Zixiu Xiang

Novel Selective Allosteric Activator of the M1 Muscarinic Acetylcholine Receptor Regulates Amyloid Processing and Produces Antipsychotic-Like Activity in Rats

Recent studies suggest that subtype-selective activators of M1/M4 muscarinic acetylcholine receptors (mAChRs) may offer a novel approach for the treatment of psychotic symptoms associated with schizophrenia and Alzheimers disease. Previously developed muscarinic agonists have provided clinical data in support of this hypothesis, but failed in clinical development because of a lack of true subtype specificity and adverse effects associated with activation of other mAChR subtypes. We now report characterization of a novel highly selective agonist for the M1 receptor with no agonist activity at any of the other mAChR subtypes, termed TBPB [1-(1′-2-methylbenzyl)-1,4′-bipiperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one]. Mutagenesis and molecular pharmacology studies revealed that TBPB activates M1 through an allosteric site rather than the orthosteric acetylcholine binding site, which is likely critical for its unprecedented selectivity. Whole-cell patch-clamp recordings demonstrated that activation of M1 by TBPB potentiates NMDA receptor currents in hippocampal pyramidal cells but does not alter excitatory or inhibitory synaptic transmission, responses thought to be mediated by M2 and M4. TBPB was efficacious in models predictive of antipsychotic-like activity in rats at doses that did not produce catalepsy or peripheral adverse effects of other mAChR agonists. Finally, TBPB had effects on the processing of the amyloid precursor protein toward the non-amyloidogenic pathway and decreased Aβ production in vitro. Together, these data suggest that selective activation of M1 may provide a novel approach for the treatment of symptoms associated with schizophrenia and Alzheimers disease.

mGluR5 Positive Allosteric Modulators Facilitate both Hippocampal LTP and LTD and Enhance Spatial Learning

Highly selective positive allosteric modulators (PAMs) of metabotropic glutamate receptor subtype 5 (mGluR5) have emerged as a potential approach to treat positive symptoms associated with schizophrenia. mGluR5 plays an important role in both long-term potentiation (LTP) and long-term depression (LTD), suggesting that mGluR5 PAMs may also have utility in improving impaired cognitive function. However, if mGluR5 PAMs shift the balance of LTP and LTD or induce a state in which afferent activity induces lasting changes in synaptic function that are not appropriate for a given pattern of activity, this could disrupt rather than enhance cognitive function. We determined the effect of selective mGluR5 PAMs on the induction of LTP and LTD at the Schaffer collateral-CA1 synapse in the hippocampus. mGluR5-selective PAMs significantly enhanced threshold θ-burst stimulation (TBS)-induced LTP. In addition, mGluR5 PAMs enhanced both DHPG-induced LTD and LTD induced by the delivery of paired-pulse low-frequency stimulation. Selective potentiation of mGluR5 had no effect on LTP induced by suprathreshold TBS or saturated LTP. The finding that potentiation of mGluR5-mediated responses to stimulation of glutamatergic afferents enhances both LTP and LTD and supports the hypothesis that the activation of mGluR5 by endogenous glutamate contributes to both forms of plasticity. Furthermore, two systemically active mGluR5 PAMs enhanced performance in the Morris water maze, a measure of hippocampus-dependent spatial learning. Discovery of small molecules that enhance both LTP and LTD in an activity-appropriate manner shows a unique action on synaptic plasticity that may provide a novel approach for the treatment of impaired cognitive function.

A selective positive allosteric modulator of metabotropic glutamate receptor subtype 2 blocks a hallucinogenic drug model of psychosis

