Characterization of vabicaserin (SCA-136), a selective 5-hydroxytryptamine 2C receptor agonist.

The 5-hydroxytryptamine 2C (5-HT(2C)) receptor subtype has received considerable attention as a target for drug discovery, having been implicated in a wide variety of disorders. Here, we describe the in vitro pharmacological profile of the novel 5-HT(2C) receptor-selective agonist vabicaserin [(-)-4,5,6,7,9,9a,10,11,12,12a-decahydrocyclopenta[c] [1,4]diazepino[6,7,1-ij]quinoline hydrochloride] (SCA-136), including a comprehensive strategy to assess 5-HT(2B) receptor selectivity using diverse preparations and assays of receptor activation. Vabicaserin displaced (125)I-(2,5-dimethoxy)phenylisopropylamine binding from human 5-HT(2C) receptor sites in Chinese hamster ovary cell membranes with a K(i) value of 3 nM and was >50-fold selective over a number of serotonergic, noradrenergic, and dopaminergic receptors. Binding affinity determined for the human 5-HT(2B) receptor subtype using [(3)H]5HT was 14 nM. Vabicaserin was a potent and full agonist (EC(50), 8 nM; E(max), 100%) in stimulating 5-HT(2C) receptor-coupled calcium mobilization and exhibited 5-HT(2A) receptor antagonism and 5-HT(2B) antagonist or partial agonist activity in transfected cells, depending on the level of receptor expression. In rat stomach fundus and human colonic longitudinal muscle endogenously expressing 5-HT(2B) receptors, vabicaserin failed to induce a 5-HT(2B) receptor-dependent contraction and produced a rightward shift of the 5-HT and α-methyl-5-HT concentration-response curves in these preparations, respectively, consistent with 5-HT(2B) competitive antagonism. Likewise, vabicaserin failed to induce a 5-HT(2B) receptor-mediated contraction in arteries from deoxycorticosterone acetate-salt-treated rats, a model of hypersensitized 5-HT(2B) receptor function, and produced a rightward shift in the 5-HT-induced response that was consistent with 5-HT(2B) receptor antagonism. In summary, vabicaserin is a novel, potent, and selective 5-HT(2C) receptor agonist.

Development of an improved IP(1) assay for the characterization of 5-HT(2C) receptor ligands.

The 5-hydroxytryptamine 2C (5-HT(2C)) receptor is a member of the serotonin 5-HT(2) subfamily of G-protein-coupled receptors signaling predominantly via the phospholipase C (PLC) pathway. Stimulation of phosphoinositide (PI) hydrolysis upon 5-HT(2C) receptor activation is traditionally assessed by measuring inositol monophosphate (IP(1)) using time-consuming and labor-intensive anion exchange radioactive assays. In this study, we have developed and optimized a cellular IP(1) assay using homogeneous time-resolved fluorescence (HTRF), a fluorescence resonance energy transfer (FRET)-based technology (Cisbio; Gif sur Yvette, France). The measurement is simple to carry out without the cumbersome steps associated with radioactive assays and may therefore be used as an alternative tool to evaluate PI hydrolysis activated by 5-HT(2C) agonists. In Chinese hamster ovary (CHO) cells stably expressing 5-HT(2C) receptors, characterization of 5-HT(2C) agonists with the HTRF platform revealed a rank order of potency (EC(50), nM) comparable to that from intracellular calcium mobilization studies measured by the fluorometric imaging plate reader (FLIPR). A similar rank order of potency was seen with conventional radioactive PI assay with the exception of 5-HT. Lastly, the new assay data correlated better with agonist-induced calcium responses in FLIPR (R(2) = 0.78) than with values determined by radioactive IP(1) method (R(2) = 0.64). Our study shows that the HTRF FRET-based assay detects IP(1) with good sensitivity and may be streamlined for high-throughput (HTS) applications.

A novel high-throughput screening assay for HCN channel blocker using membrane potential-sensitive dye and FLIPR.

Hyperpolarization-activated cation nonselective (HCN) channels represent an interesting group of targets for drug development. In this study, the authors report the development of a novel membrane potential-sensitive dye (MPSD) assay for HCN channel modulators that has been miniaturized into 384-well fluorescent imaging plate reader (FLIPR) high-throughput screening (HTS) format. When optimized (by cell plating density, plate type, cell recovery from cryopreservation), the well-to-well signal variability was low, with a Z' = 0.73 and coefficient of variation = 6.4%, whereas the MPSD fluorescence signal amplitude was -23,700 +/- 1500 FLIPR(3) relative fluorescence units (a linear relationship was found between HCN1 MPSD fluorescence signal and the cell plating density) and was completely blocked by 30 microM ZD7288. The assay tolerated up to 1% DMSO, inclusion of which did not significantly change the signal kinetics or amplitude. A single-concentration screening of an ion channel-focused library composed of 4855 compounds resulted in 89 HCN1 blocker hits, 51 of which were subsequently analyzed with an 8-point concentration-response analysis on the IonWorks HT electrophysiology platform. The correlation between MPSD and the electrophysiology assay was moderate, as shown by the linear regression analysis (r(2) = 0.56) between the respective IC(50)s obtained using these 2 assays. The reported HTS-compatible HCN channel blocker assay can serve as a tool in drug discovery in the pursuit of HCN channel isoform-selective small molecules that could be used in the development of clinically relevant compounds.

