Fluorometric Imaging Plate Reader Assay Research Articles

Pharmacological Comparative Characterization of REL-1017 (Esmethadone-HCl) and Other NMDAR Channel Blockers in Human Heterodimeric N-Methyl-D-Aspartate Receptors.

Excessive Ca2+ currents via N-methyl-D-aspartate receptors (NMDARs) have been implicated in many disorders. Uncompetitive NMDAR channel blockers are an emerging class of drugs in clinical use for major depressive disorder (MDD) and other neuropsychiatric diseases. The pharmacological characterization of uncompetitive NMDAR blockers in clinical use may improve our understanding of NMDAR function in physiology and pathology. REL-1017 (esmethadone-HCl), a novel uncompetitive NMDAR channel blocker in Phase 3 trials for the treatment of MDD, was characterized together with dextromethorphan, memantine, (±)-ketamine, and MK-801 in cell lines over-expressing NMDAR subtypes using fluorometric imaging plate reader (FLIPR), automated patch-clamp, and manual patch-clamp electrophysiology. In the absence of Mg2+, NMDAR subtypes NR1-2D were most sensitive to low, sub-μM glutamate concentrations in FLIPR experiments. FLIPR Ca2+ determination demonstrated low μM affinity of REL-1017 at NMDARs with minimal subtype preference. In automated and manual patch-clamp electrophysiological experiments, REL-1017 exhibited preference for the NR1-2D NMDAR subtype in the presence of 1 mM Mg2+ and 1 μM L-glutamate. Tau off and trapping characteristics were similar for (±)-ketamine and REL-1017. Results of radioligand binding assays in rat cortical neurons correlated with the estimated affinities obtained in FLIPR assays and in automated and manual patch-clamp assays. In silico studies of NMDARs in closed and open conformation indicate that REL-1017 has a higher preference for docking and undocking the open-channel conformation compared to ketamine. In conclusion, the pharmacological characteristics of REL-1017 at NMDARs, including relatively low affinity at the NMDAR, NR1-2D subtype preference in the presence of 1 mM Mg2+, tau off and degree of trapping similar to (±)-ketamine, and preferential docking and undocking of the open NMDAR, could all be important variables for understanding the rapid-onset antidepressant effects of REL-1017 without psychotomimetic side effects.

Identification and target-pathway deconvolution of FFA4 agonists with anti-diabetic activity from Arnebia euchroma (Royle) Johnst

FFA4 is a novel therapeutic target for the treatment of metabolic diseases, such as type II diabetes. However, there are still few ligands with structural diversity, selectivity and high potency, and the signaling pathway downstream of FFA4 remains to be poorly characterized. In this study, a high performance liquid chromatography-corona charged aerosol detector (HPLC-CAD) combined with label-free dynamic mass redistribution (DMR) method was introduced to guide the discovery of FFA4 agonists from Arnebia euchroma (Royle) Johnst. Ten compounds were identified as FFA4 agonists and structure-activity relationship was obtained. Among them, shikonin displayed the most potent activity with pEC50 value of 6.02 ± 0.19. The activity of shikonin was confirmed by FLIPR (fluorometric imaging plate reader) assay. Signaling pathways of FFA4 were explored in HT-29 cells endogenously expressing FFA4 using shikonin and known FFA4 agonists α-linolenic acid (ALA) and TUG891. Multiple pathways included Gq/11-PLC-Ca2+-PKC, RohA, JNK, p38 MAPK, Gi/o and PI3K signaling but may not involve Gs signaling triggered by shikonin, ALA and TUG891. Besides, shikonin, TUG891 and ALA could induce ERK1/2 and AKT phosphorylation in HT-29 cells. Moreover, anti-diabetes effects of shikonin were evaluated on the glucose intolerance in diabetic db/db mice. Shikonin reduced plasma glucose level, suggesting that it had the potential in treatment of type II diabetes. The agonists identified in this study provided structure guidance for FFA4 drug design. This study was also useful for understanding FFA4 pharmacology and its biological function.

Generation and phenotype analysis of CysLTR1 L118F mutant mice.

