The metabotropic glutamate receptor 7 allosteric modulator AMN082: a monoaminergic agent in disguise?

Metabotropic glutamate receptor 7 (mGluR7) remains the most elusive of the eight known mGluRs primarily because of the limited availability of tool compounds to interrogate its potential therapeutic utility. The discovery of N,N'-dibenzhydrylethane-1,2-diamine dihydrochloride (AMN082) as the first orally active, brain-penetrable, mGluR7-selective allosteric agonist by Mitsukawa and colleagues (Proc Natl Acad Sci USA 102:18712-18717, 2005) provides a means to investigate this receptor system directly. AMN082 demonstrates mGluR7 agonist activity in vitro and interestingly has a behavioral profile that supports utility across a broad spectrum of psychiatric disorders including anxiety and depression. The present studies were conducted to extend the in vitro and in vivo characterization of AMN082 by evaluating its pharmacokinetic and metabolite profile. Profiling of AMN082 in rat liver microsomes revealed rapid metabolism (t(1/2) < 1 min) to a major metabolite, N-benzhydrylethane-1,2-diamine (Met-1). In vitro selectivity profiling of Met-1 demonstrated physiologically relevant transporter binding affinity at serotonin transporter (SERT), dopamine transporter (DAT), and norepinephrine transporter (NET) (323, 3020, and 3410 nM, respectively); whereas the parent compound AMN082 had appreciable affinity at NET (1385 nM). AMN082 produced antidepressant-like activity and receptor occupancy at SERT up to 4 h postdose, a time point at which AMN082 is significantly reduced in brain and plasma while the concentration of Met-1 continues to increase in brain. Acute Met-1 administration produced antidepressant-like activity as would be expected from its in vitro profile as a mixed SERT, NET, DAT inhibitor. Taken together, these data suggest that the reported in vivo actions of AMN082 should be interpreted with caution, because they may involve other mechanisms in addition to mGluR7.

Identification of 3-sulfonylindazole derivatives as potent and selective 5-HT(6) antagonists.

As part of our efforts to develop agents for cognitive enhancement, we have been focused on the 5-HT(6) receptor in order to identify potent and selective ligands for this purpose. Herein we report the identification of a novel series of 3-sulfonylindazole derivatives with acyclic amino side chains as potent and selective 5-HT(6) antagonists. The synthesis and detailed SAR of this class of compounds are reported.

Monoacylglycerol lipase activity is a critical modulator of the tone and integrity of the endocannabinoid system.

Endocannabinoids are lipid molecules that serve as natural ligands for the cannabinoid receptors CB1 and CB2. They modulate a diverse set of physiological processes such as pain, cognition, appetite, and emotional states, and their levels and functions are tightly regulated by enzymatic biosynthesis and degradation. 2-Arachidonoylglycerol (2-AG) is the most abundant endocannabinoid in the brain and is believed to be hydrolyzed primarily by the serine hydrolase monoacylglycerol lipase (MAGL). Although 2-AG binds and activates cannabinoid receptors in vitro, when administered in vivo, it induces only transient cannabimimetic effects as a result of its rapid catabolism. Here we show using a mouse model with a targeted disruption of the MAGL gene that MAGL is the major modulator of 2-AG hydrolysis in vivo. Mice lacking MAGL exhibit dramatically reduced 2-AG hydrolase activity and highly elevated 2-AG levels in the nervous system. A lack of MAGL activity and subsequent long-term elevation of 2-AG levels lead to desensitization of brain CB1 receptors with a significant reduction of cannabimimetic effects of CB1 agonists. Also consistent with CB1 desensitization, MAGL-deficient mice do not show alterations in neuropathic and inflammatory pain sensitivity. These findings provide the first genetic in vivo evidence that MAGL is the major regulator of 2-AG levels and signaling and reveal a pivotal role for 2-AG in modulating CB1 receptor sensitization and endocannabinoid tone.