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.

Tissue distribution and functional analyses of the constitutively active orphan G protein coupled receptors, GPR26 and GPR78.

GPR26 and GPR78 are orphan GPCRs (oGPCRs) that share 51% amino acid sequence identity and are widely expressed in selected tissues of the human brain as well as the developing and adult mouse brain. Investigation of the functional activity of GPR26 and GPR78 via expression in HEK293 cells showed that both proteins are constitutively active and coupled to elevated cAMP production. Accordingly, in yeast, GPR26 demonstrated apparent agonist-independent coupling to a chimeric Gpa1 protein in which the 5 C-terminal amino acids were from Galphas. A comparison of the proteins revealed an atypical glutamine residue in GPR78 in place of the conserved arginine residue (R3.50) in the so-called DRY box. Site-directed mutants R3.50 in GPR26 were constructed and retained their constitutive activity suggesting that these 2 receptors activate G proteins in a manner that is distinct from other group 1 GPCRs.

Stable expression of adenylyl cyclase 2 leads to the functional rescue of human 5-HT6 receptor in a CHODUKX cell line.

INTRODUCTION: The generation and selection of recombinant cell lines specifically designed to express high picomolar levels of heterologous G-protein-coupled receptors can lead to loss of ligand-dependent functional activity. As a result, the clonal selection of a suitable host model and/or lower receptor expression levels within the same cell system becomes important especially when a functional assay is necessary to evaluate the pharmacological potencies of ligands at the receptor site. To address this question, we examined the utility of various signal transducers to restore the functional capacity of a high expressing human 5-HT(6) receptor CHODUKX system. METHODS: The plasmids for human 5-HT(6) receptor and full-length human G(s), G(olf) and rat adenylyl cyclase isoforms 2 (rAC2) and 5 were obtained by PCR. The h5-HT(6) receptor pHTop plasmid was stably transfected into a CHODUKX cell line to generate an h5-HT(6) expressing clone. h5-HT(6) CHODUKX cells were transfected with signaling components and functional cAMP responses measured. rAC2 was selected to generate a double stable h5-HT(6) receptor/rAC2 pHTop CHODUKX line. RESULTS: The h5-HT(6) receptor CHODUKX line was a high receptor expressor (>2 pmol/mg protein) but an extremely poor ligand-dependent functional responder, failing to produce the appropriate cAMP signal upon addition of selective agonists. We found that stable co-expression of rAC2 with h5-HT(6) receptor in the CHODUKX cell line displayed dose-dependent cAMP accumulation following agonist treatment. The pharmacological profile of several agonists in the h5-HT(6) receptor/rAC2 cell line was consistent with an h5-HT(6)-like receptor-mediated event. DISCUSSION: We provide evidence for restoration of functional capacity in a heterologous G(s)-coupled 5-HT(6)/AC2 CHODUKX expression system. We discuss the broader value of a stable AC2-expressing CHODUKX cell line in which the generation of high expressing GPCR receptor/AC2 lines can retain their functional responsiveness and provide pharmacological drug comparisons between the same host line for screening purposes and measurement of multiple cellular parameters.

WAY-100635 antagonist-induced plasticity of 5-HT receptors: regulatory differences between a stable cell line and an in vivo native system.