Recent clinical studies reveal that selective agonists of group II metabotropic glutamate (mGlu) receptors have robust efficacy in treating positive and negative symptoms in patients with schizophrenia. Group II mGlu receptor agonists also modulate the in vivo activity of psychotomimetic drugs and reduce the ability of psychotomimetic hallucinogens to increase glutamatergic transmission. Because increased excitation of the medial prefrontal cortex (mPFC) has been implicated in pathophysiology of schizophrenia, the ability of group II mGlu receptor agonists to reduce hallucinogenic drug action in this region is believed to be directly related to their antipsychotic efficacy. A novel class of ligands, termed positive allosteric modulators, has recently been identified, displaying exceptional mGlu2 receptor selectivity. These compounds do not activate mGlu2 receptors directly but potentiate the ability of glutamate and other agonists to activate this receptor. We now report that the mGlu2 receptor-selective positive allosteric modulator biphenyl-indanone A (BINA) modulates excitatory neurotransmission in the mPFC and attenuates the in vivo actions of the hallucinogenic 5-HT2A/2C receptor agonist (-)2,5-dimethoxy-4-bromoamphetamine [(-)DOB]. BINA attenuates serotonin-induced increases in spontaneous excitatory postsynaptic currents in the mPFC, mimicking the effect of the mGlu2/3 receptor agonist (2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl)glycine (DCG-IV). In addition, BINA reduced (-)DOB-induced head twitch behavior and Fos expression in mPFC, effects reversed by pretreatment with the mGlu2/3 receptor antagonist 2S-2-amino-2-(1S,2S-2-carboxycyclopropan-1-yl) -3 - (xanth-9-yl-)propionic acid (LY341495). These data confirm the relevance of excitatory signaling in the mPFC to the behavioral actions of hallucinogens and further support the targeting of mGlu2 receptors as a novel strategy for treating glutamatergic dysfunction in schizophrenia.

Discovery, characterization, and antiparkinsonian effect of novel positive allosteric modulators of metabotropic glutamate receptor 4

Parkinsons disease (PD) is caused by the death of dopamine neurons in the basal ganglia and results in motor symptoms such as tremor and bradykinesia. Activation of metabotropic glutamate receptor 4 (mGluR4) has been shown to modulate neurotransmission in the basal ganglia and results in antiparkinsonian effects in rodent PD models. N-Phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC) is a positive allosteric modulator (PAM) of mGluR4 that has been used to further validate the role of mGluR4 in PD, but the compound suffers from a lack of selectivity, relatively low potency, and poor solubility. Via high-throughput screening, we discovered more than 400 novel PAMs of mGluR4. Compounds derived from a novel chemical scaffold were characterized in vitro at both rat and human mGluR4 using two distinct assays of mGluR4 function. The lead compound was approximately 8-fold more potent than PHCCC, enhanced the potency of glutamate at mGluR4 by 8-fold, and did not show any significant potentiator or antagonist activity at other mGluR subtypes. Resolution of the regioisomers of the lead revealed that the cis regioisomer, (±)-cis-2-(3,5-dichlorphenylcarbamoyl)cyclohexanecarboxylic acid (VU0155041), contained the majority of the mGluR4 PAM activity and also exhibited partial agonist activity at mGluR4 at a site that was distinct from the glutamate binding site, suggesting that this compound is a mixed allosteric agonist/PAM of mGluR4. VU0155041 was soluble in an aqueous vehicle, and intracerebroventricular administration of 31 to 316 nmol of VU0155041 dose-dependently decreased haloperidol-induced catalepsy and reserpine-induced akinesia in rats. These exciting results provide continued support for mGluR4 as a therapeutic target in PD.

A selective allosteric potentiator of the M1 muscarinic acetylcholine receptor increases activity of medial prefrontal cortical neurons and restores impairments in reversal learning

