Parkinson's 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, (Ϯ)-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.Metabotropic glutamate receptors (mGluRs) play important roles in a broad range of central nervous system functions and have therapeutic potential in a variety of neurological and psychiatric disorders (Niswender et al., 2005). mGluRs are G protein-coupled receptors (GPCRs) classified into three major groups, groups I, II, and III, based on their sequence homology, signal transduction profile, and ligand binding specificity. The group III mGluRs (mGluRs 4, 6, 7,

Modulators of metabotropic glutamate receptor subtype 5 (mGluR5) may provide novel treatments for multiple central nervous system (CNS) disorders, including anxiety and schizophrenia. Although compounds have been developed to better understand the physiological roles of mGluR5 and potential usefulness for the treatment of these disorders, there are limitations in the tools available, including poor selectivity, low potency, and limited solubility. To address these issues, we developed an innovative assay that allows simultaneous screening for mGluR5 agonists, antagonists, and potentiators. We identified multiple scaffolds that possess diverse modes of activity at mGluR5, including both positive and negative allosteric modulators (PAMs and NAMs, respectively). 3-Fluoro-5-(3-(pyridine-2-yl)-1,2,4-oxadiazol-5-yl)benzonitrile (VU0285683) was developed as a novel selective mGluR5 NAM with high affinity for the 2-methyl-6-(phenylethynyl)-pyridine (MPEP) binding site. VU0285683 had anxiolytic-like activity in two rodent models for anxiety but did not potentiate phencyclidine-induced hyperlocomotor activity. (4-Hydroxypiperidin-1-yl)(4-phenylethynyl)phenyl)methanone (VU0092273) was identified as a novel mGluR5 PAM that also binds to the MPEP site. VU0092273 was chemically optimized to an orally active analog, N-cyclobutyl-6-((3-fluorophenyl)ethynyl)nicotinamide hydrochloride (VU0360172), which is selective for mGluR5. This novel mGluR5 PAM produced a dose-dependent reversal of amphetamine-induced hyperlocomotion, a rodent model predictive of antipsychotic activity. Discovery of structurally and functionally diverse allosteric modulators of mGluR5 that demonstrate in vivo efficacy in rodent models of anxiety and antipsychotic activity provide further support for the tremendous diversity of chemical scaffolds and modes of efficacy of mGluR5 ligands. In addition, these studies provide strong support for the hypothesis that multiple structurally distinct mGluR5 modulators have robust activity in animal models that predict efficacy in the treatment of CNS disorders.

The G-protein activated, inward-rectifying potassium (K + ) channels, "GIRKs", are a family of ion channels (K ir 3.1-K ir 3.4) that has been the focus of intense research interest for nearly two decades. GIRKs are comprised of various homo-and heterotetrameric combinations of four different subunits. These subunits are expressed in different combinations in a variety of regions throughout the central nervous system and in the periphery. The body of GIRK research implicates GIRK in processes as diverse as controlling heart rhythm, to effects on reward/addiction, to modulation of response to analgesics. Despite years of GIRK research, very few tools exist to selectively modulate GIRK channels' activity and until now no tools existed that potently and selectively activated GIRKs. Here we report the development and characterization of the first truly potent, effective, and selective GIRK activator, ML297 (VU0456810). We further demonstrate that ML297 is active in two in vivo models of epilepsy, a disease where up to 40% of patients remain with symptoms refractory to present treatments. The development of ML297 represents a truly significant advancement in our ability to selectively probe GIRK's role in physiology as well as providing the first tool for beginning to understand GIRK's potential as a target for a diversity of therapeutic indications.