Preclinical characterization of BRL 44408: antidepressant- and analgesic-like activity through selective alpha2A-adrenoceptor antagonism.

Biogenic amines such as norepinephrine, dopamine, and serotonin play a well-described role in the treatment of mood disorders and some types of pain. As alpha2A-adrenoceptors regulate the release of these neurotransmitters, we examined the therapeutic potential of BRL 44408, a potent (Ki=8.5 nM) and selective (>50-fold) alpha2A-adrenoceptor antagonist (K(B)=7.9 nM). In rats, BRL 44408 penetrated the central nervous system resulting in peak brain and plasma concentrations of 586 ng/g and 1124 ng/ml, respectively. In a pharmacodynamic assay, pretreatment with BRL 44408 to rats responding under a fixed-ratio 30 operant response paradigm resulted in a rightward shift of the clonidine dose-response curve, an effect indicative of alpha2-adrenoceptor antagonism in vivo. Consistent with presynaptic autoreceptor antagonism and tonic regulation of neurotransmitter release, acute administration of BRL 44408 elevated extracellular concentrations of norepinephrine and dopamine, but not serotonin, in the medial prefrontal cortex. Additionally, BRL 44408, probably by inhibiting alpha2A heteroceptors, produced a significant increase in cortical levels of acetylcholine. In the forced swim test and schedule-induced polydipsia assay, BRL 44408 produced an antidepressant-like response by dose-dependently decreasing immobility time and adjunctive water intake, respectively, while in a model of visceral pain, BRL 44408 exhibited analgesic activity by decreasing para-phenylquinone (PPQ)-induced abdominal stretching. Finally, BRL 44408 did not produce deficits in overall motor coordination nor alter general locomotor activity. This preclinical characterization of the neurochemical and behavioural profile of BRL 44408 suggests that selective antagonism of alpha2A-adrenoceptors may represent an effective treatment strategy for mood disorders and visceral pain.

Potent dihydroquinolinone dopamine D2 partial agonist/serotonin reuptake inhibitors for the treatment of schizophrenia.

A dihydroquinolinone moiety was found to be a potent serotonin reuptake inhibitor pharmacophore when combined with certain amines. This fragment was coupled with selected D(2) ligands to prepare a series of dual acting compounds with attractive in vitro profiles as dopamine D(2) partial agonists and serotonin reuptake inhibitors. Structure-activity studies revealed that the linker plays a key role in contributing to D(2) affinity, function, and SRI activity.

Old and new pharmacology: positive allosteric modulation of the alpha7 nicotinic acetylcholine receptor by the 5-hydroxytryptamine(2B/C) receptor antagonist SB-206553 (3,5-dihydro-5-methyl-N-3-pyridinylbenzo[1,2-b:4,5-b']di pyrrole-1(2H)-carboxamide).

The alpha7 nicotinic acetylcholine receptor (nAChR) has been implicated in Alzheimer's disease and schizophrenia, leading to efforts targeted toward discovering agonists and positive allosteric modulators (PAMs) of this receptor. In a Ca2+ flux fluorometric imaging plate reader assay, SB-206553 (3,5-dihydro-5-methyl -N-3-pyridinylbenzo [1, 2-b:4,5 -b']-di pyrrole-1(2H)-carboxamide), a compound known as a 5-hydroxytryptamine(2B/2C) receptor antagonist, produced an 8-fold potentiation of the evoked calcium signal in the presence of an EC(20) concentration of nicotine and a corresponding EC(50) of 1.5 muM for potentiation of EC(20) nicotine responses in GH4C1 cells expressing the alpha7 receptor. SB-206553 was devoid of direct alpha7 receptor agonist activity and selective against other nicotinic receptors. Confirmation of the PAM activity of SB-206553 on the alpha7 nAChR was obtained in patch-clamp electrophysiological experiments in GH4C1 cells, where it failed to evoke any detectable currents when applied alone, yet dramatically potentiated the currents evoked by an EC(20) (17 microM) and EC(100) (124 microM) of acetylcholine (ACh). Native nicotinic receptors in CA1 stratum radiatum interneurons of rat hippocampal slices could also be activated by ACh (200 microM), an effect that was entirely blocked by the alpha7-selective antagonist methyllycaconitine (MLA). These ACh currents were potentiated by SB-206553, which increased the area of the current response significantly, resulting in a 40-fold enhancement at 100 microM. In behavioral experiments in rats, SB-206553 reversed an MK-801 (dizocilpine maleate)-induced deficit in the prepulse inhibition of acoustic startle response, an effect attenuated in the presence of MLA. This latter observation provides further evidence in support of the potential therapeutic utility of alpha7 nAChR PAMs in schizophrenia.