Cysteinyl leukotrienes (CysLTs) are a group of eicosanoids that regulate the pathogenesis of various human diseases, mainly by signaling through the cysteinyl leukotriene receptor 1 (CysLTR1). The aim of this study was to generate and examine the phenotype of CysLTR1 L118F mutant mice. CysLTR1 L118F mutant mice were generated by the simultaneous microinjection of single guide RNA, Cas9 messenger RNA, and donor plasmid into fertilized mouse eggs. The morphological and behavioral characteristics of the resultant CysLTR1 L118F mutant mice were analyzed using an animal phenotype analysis platform, which included the assessment of body length, tail length, grip strength, and locomotor activity. Immunoprecipitation coupled with mass spectrometry was performed to identify CysLTR1-interacting proteins, and the intracellular calcium levels were determined using fluorometric imaging plate reader assays. The body length and tail length of CysLTR1 L118F mutant mice were significantly increased compared with wild-type mice. In addition, the grip strength and locomotor activity were remarkably elevated in L118F mutant mice compared with wild-type mice. Only three proteins were found to interact with both wild-type and CysLTR1 L118F proteins, whereas 4 and 13 additional proteins interacted exclusively with wild-type and mutant CysLTR1, respectively. Lastly, the responsiveness of cardiac muscle cells to CysLTs were significantly impaired by the L118F substitution in CysLTR1 proteins. The CysLTR1 L118F point mutation induced significant changes in the mouse morphology and behavior, which might be mediated by alterations of its protein interaction profile.

A FLIPR Assay for Discovery of GABAA Receptor Modulators of Natural Origin.

A fluorometric imaging plate reader (FLIPR) assay utilizing Chinese hamster ovary (CHO) cells stably transfected with GABAA receptors of α 1 β 2 γ 2 subunit composition was evaluated and validated for rapid screening of plant extract libraries and efficient localization of active compounds in extracts. Validation was performed with pure compounds and extracts known to contain allosteric GABAA receptor modulators. Plants extracts that had been previously reported as active in an assay using Xenopus laevis oocytes transiently expressing GABAA receptors of α 1 β 2 γ 2 subunit composition were also active in the FLIPR assay. A protocol for HPLC-based activity profiling was developed, whereby separations of 0.4 - 1.2 mg of extracts on an analytical HPLC column were found to be sufficient for the sensitivity of the bioassay. The protocol successfully localized the activity of known GABAergic natural products, such as magnolol in Magnolia officinalis, valerenic acid in Valeriana officinalis, and piperine in Piper nigrum extract. EC50 values of compounds (magnolol: 4.81 ± 1.0 µM, valerenic acid: 12.56 ± 1.2 µM, and piperine: 5.76 ± 0.7 µM) were found to be comparable or lower than those reported using Xenopus oocyte assays.

Gonadotropin-releasing hormone receptor-targeted paclitaxel-degarelix conjugate: synthesis and in vitro evaluation.

To increase the selectivity of chemotherapeutic agents, receptor-mediated tumor-targeting approaches have been developed. Here, degarelix [Ac-D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(Cbm)-Leu-ILys-Pro-D-Ala-NH2], a gonadotropin-releasing hormone antagonist, was employed as a targeting moiety for paclitaxel (PTX). Five PTX-degarelix conjugates were synthesized, in which PTX was attached via disulfide bond to the different position in the degarelix sequence. All of the PTX-degarelix conjugates exhibited a half-life greater than 10 h determined in human serum. A fluorometric imaging plate reader assay showed that the conjugates LK-MY-9 and LK-MY-10 had an antagonism efficacy similar to that of degarelix. The in vitro cytostatic effects of the conjugates were determined by a (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay, and the 50% inhibitory concentration value of the conjugates on 3T3 mouse embryonic fibroblast cells were one order of magnitude higher than the 50% inhibitory concentration values of the conjugates on MCF-7 human breast cancer cells and HT-29 human colon cancer cells. Receptor saturation tests further demonstrated that pre-incubation of the cells with degarelix reduced the efficacy of LK-MY-10 in a concentration-dependent manner. In conclusion, degarelix is a valid and stable moiety that has great potential for targeting chemotherapy drugs.