Activators of M 1 muscarinic acetylcholine receptors (mAChRs) may provide novel treatments for schizophrenia and Alzheimer's disease. Unfortunately, the development of M 1 -active compounds has resulted in nonselective activation of the highly related M 2 to M 5 mAChR subtypes, which results in doselimiting side effects. Using a functional screening approach, we identified several novel ligands that potentiated agonist activation of M 1 with low micromolar potencies and induced 5-fold or greater leftward shifts of the acetylcholine (ACh) concentrationresponse curve. These ligands did not compete for binding at the ACh binding site, indicating that they modulate receptor activity by binding to allosteric sites. The two most selective compounds, cyclopentyl 1,progressive shifts in ACh affinity at M 1 that were consistent with their effects in a functional assay, suggesting that the mechanism for enhancement of M 1 activity by these compounds is by increasing agonist affinity. These compounds were strikingly different, however, in their ability to potentiate responses at a mutant M 1 receptor with decreased affinity for ACh and in their ability to affect responses of the allosteric M 1 agonist, 1-[1Ј-(2-tolyl)-1,4Ј-bipiperidin-4-yl]-1,3-dihydro-2H-benzimidazol-2-one. Furthermore, these two compounds were distinct in their abilities to potentiate M 1 -mediated activation of phosphoinositide hydrolysis and phospholipase D. The discovery of multiple structurally distinct positive allosteric modulators of M 1 is an exciting advance in establishing the potential of allosteric modulators for selective activation of this receptor. These data also suggest that structurally diverse M 1 potentiators may act by distinct mechanisms and differentially regulate receptor coupling to downstream signaling pathways.The psychotic and cognitive symptoms of neuropsychiatric disorders such as schizophrenia and Alzheimer's disease (AD) remain serious unmet medical challenges. Patients with schizophrenia exhibit a constellation of symptoms that include positive, negative, and cognitive symptom clusters. Although current antipsychotic agents are effective in reducing positive symptoms such as hallucinations and delusions in most patients, negative symptoms such as anhedonia and blunted affect, as well as deficits in cognitive function, are not effectively treated with current medications (Vohora, 2007). In addition to the unmet medical needs of schizophrenia, the devastating cognitive and neuropsychiatric symptoms characteristic of AD present urgent needs for new therapeutic interventions (Saddichha and Pandey, 2008).

Positive allosteric modulators (PAMs) of metabotropic glutamate receptor subtype 5 (mGlu 5 ) have emerged as an exciting new approach for the treatment of schizophrenia and other central nervous system (CNS) disorders. Of interest, some mGlu 5 PAMs act as pure PAMs, only potentiating mGlu 5 responses to glutamate whereas others [allosteric agonists coupled with PAM activity (ago-PAMs)] potentiate responses to glutamate and have intrinsic allosteric agonist activity in mGlu 5 -expressing cell lines. All mGlu 5 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 mGlu 5 pure PAMs that are devoid of detectable agonist activity and structurally related mGlu 5 ago-PAMs that activate mGlu 5 alone in cell lines. Studies of mGlu 5 PAMs in cell lines revealed that ago-PAM activity is dependent on levels of mGlu 5 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 mGlu 5 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 mGlu 5 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.

The group III metabotropic glutamate receptors (mGluRs) represent a family of presynaptically expressed G-protein-coupled receptors (GPCRs) with enormous therapeutic potential; however, robust cellular assays to study their function have been difficult to develop. We present here a new assay, compatible with traditional high-throughput screening platforms, to detect activity of pharmacological ligands interacting with G i/o -coupled GPCRs, including the group III mGluRs 4, 7, and 8. The assay takes advantage of the ability of the G␤␥ subunits of G i and G o heterotrimers to interact with G-protein regulated inwardly rectifying potassium channels (GIRKs), and we show here that we are able to detect the activity of multiple types of pharmacophores including agonists, antagonists, and allosteric modulators of several distinct GPCRs. Using GIRK-mediated thallium flux, we perform a side-by-side comparison of the activity of a number of commercially available compounds, some of which have not been extensively evaluated because of the previous lack of robust assays at each of the three major group III mGluRs. It is noteworthy that several compounds previously considered to be general group III mGluR antagonists have very weak activity using this assay, suggesting the possibility that these compounds may not effectively inhibit these receptors in native systems. We anticipate that the GIRKmediated thallium flux strategy will provide a novel tool to advance the study of G i/o -coupled GPCR biology and promote ligand discovery and characterization.The metabotropic glutamate receptors (mGluRs) are among the most abundantly expressed receptors in the mammalian central nervous system and are thought to play critical roles in regulating activity in a broad range of CNS circuits (Conn and Pin, 1997). Despite an increasing appreciation of the involvement of mGluRs in CNS function, however, progress in understanding the signaling pathways and functional properties of some members of the mGluR family has been relatively slow. This is especially true for the group III mGluRs, which include mGluRs 4, 6, 7, and 8.