M1 muscarinic acetylcholine receptors (mAChRs) may represent a viable target for treatment of disorders involving impaired cognitive function. However, a major limitation to testing this hypothesis has been a lack of highly selective ligands for individual mAChR subtypes. We now report the rigorous molecular characterization of a novel compound, benzylquinolone carboxylic acid (BQCA), which acts as a potent, highly selective positive allosteric modulator (PAM) of the rat M1 receptor. This compound does not directly activate the receptor, but acts at an allosteric site to increase functional responses to orthosteric agonists. Radioligand binding studies revealed that BQCA increases M1 receptor affinity for acetylcholine. We found that activation of the M1 receptor by BQCA induces a robust inward current and increases spontaneous EPSCs in medial prefrontal cortex (mPFC) pyramidal cells, effects which are absent in acute slices from M1 receptor knock-out mice. Furthermore, to determine the effect of BQCA on intact and functioning brain circuits, multiple single-unit recordings were obtained from the mPFC of rats that showed BQCA increases firing of mPFC pyramidal cells in vivo. BQCA also restored discrimination reversal learning in a transgenic mouse model of Alzheimers disease and was found to regulate non-amyloidogenic APP processing in vitro, suggesting that M1 receptor PAMs have the potential to provide both symptomatic and disease modifying effects in Alzheimers disease patients. Together, these studies provide compelling evidence that M1 receptor activation induces a dramatic excitation of PFC neurons and suggest that selectively activating the M1 mAChR subtype may ameliorate impairments in cognitive function.

GABAA receptor‐mediated currents in interneurons and pyramidal cells of rat visual cortex

1 We compared γ‐aminobutyric acid (GABA)‐mediated responses of identified pyramidal cells and fast spiking interneurons in layer V of visual cortical slices from young rats (P11‐14). 2 The frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) was similar in pyramidal cells and interneurons (1.7 vs. 1.9 Hz). For events with 10‐90 % rise times less than 0.9 ms, no significant differences were found in mean amplitude (61 vs. 65 pA), mean rise time (0.58 vs. 0.61 ms), or the first time constant of decay (τ1, 6.4 vs. 6.5 ms) between pyramidal cells and interneurons. The second decay time constant (τ2) was significantly longer in interneurons than in pyramidal cells (49 vs. 22 ms). The difference in sIPSC decay kinetics between two cell types also existed in adult rats (P36‐42), suggesting the kinetic difference is not due to differential development of GABAA receptors in these cell types. 3 The decay kinetics of monosynaptic evoked IPSCs were also longer in interneurons. As in the case of sIPSCs, the difference was accounted for by the second decay time constant. τ1 and τ2 were, respectively, 13 and 64 ms for interneurons and 12 and 47 ms for pyramidal cells. 4 Cell‐attached patch recordings revealed that the mean open time for single Cl− channels in response to 2 μM GABA was significantly longer in interneurons than pyramidal cells (5.0 vs. 2.8 ms). The chord conductance of these channels in interneurons (12 pS) was significantly smaller than in pyramidal cells (15 pS). Single channel currents reversed polarity when the pipette potential was approximately ‐10 mV for both cell types. 5 These results show that there is a functional diversity of GABAA receptors in electrophysiologically and morphologically identified cortical pyramidal cells and interneurons. This diversity might derive from the different molecular composition of the receptors in these two cell types.

Functional impact of allosteric agonist activity of selective positive allosteric modulators of metabotropic glutamate receptor subtype 5 in regulating central nervous system function.

Positive allosteric modulators (PAMs) of metabotropic glutamate receptor subtype 5 (mGlu5) have emerged as an exciting new approach for the treatment of schizophrenia and other central nervous system (CNS) disorders. Of interest, some mGlu5 PAMs act as pure PAMs, only potentiating mGlu5 responses to glutamate whereas others [allosteric agonists coupled with PAM activity (ago-PAMs)] potentiate responses to glutamate and have intrinsic allosteric agonist activity in mGlu5-expressing cell lines. All mGlu5 PAMs previously shown to have efficacy in animal models act as ago-PAMs in cell lines, raising the possibility that allosteric agonist activity is critical for in vivo efficacy. We have now optimized novel mGlu5 pure PAMs that are devoid of detectable agonist activity and structurally related mGlu5 ago-PAMs that activate mGlu5 alone in cell lines. Studies of mGlu5 PAMs in cell lines revealed that ago-PAM activity is dependent on levels of mGlu5 receptor expression in human embryonic kidney 293 cells, whereas PAM potency is relatively unaffected by levels of receptor expression. Furthermore, ago-PAMs have no agonist activity in the native systems tested, including cortical astrocytes and subthalamic nucleus neurons and in measures of long-term depression at the hippocampal Schaffer collateral-CA1 synapse. Finally, studies with pure PAMs and ago-PAMs chemically optimized to provide comparable CNS exposure revealed that both classes of mGlu5 PAMs have similar efficacy in a rodent model predictive of antipsychotic activity. These data suggest that the level of receptor expression influences the ability of mGlu5 PAMs to act as allosteric agonists in vitro and that ago-PAM activity observed in cell-based assays may not be important for in vivo efficacy.