Procognitive and neuroprotective activity of a novel alpha7 nicotinic acetylcholine receptor agonist for treatment of neurodegenerative and cognitive disorders.

The alpha7 nicotinic acetylcholine receptor (nAChR) is a promising target for treatment of cognitive dysfunction associated with Alzheimer's disease and schizophrenia. Here, we report the pharmacological properties of 5-morpholin-4-yl-pentanoic acid (4-pyridin-3-yl-phenyl)-amide [SEN12333 (WAY-317538)], a novel selective agonist of alpha7 nAChR. SEN12333 shows high affinity for the rat alpha7 receptor expressed in GH4C1 cells (K(i) = 260 nM) and acts as full agonist in functional Ca(2+) flux studies (EC(50) = 1.6 microM). In whole-cell patch-clamp recordings, SEN12333 activated peak currents and maximal total charges similar to acetylcholine (EC(50) = 12 microM). The compound did not show agonist activity at other nicotinic receptors tested and acted as a weak antagonist at alpha3-containing receptors. SEN12333 treatment (3 mg/kg i.p.) improved episodic memory in a novel object recognition task in rats in conditions of spontaneous forgetting as well as cognitive disruptions induced via glutamatergic [5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate); MK-801] or cholinergic (scopolamine) mechanisms. This improvement was blocked by the alpha7-selective antagonist methyllycaconitine, indicating that it is mediated by alpha7 activation. SEN12333 also prevented a scopolamine-induced deficit in a passive avoidance task. In models targeting other cognitive domains, including attention and perceptual processing, SEN12333 normalized the apomorphine-induced deficit of prepulse inhibition. Neuroprotection of SEN12333 was demonstrated in quisqualate-lesioned animals in which treatment with SEN12333 (3 mg/kg/day i.p.) resulted in a significant protection of choline acetyltransferase-positive neurons in the lesioned hemisphere. Cumulatively, our results demonstrate that the novel alpha7 nAChR agonist SEN12333 has procognitive and neuroprotective properties, further demonstrating utility of alpha7 agonists for treatment of neurodegenerative and cognitive disorders.

Identification of a series of benzoxazoles as potent 5-HT6 ligands.

As part of our continuing efforts to identify therapeutics for CNS diseases such as schizophrenia and Alzheimer's disease (AD), we have been focused on the 5-HT(6) receptor in order to identify potent and selective ligands as a potential treatment for cognitive dysfunction. Herein we report the identification of a novel series of benzoxazole derivatives as potent 5-HT(6) ligands. The synthesis and detailed SAR of this class of compounds are reported. The compounds have been shown to be full antagonists in a cyclic AMP functional assay.

Identification of a novel series of 3-piperidinyl-5-sulfonylindazoles as potent 5-HT6 ligands.

Cognitive dysfunction is a characteristic of various forms of dementia such as Alzheimer's disease (AD) and a core feature of schizophrenia. As part of our continuing efforts to develop agents for cognitive enhancement, we have been focused on the 5-HT(6) receptor-one of the emerging therapeutic targets in this area. Herein, we report the identification of a novel series of 3-piperidinyl-5-sulfonylindazole derivatives as potent 5-HT(6) antagonists. The synthesis and SAR of this class of compounds are reported.