Discovery of Novel P2Y14 Agonist and Antagonist Using Conventional and Nonconventional Methods

P2Y14 is a member of the pyrimidinergic GPCR family. UDP-Glc has been previously shown to activate human P2Y14, whereas UDP was unable to activate the receptor. In this study, the authors used conventional and nonconventional methods to further characterize P2Y14 and its ligands. Conventional calcium mobilization and nonconventional cellular impedance functional assays revealed that UMP and UDP selectively activated HEK cells coexpressing P2Y14 and Gα(qi5). In the impedance assays, the presence of exogenous Gα(qi5) resulted in agonist-induced Gq signaling, whereas in the absence of exogenous Gα(qi5), the signal was indicative of Gi. The authors established the first P2Y14 membrane filtration binding assay using a novel optimized expression vector and [(3)H]UDP as radioligand. UDP-Glc, UMP, and UDP dose dependently inhibited [(3)H]UDP binding in the binding assay, and saturation analysis revealed that UDP bound P2Y14 with a K(D) = 10 nM and a B(max) = 110 pmol/mg. The authors screened a phosphonate library and identified compound A, which inhibited UDP-Glc-mediated calcium signaling in the fluorometric imaging plate reader assay (IC(50) = 2.3 µM) and competed for [(3)H]UDP binding in the novel binding assay with a K(i) = 1280 nM.

Pharmacological characterization of the ghrelin receptor antagonist, GSK1614343 in rat RC-4B/C cells natively expressing GHS type 1a receptors

A novel growth hormone secretagogues type 1a (GHS1a) receptors antagonist (2R)-N'-[3,5- bis(trifluoromethyl)phenyl]-2-[(8aR)-hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl]-2-(3- pyridinyl)ethanohydrazide (GSK1614343) was functionally characterised in rat pituitary adenoma cell line, RC-4B/C endogenously expressing GHS1a receptors. The antagonism profile of GSK1614343 was compared with that of 6-[(4-fluorophenyl)oxy]-2-methyl-3-{[(3S)-1-(1-methylethyl)-3- piperidinyl]methyl}-4(3H)-quinazolinone (YIL-781) another ghrelin receptor antagonist recently published. The activity of both compounds was also evaluated at rat recombinant GHS1a receptors. The characterization of the two antagonists was performed by intracellular calcium mobilization measurements by using fluorometric imaging plate reader (FLIPR) technology and inositol phosphate (IP) turnover measurements by [3H]-IP accumulation assay. RC-4B/C and U2-OS cells transiently transduced with rat GHS1a receptors virus were used. In RC-4B/C cells, GSK1614343 and YIL-781, depressed the ghrelin maximal response in FLIPR assay as result of hemi-equilibria phenomenon. When using the [3H]-IP accumulation assay both compounds behaved as competitive antagonist with pKB values of 8.03 for GSK1614343 and 7.54 for YIL-781. In rat recombinant receptor, GSK1614343 and YIL-781 inhibited the calcium response induced by ghrelin with pIC50 values of 7.90 and 8.27, respectively. GSK1614343 and YIL-781 did not show intrinsic activity in both endogenously expressed and recombinant rat GHS1a receptors. The new ghrelin receptor antagonist GSK1614343 is a potent competitive antagonist in rat pituitary RC-4B/C cells endogenously expressing GHS1a receptors when equilibrium conditions between ligand and receptor are reached in the test assay. GSK1614343 represents a useful tool to investigate the physiological relevance of the ghrelin system in rat models.

High-Throughput Screening for Kv1.3 Channel Blockers Using an Improved FLIPR-Based Membrane-Potential Assay

Voltage-gated K(+) channels are potential drug targets for an increasing number of disease indications. Searching for compounds that modulate K(+) channel activities by high-throughput screening (HTS) is becoming a standard approach in the drug discovery effort. Here the authors report an improved fluorometric imaging plate reader (FLIPR) membrane potential assay for Kv1.3 K(+) channel HTS. They have found that the Chinese hamster ovary (CHO) cells have endogenous membrane electrogenic transporters that contribute to maintaining membrane potential. Blocking the recombinant K(+) channels in the overexpressing CHO cell line hardly changed the membrane potential. Inhibition of the endogenous transporters is essential to achieve the required assay robustness. The authors identified the optimal assay conditions and designed a simple assay format. After an HTS campaign using this assay, various chemical series of Kv1.3 channel blockers have been identified and confirmed by the automated electrophysiological IonWorks assay. The correlation in dose response between FLIPR and IonWorks was established by biophysical modeling and experimental data. After characterization using patch-clamp recording, both use-dependent and use-independent compounds were identified. Some compounds possess nanomolar potency, indicating that the FLIPR assay is effective for successfully identifying K(+) channel blockers as novel drug candidates.