A Novel Selective Muscarinic Acetylcholine Receptor Subtype 1 Antagonist Reduces Seizures without Impairing Hippocampus-Dependent Learning

Previous studies suggest that selective antagonists of specific subtypes of muscarinic acetylcholine receptors (mAChRs) may provide a novel approach for the treatment of certain central nervous system (CNS) disorders, including epileptic disorders, Parkinsons disease, and dystonia. Unfortunately, previously reported antagonists are not highly selective for specific mAChR subtypes, making it difficult to definitively establish the functional roles and therapeutic potential for individual subtypes of this receptor subfamily. The M1 mAChR is of particular interest as a potential target for treatment of CNS disorders. We now report the discovery of a novel selective antagonist of M1 mAChRs, termed VU0255035 [N-(3-oxo-3-(4-(pyridine-4-yl)piperazin-1-yl)propyl)-benzo[c][1,2,5]thiadiazole-4 sulfonamide]. Equilibrium radioligand binding and functional studies demonstrate a greater than 75-fold selectivity of VU0255035 for M1 mAChRs relative to M2-M5. Molecular pharmacology and mutagenesis studies indicate that VU0255035 is a competitive orthosteric antagonist of M1 mAChRs, a surprising finding given the high level of M1 mAChR selectivity relative to other orthosteric antagonists. Whole-cell patch-clamp recordings demonstrate that VU0255035 inhibits potentiation of N-methyl-d-aspartate receptor currents by the muscarinic agonist carbachol in hippocampal pyramidal cells. VU0255035 has excellent brain penetration in vivo and is efficacious in reducing pilocarpine-induced seizures in mice. We were surprised to find that doses of VU0255035 that reduce pilocarpine-induced seizures do not induce deficits in contextual freezing, a measure of hippocampus-dependent learning that is disrupted by nonselective mAChR antagonists. Taken together, these data suggest that selective antagonists of M1 mAChRs do not induce the severe cognitive deficits seen with nonselective mAChR antagonists and could provide a novel approach for the treatment certain of CNS disorders.

Unique signaling profiles of positive allosteric modulators of metabotropic glutamate receptor subtype 5 determine differences in in vivo activity

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.

Novel allosteric agonists of M1 muscarinic acetylcholine receptors induce brain region-specific responses that correspond with behavioral effects in animal models.

M1 muscarinic acetylcholine receptors (mAChRs) represent a viable target for treatment of multiple disorders of the central nervous system (CNS) including Alzheimers disease and schizophrenia. The recent discovery of highly selective allosteric agonists of M1 receptors has provided a major breakthrough in developing a viable approach for the discovery of novel therapeutic agents that target these receptors. Here we describe the characterization of two novel M1 allosteric agonists, VU0357017 and VU0364572, that display profound differences in their efficacy in activating M1 coupling to different signaling pathways including Ca2+ and β-arrestin responses. Interestingly, the ability of these agents to differentially activate coupling of M1 to specific signaling pathways leads to selective actions on some but not all M1-mediated responses in brain circuits. These novel M1 allosteric agonists induced robust electrophysiological effects in rat hippocampal slices, but showed lower efficacy in striatum and no measureable effects on M1-mediated responses in medial prefrontal cortical pyramidal cells in mice. Consistent with these actions, both M1 agonists enhanced acquisition of hippocampal-dependent cognitive function but did not reverse amphetamine-induced hyperlocomotion in rats. Together, these data reveal that M1 allosteric agonists can differentially regulate coupling of M1 to different signaling pathways, and this can dramatically alter the actions of these compounds on specific brain circuits important for learning and memory and psychosis.