1-Sulfonylindazoles as potent and selective 5-HT6 ligands.

As part of our continuing efforts to identify therapeutics for CNS diseases, such as schizophrenia and Alzheimer's disease (AD), we have been focused on the 5-HT(6) receptor in an attempt to identify ligands as a potential treatment for cognitive dysfunction. Herein we report the identification of a novel series of 1-sulfonylindazole derivatives as potent and selective 5-HT(6) antagonists. The synthesis and SAR of this class of compounds are reported. Several potent compounds in both binding and cyclase functional assays also display good selectivity, microsomal stability, solubility, and brain penetration as well as low cytochrome P450 inhibition. One compound exemplified in this series showed 24% oral bioavailability and in vivo efficacy in a NOR cognition model at 10mg/kg following an oral administration in rats.

Tetrahydrocarbazole-based serotonin reuptake inhibitor/dopamine D2 partial agonists for the potential treatment of schizophrenia.

A 5-fluoro-tetrahydrocarbazole serotonin reuptake inhibitor (SRI) building block was combined with a variety of linkers and dopamine D2 receptor ligands in an attempt to identify potent D2 partial agonist/SRI molecules for treatment of schizophrenia. This approach has the potential to treat a broader range of symptoms compared to existing therapies. Selected compounds in this series demonstrate high affinity for both targets and D2 partial agonism in cell-based and in vivo assays.

Development of a scintillation proximity assay binding method for the human 5-hydroxytryptamine 6 receptor using intact cells.

We describe the first validated scintillation proximity assay (SPA) binding method for quantitation of (3)H-labeled d-lysergic acid diethylamide (LSD) binding to recombinant human 5-hydroxytryptamine 6 (5-HT(6)) receptors expressed in Chinese hamster ovary (CHO)-Dukx and HeLa cells. The assay was developed using intact cells as a receptor source because membrane fractions derived from these cells failed to discern specific binding from a high level of nonspecific binding. The pharmacological binding profile of seven 5-HT(6) agonists and antagonists using intact CHO-Dukx/5-HT(6) cells in the SPA format was similar to data obtained from a filtration binding assay using HeLa/5-HT(6) membranes. K(i) values and rank order of potencies obtained in the SPA format were consistent with published filtration data as follows: SB-271046 (K(i)=1.9 nM)>methiothepin (K(i)=6.2 nM)>mianserin (K(i)=74.3 nM)>5-methoxytryptamine (5-MeOT, K(i)=111 nM)>5-HT (K(i)=150 nM)>ritanserin (K(i)=207 nM)>5-carboxamidotryptamine (5-CT, K(i)=704 nM). Additional evaluation with four antipsychotics demonstrated strong agreement with previous literature reports. A high specific binding signal and low assay variability, as determined by Z'=0.81+/-0.017, make the SPA format amenable to automation and higher throughput; hence, this assay can be a viable alternative to the more labor-intensive filtration and centrifugation methods.

A regiospecific synthesis of a series of 1-sulfonyl azepinoindoles as potent 5-HT6 ligands.

A regiospecific synthesis of a series of 1-sulfonyl azepinoindoles as potent 5-HT6 ligands is reported.

In vitro screening strategies for nicotinic receptor ligands.

