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.

Insecticide resistance is a growing threat to mosquito control programs around the world, thus creating the need to discover novel target sites and target-specific compounds for insecticide development. Emerging evidence suggests that mosquito inward rectifier potassium (Kir) channels represent viable molecular targets for developing insecticides with new mechanisms of action. Here we describe the discovery and characterization of VU041, a submicromolar-affinity inhibitor of Anopheles (An.) gambiae and Aedes (Ae.) aegypti Kir1 channels that incapacitates adult female mosquitoes from representative insecticide-susceptible and -resistant strains of An. gambiae (G3 and Akron, respectively) and Ae. aegypti (Liverpool and Puerto Rico, respectively) following topical application. VU041 is selective for mosquito Kir channels over several mammalian orthologs, with the exception of Kir2.1, and is not lethal to honey bees. Medicinal chemistry was used to develop an analog, termed VU730, which retains activity toward mosquito Kir1 but is not active against Kir2.1 or other mammalian Kir channels. Thus, VU041 and VU730 are promising chemical scaffolds for developing new classes of insecticides to combat insecticide-resistant mosquitoes and the transmission of mosquito-borne diseases, such as Zika virus, without harmful effects on humans and beneficial insects.

KCC2, a neuronal-specific K-Cl cotransporter, plays a major role in maintaining intracellular Cl ؊ concentration in neurons below its electrochemical equilibrium potential, thus favoring robust GABA hyperpolarizing or inhibitory responses. The pharmacology of the K-Cl cotransporter is dominated by loop diuretics such as furosemide and bumetanide, molecules used in clinical medicine because they inhibit the loop of Henle Na-K-2Cl cotransporter with much higher affinity. To identify molecules that affect KCC2 activity, we developed a fluorescence-based assay suitable for highthroughput screening (HTS) and used the assay to screen a library of 234,000 small molecules. We identified a large number of molecules that either decrease or increase the activity of the cotransporter. Here, we report the characterization of a small number of inhibitors, some of which inhibit KCC2 activity in the submicomolar range without substantially affecting NKCC1 activity. Using medicinal chemistry, we synthesized a number of variants, tested their effect on KCC2 function, and provide an analysis of structure/activity relationships. We also used one of the compounds to demonstrate competitive inhibition in regard to external [K ؉ ] versus noncompetitive inhibition in respect to external [Cl ؊ ].fluorescence ͉ high-througput screening ͉ Na-K-2Cl cotransporter ͉ thallium C ation-chloride cotransporters have received much attention in the past decade for the role they play in the nervous system. In particular KCC2, a neuronal-specific K-Cl cotransporter, has been shown to modulate inhibitory neurotransmission both in the brain and in the spinal cord. By reducing the intracellular Cl Ϫ concentration below its thermodynamic equilibrium potential in central neurons, KCC2 strengthens synaptic inhibition. Several studies have shown that loss of KCC2 function in central neurons results in the development of CNS hyperexcitability (1-3). Moreover, a paper from Coull and coworkers also showed that disinhibition in the dorsal horn of the spinal cord triggered by peripheral nerve injury was mediated by a significant decrease in KCC2 expression (4). Local blockade or knockdown of spinal KCC2 in intact rats markedly reduced the nocicepetive threshold. By linking change in KCC2 function to disruption of Cl Ϫ homeostasis in lamina I neurons, the study clearly linked the loss of KCC2 function to a loss of inhibition in this region of the spinal cord and to an increase in chronic pain. The relationship between KCC2 and nerve or spinal cord injury has been confirmed in subsequent studies that have shown that inflammatory response due to intraplantar injection of formalin (5, 6) or hind paw injection of complete Freund's adjuvant (7), or to loose ligation of the sciatic nerve (8), or contusive spinal cord injury at T9 (9), all led to a down-regulation of cotransporter expression. Altogether, these studies also point to a role of KCC2 in neuropathic pain.The pharmacology of the cation-chloride cotransporters is dominated by 2 classes of drugs: the thiazide a...

Metabotropic glutamate receptor 7 (mGlu7) is a member of the group III mGlu receptors (mGlus), encompassed by mGlu4, mGlu6, mGlu7, and mGlu8. mGlu7 is highly expressed in the presynaptic active zones of both excitatory and inhibitory synapses, and activation of the receptor regulates the release of both glutamate and GABA. mGlu7 is thought to be a relevant therapeutic target for a number of neurological and psychiatric disorders, and polymorphisms in the GRM7 gene have been linked to autism, depression, ADHD, and schizophrenia. Here we report two new pan-group III mGlu positive allosteric modulators, VU0155094 and VU0422288, which show differential activity at the various group III mGlus. Additionally, both compounds show probe dependence when assessed in the presence of distinct orthosteric agonists. By pairing studies of these nonselective compounds with a synapse in the hippocampus that expresses only mGlu7, we have validated activity of these compounds in a native tissue setting. These studies provide proof-of-concept evidence that mGlu7 activity can be modulated by positive allosteric modulation, paving the way for future therapeutics development.