We present evidence that the 5-hydroxytryptamine(1A) (5-HT(1A)) receptor antagonist, N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]-ethyl}-N-(2-pyridinyl)cyclohexanecarboxamide (WAY-100635), can induce receptor internalization in a human (h)5-HT(1A) receptor Chinese hamster ovary (CHO-K1) cell system. Exposure of h5-HT(1A) CHO cells to WAY-100635 decreased the cell-surface h5-HT(1A) receptor density in a way that was both time (24-72 h) and concentration (1-100 nm) dependent.[(3)H]WAY-100635 and [(3)H]8-hydroxy-dipropylaminotetralin ([(3)H]8-OH-DPAT) saturation analyses demonstrated a significant reduction (50-60%) in total h5-HT(1A) receptor number in the WAY-100635-treated (100 nm; 72 h) compared with control cells. In WAY-100635-treated cells, the 8-OH-DPAT-mediated inhibition of forskolin (FSK)-stimulated cAMP accumulation was right-shifted and the maximal inhibitory response of 8-OH-DPAT was impaired compared with control cells. Similar results were obtained for 8-OH-DPAT-mediated Ca(2+) mobilization after WAY-100635 treatment. h5-HT(1A) receptors labeled with [(3)H]WAY-100635, as well as [(3)H]4-(2'-Methoxy)-phenyl-1-[2'-(N-2''-pyridinyl)-p-fluorobenzamido]ethyl-piperazine (MPPF), exhibited a time-dependent rate of cellular internalization that was blocked by endocytotic suppressors and was pertussis-toxin insensitive. In contrast, quantitative autoradiographic studies demonstrated that chronic treatment of rats with WAY-100635 for two weeks produced a region-specific increase in the 5-HT(1A) receptor density. In conclusion, prolonged exposure of an h5-HT(1A) cell-based system to the 5-HT(1A) antagonist, WAY-100635, induced a paradoxical internalization of cell surface receptor resulting in depressed functional activity. This suggests that an antagonist can influence 5-HT(1A) receptor recycling in vitro differently to in vivo regulatory conditions.

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.

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.

Characterization of the 5-HT6 receptor coupled to Ca2+ signaling using an enabling chimeric G-protein.

We examined the feasibility of coupling the 5-HT(6) receptor to a Ca(2+) signaling read-out using a chimeric G-protein, comprising of G(alphaq) with the C-terminal five amino acids from G(alphas), to facilitate assays on the fluorometric imaging plate reader (FLIPR). Using a transient transfection assay in human embryonic kidney (HEK) cells, Ca(2+) signaling in response to serotonin (5-HT) was facilitated by co-transfection of the 5-HT(6) receptor with the G(alphaq)/G(alphas) chimera, but not with the 5-HT(6) receptor alone or with a similar chimera incorporating the C-terminal five amino acids of G(alphai3). A series of agonist concentration-response curves were constructed using the 5-HT(6)-G(alphaq)/G(alphas) signaling assay generating the following rank order of agonist potency; 5-methoxytryptamine (EC(50), 9 nM)=5-HT (12 nM)=2-methyl 5-HT (13 nM)>tryptamine (86 nM)=5-carboxamidotryptamine (5-CT) (119 nM)>>lisuride (>1 microM). In comparison, essentially identical EC(50) values were observed for the stimulation of cAMP accumulation with the same compounds; 5-methoxytryptamine (EC(50), 6 nM)=5-HT (6 nM)=2-methyl 5-HT (15 nM)>tryptamine (91 nM)=5-CT (153 nM)>lisuride (>350 nM). Clozapine and SB 271046 both produced a concentration-dependent antagonism of the 5-HT-stimulated Ca(2+) response with IC(50) values of 45 and 11 nM, respectively. In contrast, aripiprazole, a recently launched atypical anti-psychotic with a novel mechanism of action described as a dopamine/serotonin stabilizer, was essentially devoid of 5-HT(6) receptor antagonist activity. Our results demonstrate that a FLIPR-based Ca(2+) signaling assay is a feasible approach to the functional characterization of 5-HT(6) receptor ligands. Moreover, the equivalent coupling efficiency, as indexed by agonist potency, observed using this system compared with the native coupling assay to cAMP suggests that the C-terminal five amino acids of G(alphas) are the major determinant for the receptor/G-protein interaction of the 5-HT(6) receptor subtype.

The C-terminus of Gi family G-proteins as a determinant of 5-HT(1A) receptor coupling.

Using a universal signaling assay employing G-protein chimeras comprising the C-terminal five amino acids of Gi1/2, Gi3, Go, and Gz fused to Gq, the calcium mobilizing G-protein, we explored the role of the C-terminus of Gi family G-proteins as a determinant for 5-HT(1A) receptor functional coupling. Co-expression of the 5-HT(1A) receptor with each of the Gq/Gi family chimeras resulted in a concentration-dependent increase in calcium upon addition of 5-HT, although the coupling efficiency differed dramatically. Gq/Gi3 resulted in the most efficient coupling based on both potency and relative maximum response to 5-HT. Gq/Go also produced efficient coupling in terms of relative 5-HT efficacy (76% of the Gq/Gi3 maximum response), although 5-HT exhibited 4-fold lower agonist potency, and Gq/Gz and Gq/Gi1/2 conferred poor functional coupling. Agonist potencies and relative efficacies determined for a number of 5-HT(1A) receptor agonists using Gq/Gi3 coupling were significantly weaker than those described previously for coupling through the native G-protein. These results indicate the C-terminus of Gi3 as an important determinant for coupling to the 5-HT(1A) receptor, while the reduced functional agonist activities suggest additional motifs participate in receptor/G-protein coupling.