In vitro and in vivo characterization of A‐940894: a potent histamine H4 receptor antagonist with anti‐inflammatory properties

The histamine H4 receptor is widely expressed in cells of immune origin and has been shown to play a role in a variety of inflammatory processes mediated by histamine. In this report, we describe the in vitro and in vivo anti-inflammatory properties of a potent histamine H4 receptor antagonist, A-940894 (4-piperazin-1-yl-6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-d]pyrimidin-2-ylamine). We have analysed the pharmacological profile of A-940894 at mouse native, rat recombinant and human recombinant and native, histamine H4 receptors by radioligand binding, calcium mobilization, mast cell shape change, eosinophil chemotaxis assays and in the mouse model of zymosan-induced peritonitis. A-940894 potently binds to both human and rat histamine H4 receptors and exhibits considerably lower affinity for the human histamine H1, H2 or H3 receptors. It potently blocked histamine-evoked calcium mobilization in the fluorometric imaging plate reader assays and inhibited histamine-induced shape change of mouse bone marrow-derived mast cells and chemotaxis of human eosinophils in vitro. In a mouse mast cell-dependent model of zymosan-induced peritonitis, A-940894 significantly blocked neutrophil influx and reduced intraperitoneal prostaglandin D2 levels. Finally, A-940894 has good pharmacokinetic properties, including half-life and oral bioavailability in rats and mice. These data suggest that A-940894 is a potent and selective histamine H4 receptor antagonist with pharmacokinetic properties suitable for long-term in vivo testing and could serve as a useful tool for the further characterization of histamine H4 receptor pharmacology.

Pharmacology of α7 nicotinic acetylcholine receptor mediated extracellular signal‐regulated kinase signalling in PC12 cells

Neuronal nicotinic acetylcholine receptors (nAChR) can modulate cell survival and memory processing. The involvement of specific nAChR subtypes in downstream signalling events has been ill defined thus far, because of a lack of subtype-selective ligands. In this study, we investigated activation and modulation of alpha7 nAChR-mediated phosphorylation of extracellular signal-regulated kinases (ERK1/2) in PC12 cells, using selective agonists and positive allosteric modulators. We used undifferentiated PC12 cells endogenously expressing alpha7 nAChR for both biochemical and functional studies. ERK phosphorylation changes were measured by using a novel In-Cell Western procedure. alpha7 nAChR-mediated Ca(2+) signalling was determined by using the fluorometric imaging plate reader assay. Robust induction of ERK phosphorylation followed exposure of PC12 cells to the selective agonist PNU-282987 in the presence of the alpha7 nAChR modulator PNU-120596. ERK phosphorylation was transient and was attenuated by the selective antagonist methyllycaconitine. Consistent with allosteric modulation of alpha7 nAChRs, PNU-120596 enhanced both the agonist potency and efficacy in activating ERK. Moreover, alpha7 nAChR agonists could be quantitatively differentiated based on their potency in activating ERK signalling. The rank order of potencies correlated fairly well with the corresponding binding K(i) values of these alpha7 nAChR agonists. The present work extends previous observations demonstrating the involvement of alpha7 nAChRs in ERK1/2 phosphorylation in PC12 cells. The In-Cell Western procedure allowed a detailed investigation of alpha7 nAChR function and downstream ERK signalling in response to agonist and allosteric modulators.

Old and New Pharmacology: Positive Allosteric Modulation of the α7 Nicotinic Acetylcholine Receptor by the 5-Hydroxytryptamine2B/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.