A common historical strategy to the discovery of nicotinic receptor ligands has involved the use of radioligand-binding assays for ligand identification in combination with two-electrode voltage clamp in Xenopus oocytes for electrophysiological characterization. More recently, higher-throughput methodologies have replaced these approaches to accommodate screening of large compound libraries and to provide increased capacity for electrophysiological profiling in mammalian cell lines. We, and others, have implemented cell-based screening assays using the fluorometric imaging plate reader (FLIPR) for primary and lead optimization screening of nicotinic receptor agonists and positive allosteric modulators (PAMs). Using GH4C1 cells expressing the rat alpha7 nicotinic receptor, both acetylcholine and nicotine produced concentration-dependent elevations of intracellular calcium with EC(50) values of 5.5 and 1.6 microM, respectively. PAM activity was robustly detected using the FLIPR assay; for example, the known alpha7 receptor PAM 5-hydroxyindole failed to directly activate the receptor but produced a leftward shift of the nicotine concentration-response curve in combination with a potentiation of the maximum evoked response to nicotine. Electrophysiological confirmation of agonist activity was achieved using the Dynaflow rapid perfusion system and patch clamp in the same GH4C1 cell expression system. Estimated EC(50) values for acetylcholine-evoked currents in GH4C1/alpha7 cells were 55 and 576 microM for area-under-the-curve (AUC) and maximum peak height calculations, respectively. Similarly, PAM activity was confirmed using electrophysiological recordings while also allowing for the mechanistic discrimination of compounds, not possible using the FLIPR assay. Specifically, PAMs capable of slowing the rapid desensitization of alpha7 receptors to different extents were discernable in these studies. Further improvements in the capacity to screen compounds using electrophysiology has been achieved by implementation of high-throughput gigaohm quality recording systems such as the QPatch and PatchXpress where agonist EC(50) values are highly comparable to those obtained using conventional manual patch clamp.

Distinct functional profiles of aripiprazole and olanzapine at RNA edited human 5-HT2C receptor isoforms.

In this study we have functionally characterized aripiprazole (OPC-14597; 7-(4-[4-(2,3-dichlorophenyl)-1-piperazinyl]butyloxy-3,4-dihydro-2-(1H)-quinolinone), the prototype of a new generation antipsychotic drug termed dopamine-serotonin-system stabilizer, in cells expressing 5-hydroxytryptamine2 (5-HT2) receptor subtypes in comparison with olanzapine. In Chinese hamster ovary (CHO) cells stably expressing 5-HT2 receptors, aripiprazole displayed a dual agonist/antagonist profile for 5-HT2C receptor (VNI isoform) mediated calcium signaling (EC50 1070 nM, IC50 281 nM). It exhibited no appreciable 5-HT2A or 5-HT2B agonism, whereas it antagonized 5-HT-stimulated calcium increase at either 5-HT2A or 5-HT2B receptor expressed in CHO cells (IC50s of 369 and 0.46 nM, respectively). In comparison, olanzapine was devoid of agonism but was an antagonist at all three subtypes, with a potency rank order of 5-HT2A (IC50, 2.5 nM)>5-HT2B (47 nM)>5-HT2C (69 nM). In human embryonic kidney (HEK) cells transiently expressing 5-HT2C receptor isoforms, aripiprazole exhibited full agonism at the unedited INI, but partial agonism at the partially edited VNI and fully edited VSV isoforms (EC50s of 571, 1086 and 2099 nM, respectively). A partial antagonism was also observed for aripiprazole at the two edited isoforms (IC50s of 1138 and 1000 nM, respectively). In contrast, while lacking agonist activity at the VNI and VSV, olanzapine showed inverse agonism at the INI isoform (IC50 594 nM), reaching a maximal attenuation of 20%. In addition, olanzapine was a full antagonist at all three isoforms, with a rank order of potency of VNI (IC50, 79 nM)>VSV (101 nM)>INI (3856 nM). The modest 5-HT2A antagonism and 5-HT2C partial agonism, along with reported D2 and 5-HT1A partial agonism, may allow aripiprazole to stabilize the disturbed dopamine-serotonin interplay in schizophrenia with a moderate yet adequate pharmacological intervention. 5-HT2C agonism may also underlie the minimal weight gain seen with aripiprazole.

Rb+ efflux through functional activation of cardiac KCNQ1/minK channels by the benzodiazepine R-L3 (L-364,373).

