Highly selective positive allosteric modulators (PAMs) of the M subtype of muscarinic acetylcholine receptor have emerged as an exciting new approach for improving cognitive function in patients suffering from Alzheimer's disease and schizophrenia. However, excessive activation of M is known to induce seizure activity and have actions in the prefrontal cortex (PFC) that could impair cognitive function. We now report a series of pharmacological, electrophysiological, and behavioral studies in which we find that recently reported M PAMs, PF-06764427 and MK-7622, have robust agonist activity in cell lines and agonist effects in the mouse PFC, and have the potential to overactivate the M receptor and disrupt PFC function. In contrast, structurally distinct M PAMs (VU0453595 and VU0550164) are devoid of agonist activity in cell lines and maintain activity dependence of M activation in the PFC. Consistent with the previously reported effect of PF-06764427, the ago-PAM MK-7622 induces severe behavioral convulsions in mice. In contrast, VU0453595 does not induce behavioral convulsions at doses well above those required for maximal efficacy in enhancing cognitive function. Furthermore, in contrast to the robust efficacy of VU0453595, the ago-PAM MK-7622 failed to improve novel object recognition, a rodent assay of cognitive function. These findings suggest that in vivo cognition-enhancing efficacy of M PAMs can be observed with PAMs lacking intrinsic agonist activity and that intrinsic agonist activity of M PAMs may contribute to adverse effects and reduced efficacy in improving cognitive function.

Fear promotes adaptive responses to threats. However, when the level of fear is not proportional to the level of threat, maladaptive fear-related behaviors characteristic of anxiety disorders result. Post-traumatic stress disorder develops in response to a traumatic event, and patients often show sensitized reactions to mild stressors associated with the trauma. Stress-enhanced fear learning (SEFL) is a rodent model of this sensitized responding, in which exposure to a 15-shock stressor nonassociatively enhances subsequent fear conditioning training with only a single trial. We examined the role of corticosterone (CORT) in SEFL. Administration of the CORT synthesis blocker metyrapone prior to the stressor, but not at time points after, attenuated SEFL. Moreover, CORT co-administered with metyrapone rescued SEFL. However, CORT alone without the stressor was not sufficient to produce SEFL. In these same animals, we then looked for correlates of SEFL in terms of changes in excitatory receptor expression. Western blot analysis of the basolateral amygdala (BLA) revealed an increase in the GluA1 AMPA receptor subunit that correlated with SEFL. Thus, CORT is permissive to trauma-induced changes in BLA function.

Support for the N-methyl-d-aspartate receptor (NMDAR) hypofunction hypothesis of schizophrenia has led to increasing focus on restoring proper glutamatergic signaling as an approach for treatment of this devastating disease. The ability of metabotropic glutamate (mGlu) receptors to modulate glutamatergic neurotransmission has thus attracted considerable attention for the development of novel antipsychotics. Consisting of eight subtypes classified into three groups based on sequence homology, signal transduction, and pharmacology, the mGlu receptors provide a wide range of targets to modulate NMDAR function as well as glutamate release. Recently, allosteric modulators of mGlu receptors have been developed that allow unprecedented selectivity among subtypes, not just groups, facilitating the investigation of the effects of subtype-specific modulation. In preclinical animal models, positive allosteric modulators (PAMs) of the group I mGlu receptor mGlu5 have efficacy across all three symptom domains of schizophrenia (positive, negative, and cognitive). The discovery and development of mGlu5 PAMs that display unique signal bias suggests that efficacy can be retained while avoiding the neurotoxic effects of earlier compounds. Interestingly, mGlu1 negative allosteric modulators (NAMs) appear efficacious in positive symptom models of the disease but are still in early preclinical development. While selective group II mGlu receptor (mGlu2/3) agonists have reached clinical trials but were unsuccessful, specific mGlu2 or mGlu3 receptor targeting still hold great promise. Genetic studies implicated mGlu2 in the antipsychotic effects of group II agonists and mGlu2 PAMs have since entered into clinical trials. Additionally, mGlu3 appears to play an important role in cognition, may confer neuroprotective effects, and thus is a promising target to alleviate cognitive deficits in schizophrenia. Although group III mGlu receptors (mGlu4/6/7/8) have attracted less attention, mGlu4 agonists and PAMs appear to have efficacy across all three symptoms domains in preclinical models. The recent discovery of heterodimers comprising mGlu2 and mGlu4 may explain the efficacy of mGlu4 selective compounds but this remains to be determined. Taken together, compounds targeting mGlu receptors, specifically subtype-selective allosteric modulators, provide a compelling alternative approach to fill the unmet clinical needs for patients with schizophrenia.

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Selective activation of the M subtype of muscarinic acetylcholine receptor, via positive allosteric modulation (PAM), is an exciting strategy to improve cognition in schizophrenia and Alzheimer's disease patients. However, highly potent M ago-PAMs, such as MK-7622, PF-06764427, and PF-06827443, can engender excessive activation of M, leading to agonist actions in the prefrontal cortex (PFC) that impair cognitive function, induce behavioral convulsions, and result in other classic cholinergic adverse events (AEs). Here, we report a fundamentally new and highly selective M PAM, VU0486846. VU0486846 possesses only weak agonist activity in M-expressing cell lines with high receptor reserve and is devoid of agonist actions in the PFC, unlike previously reported ago-PAMs MK-7622, PF-06764427, and PF-06827443. Moreover, VU0486846 shows no interaction with antagonist binding at the orthosteric acetylcholine (ACh) site (e.g., neither bitopic nor displaying negative cooperativity with [H]-NMS binding at the orthosteric site), no seizure liability at high brain exposures, and no cholinergic AEs. However, as opposed to ago-PAMs, VU0486846 produces robust efficacy in the novel object recognition model of cognitive function. Importantly, we show for the first time that an M PAM can reverse the cognitive deficits induced by atypical antipsychotics, such as risperidone. These findings further strengthen the argument that compounds with modest in vitro M PAM activity (EC > 100 nM) and pure-PAM activity in native tissues display robust procognitive efficacy without AEs mediated by excessive activation of M. Overall, the combination of compound assessment with recombinant in vitro assays (mindful of receptor reserve), native tissue systems (PFC), and phenotypic screens (behavioral convulsions) is essential to fully understand and evaluate lead compounds and enhance success in clinical development.