rats and reversed behavioral deficits in a mouse model of NMDAR hypofunction. In contrast, DPFE had no effect on reversing apomorphine-induced disruptions of prepulse inhibition of the acoustic startle reflex. These mGlu 5 PAMs also increased monoamine levels in the prefrontal cortex, enhanced performance in a hippocampal-mediated memory task, and elicited changes in electroencephalogram dynamics commensurate with procognitive effects. Collectively, these data support and extend the role for the development of novel mGlu 5 PAMs for the treatment of psychosis and cognitive deficits observed in individuals with schizophrenia.