The slow delayed rectifier K+ current, Iks, encoded by KCNQ1 (KvLQT1)/KCNE1 (mink) genes, contributes to cardiac action potential repolarization and determines the heartbeat rate. Mutations in either KCNQ1 or KCNE1 that reduce Iks cause long-QT syndrome (LQTS), a disorder of ventricular repolarization that results in cardiac arrhythmia and sudden death. A well-recognized potential treatment for LQTS caused by reduction of Iks is to enhance functional activation of cardiac KCNQ1/KCNE1 channels. In the present study, we generated a stable Chinese hamster ovary cell line that expresses KCNQ1/KCNE1 channels confirmed by electrophysiology. Using a pharmacological tool compound R-L3 (L-364,373 [(3-R)-1,3-dihydro-5-(2-fluorophenyl)-3-(1H-indol- 3-ylmethyl)-1-methyl-2H-1,4-benzodiazepin-2-one]), which activates KCNQ1/mink channels, we then developed and validated a non-radioactive rubidium (Rb+) efflux assay that directly measures the functional activity of KCNQ1/KCNE1 channels by atomic absorption spectroscopy. Our results show that the validated Rb+ efflux assay can be used for screening of KCNQ1/KCNE1 openers that potentially treat LQTS in both inherited and acquired forms. In addition, the assay also can be used for evaluation of possible long-QT liability during cardiac selectivity of new chemical entities.

Evaluation of FLIPR Calcium 3 Assay Kit--a new no-wash fluorescence calcium indicator reagent.

We have evaluated the FLIPR Calcium 3 Assay Kit (Calcium 3), a new no-wash fluorescence calcium indicator dye reagent, for the measurement of agonist-stimulated calcium signaling in cells expressing the serotonin 2C (5-HT(2C)), metabotropic glutamate receptor 5 (mGluR5) and the vasopressin 2 (V2) G-protein-coupled receptors. Calcium 3 yielded equivalent (5-HT(2C)) or superior (mGluR5 and V2) sensitivity to FLUO-4 as indexed by the change in fluorescence counts following agonist application. Assay variability, indexed by CV, using Calcium 3 or FLUO-4 was equivalent with 5-HT(2C) receptor responses although CVs were reduced using Calcium 3 in the examples of the mGluR5 and V2 receptors. Receptor pharmacologies based on agonist EC(50) values were identical when either Calcium 3 or FLUO-4 were utilized. Our results validate Calcium 3 as a compelling alternative to FLUO-4 in the choice of fluorescent dye reagent for studying G-protein-coupled receptors, providing the advantage of a homogenous, no-wash assay format.

WAY-855 (3-amino-tricyclo[2.2.1.02.6]heptane-1,3-dicarboxylic acid): a novel, EAAT2-preferring, nonsubstrate inhibitor of high-affinity glutamate uptake.

The pharmacological profile of a novel glutamate transport inhibitor, WAY-855 (3-amino-tricyclo[2.2.1.0(2.6)]heptane-1,3-dicarboxylic acid), on the activity of the human forebrain glutamate transporters EAAT1, EAAT2 and EAAT3 expressed in stable mammalian cell lines and in Xenopus laevis oocytes is presented. WAY-855 inhibited glutamate uptake mediated by all three subtypes in a concentration-dependent manner, with preferential inhibition of the CNS-predominant EAAT2 subtype in both cells and oocytes. IC50 values for EAAT2 and EAAT3 inhibition in cells were 2.2 and 24.5 microM, respectively, while EAAT1 activity was inhibited by 50% at 100 microM (IC50 values determined in oocytes were 1.3 microM (EAAT2), 52.5 microM (EAAT3) and 125.9 microM (EAAT1)). Application of WAY-855 to EAAT-expressing oocytes failed to induce a transporter current, and the compound failed to exchange with accumulated [3H]d-aspartate in synaptosomes consistent with a nonsubstrate inhibitor. WAY-855 inhibited d-aspartate uptake into cortical synaptosomes by a competitive mechanism, and with similar potency to that observed for the cloned EAAT2. WAY-855 failed to agonise or antagonise ionotropic glutamate receptors in cultured hippocampal neurones, or the human metabotropic glutamate receptor subtype 4 expressed in a stable cell line. WAY-855 represents a novel structure in glutamate transporter pharmacology, and exploration of this structure might provide insights into the discrimination between EAAT2 and other EAAT subtypes.