ML297 was recently identified as a potent and selective small molecule agonist of G-protein-gated inwardly rectifying K + (GIRK/Kir3) channels. Here, we show ML297 selectively activates recombinant neuronal GIRK channels containing the GIRK1 subunit in a manner that requires phosphatidylinositol-4,5-bisphosphate (PIP 2 ), but is otherwise distinct from receptor-induced, G-protein-dependent channel activation. Two amino acids unique to the pore helix (F137) and second membrane-spanning (D173) domain of GIRK1 were identified as necessary and sufficient for the selective activation of GIRK channels by ML297. Further investigation into the behavioral effects of ML297 revealed that in addition to its known antiseizure efficacy, ML297 decreases anxiety-related behavior without sedative or addictive liabilities. Importantly, the anxiolytic effect of ML297 was lost in mice lacking GIRK1. Thus, activation of GIRK1-containing channels by ML297 or derivatives may represent a new approach to the treatment of seizure and/or anxiety disorders.electrophysiology | structure-activity relationship S ignal transduction involving inhibitory (G i/o ) G proteins titrates the excitability of neurons, cardiac myocytes, and endocrine cells, influencing behavior, cardiac output, and energy homeostasis (1). G-protein-gated inwardly rectifying potassium (K + ) (GIRK/Kir3) channels are a common effector for G i/odependent signaling pathways in the heart and nervous system (2, 3). Polymorphisms and mutations in human GIRK channels have been linked to arrhythmias, hyperaldosteronism (and associated hypertension), schizophrenia, sensitivity to analgesics, and alcohol dependence (1).GIRK channels are activated by binding of the G protein Gβγ subunit (1-3). Gβγ binding strengthens channel affinity for phosphatidylinositol-4,5-bisphosphate (PIP 2 ), a necessary cofactor for channel gating (4, 5). GIRK channels are also activated in a G-protein-independent manner by ethanol (6, 7), volatile anesthetics (8, 9), and naringin (10). Many psychoactive and clinically relevant compounds with other primary molecular targets inhibit GIRK channels, albeit at relatively high doses (1, 11). The lack of selective GIRK channel modulators, and in particular, drugs that discriminate among GIRK channel subtypes, has hampered investigation into their physiological relevance and therapeutic potential.GIRK channels are homo-and heterotetramers formed by GIRK1, GIRK2, GIRK3, and GIRK4 subunits (2, 3). GIRK subunits exhibit overlapping but distinct cellular expression patterns, potentially yielding multiple channel subtypes (1). Although it cannot form functional homomers (12), GIRK1 is an integral subunit of the cardiac GIRK channel and most neuronal GIRK channels (13,14). GIRK1 confers robust basal and receptordependent activity to GIRK heteromers, attributable in part to unique residues in the pore and second transmembrane domain (15-17). The intracellular C-terminal domain also contributes to the potentiating influence of GIRK1 on channel activity, likely due to th...

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, Parkinson's 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 M 1 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 M 1 mAChRs, termed VU0255035 [N-(3-oxo-3-(4-(pyridine-4-yl) 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 M 1 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.Muscarinic acetylcholine receptors (mAChRs) are G protein-coupled receptors (GPCRs) that are widely expressed in the central nervous system (CNS) and are critical for the modulation of activity in multiple brain circuits (Langmead et al., 2008). Previous studies suggest that mAChRs play important roles in a broad range of CNS functions, including