Functional calcium coupling with the human metabotropic glutamate receptor subtypes 2 and 4 by stable co-expression with a calcium pathway facilitating G-protein chimera in Chinese hamster ovary cells.

The objective of the current study was to facilitate functional calcium assays, compatible with the fluorometric imaging plate reader platform, for the human metabotropic glutamate receptor (mGluR) subtypes 2 and 4, by co-expressing each receptor with a G-protein chimera comprising Galphaq with the C-terminal five amino acids replaced with those from Galphai3 (GqGi3). Transfection of GqGi3 into previously validated stable CHO cell lines expressing mGluR2 or mGluR4 allowed for the selection of new double transfectants in which application of L-glutamate and other mGluR agonists resulted in calcium coupling with a high signal:noise ratio (maximal changes in relative fluorescence units up to 20,000). The rank order of agonist potency for the stimulation of calcium mobilization in the mGluR2/GqGi3 stable cell line was LY354740>L-CCG-I=DCG-IV>L-glutamate>/=(2R,4R)-APDC>/=(1S,3R)-ACPD. In the mGluR4/GqGi3 stable cell line the rank order of agonist potency was L-AP4>L-SOP>/=ACPT-I=L-CCG-I>/=L-glutamate=(R,S)-PPG. By comparison, equivalent potency orders and a significant correlation in functional activities were observed when the same compounds were profiled in [35S]GTPgammaS binding assays for each mGluR subtype. These results validate the use of functional calcium assays, amenable to high-throughput applications on the fluorometric imaging plate reader, for the mGluR2 and mGluR4 subtypes when co-expressed in stable cell lines with the GqGi3 chimera.

Validation of an atomic absorption rubidium ion efflux assay for KCNQ/M-channels using the ion Channel Reader 8000.

M-channels (M-current), encoded by KCNQ2/3 K(+) channel genes, have emerged as novel drug targets for a number of neurological disorders. The lack of direct high throughput assays combined with the low throughput of conventional electrophysiology (EP) has impeded rapid screening and evaluation of K(+)-channel modulators. Development of a sensitive and efficient assay for the direct measurement of M-current activity is critical for identifying novel M-channel modulators and subsequent investigation of their therapeutic potential. Using a stable CHO cell line expressing rat KCNQ2/3 K(+) channels confirmed by EP, we have developed and validated a nonradioactive rubidium (Rb(+)) efflux assay in a 96-well plate format. The Rb(+) efflux assay directly measures the activity of functional channels by atomic absorption spectroscopy using the automated Ion Channel Reader (ICR) 8000. The stimulated Rb(+) efflux from KCNQ2/3-expressing cells was blocked by the channel blockers XE991 and linopirdine with IC(50) values of 0.15 microM and 1.3 microM, respectively. Twelve compounds identified as KCNQ2/3 openers were further assessed in this assay, and their EC(50) values were compared with those obtained with EP. A higher positive correlation coefficient between these two assays (r = 0.60) was observed than that between FlexStation membrane potential and EP assays (r = 0.23). To simplify the assay and increase the throughput, we demonstrate that EC(50) values obtained by measuring Rb(+) levels in the supernatant are as robust and consistent as those obtained from the ratio of Rb(+) in supernatant/lysate. By measuring the supernatant only, the throughput of ICR8000 in an eight-point titration is estimated to be 40 compounds per day, which is suitable for a secondary confirmation assay.