Use of FLIPR Membrane Potential Dyes for Validation of High-Throughput Screening with the FLIPR and μARCS Technologies: Identification of Ion Channel Modulators Acting on the GABAA Receptor

Fluorometric imaging plate reader (FLIPR) membrane potential dyes (FMP-Red-Dye and FMP-Blue-Dye) were evaluated for the detection of compounds acting either as positive allosteric modulators or agonists on the GABA(A) receptor (GABA(A)R). A stable HEK293 cell line with constitutive expression of the rat GABA(A)R alpha1, beta2, and gamma2 genes was used to establish a functional high-throughput screening (HTS) assay based on measurement of the membrane potential change in living cells. The assay was validated with the FLIPR technology for identification of agonists and positive allosteric modulators using GABA and diazepam as model compounds. The FMP-Red-Dye showed better performance than the FMP-Blue-Dye, and the effects induced by GABA and diazepam were comparable to electrophysiology data. Subsequently, the assay was also validated with an ultra-HTS approach known as microarrayed compound screening (microARCS). The LOPAC library was used in a test screen for an initial assessment of the technology. Finally, the FLIPR and microARCS technologies were tested with a larger screening campaign. A focused library of 3520 putative positive modulators was tested with the FLIPR assay, and a diverse subset of 84,480 compounds was selected for screening with the microARCS technology. All hits were subjected to verification using the FLIPR technology, and confirmed hits were subsequently evaluated by EC50 determination. Finally, selected hits were further confirmed with electrophysiology testing.

Functional Selectivity of Melanocortin 4 Receptor Peptide and Nonpeptide Agonists: Evidence for Ligand-Specific Conformational States

Agonists of the melanocortin 4 (MC4) receptor have potential pharmaceutical benefit in the treatment of obesity and sexual dysfunction. In this study, we have compared the ability of a number of peptide and nonpeptide agonists to activate a FLAG-tagged human MC4 (FMC4) receptor, as measured by both cAMP accumulation and calcium mobilization using a fluorometric imaging plate reader (FLIPR). In addition, we have analyzed the ability of these agonists to cause receptor internalization, as measured by fluorescence-activated cell sorting analysis. The endogenous agonist alpha-melanocortin-stimulating hormone (alpha-MSH) increased cAMP accumulation, calcium mobilization, and receptor internalization in a dose-dependent manner in human embryonic kidney 293 cells expressing the FMC4 receptor. The activity of the other agonists varied considerably in these assays, and overall, the potency and intrinsic activity of the agonists in the cAMP accumulation assays did not correlate with their potency or intrinsic activity in either the FLIPR or receptor internalization assays. Agonists could be clearly separated into two functional classes based on their structure. Peptide agonists beta-MSH, des-acetyl-alpha-MSH, and [Nle(4), D-Phe(7)]-alpha-melanocortin-stimulating hormone exhibited 80 to 112% of the maximal alpha-MSH response in cAMP accumulation and 62 to 96% in FLIPR assays and were able to cause 75 to 118% of receptor internalization induced by alpha-MSH. Conversely, although the nonpeptide agonists exhibited 73 to 149% of the alpha-MSH response in the cAMP accumulation assays, they were significantly impaired in the FLIPR (7-40%) and receptor internalization (-5-38%) assays. These findings demonstrate an important difference in activation and internalization of the MC4 receptor by nonpeptide versus peptide agonists and provides evidence of agonist-specific conformational states.

Miniaturization of Intracellular Calcium Functional Assays to 1536-Well Plate Format Using a Fluorometric Imaging Plate Reader

The measurement of intracellular calcium response transients in living mammalian cells is a popular functional assay for identification of agonists and antagonists to receptors or channels of pharmacological interest. In recent years, advances in fluorescence-based detection techniques and automation technologies have facilitated the adaptation of this assay to 384-well microplate format high-throughput screening (HTS) assays. However, the cost and time required performing the intracellular calcium HTS assays in the 384-well format can be prohibitive for HTS campaigns of greater than 1 x 10(6) wells. For these reasons, it is attractive to miniaturize intracellular calcium functional assays to the 1536-well microplate format, where assay volumes and plate throughput can be decreased by several fold. The focus of the research described in this article is the miniaturization of an intracellular calcium assay to 1536-well plate format. This was accomplished by modifying the hardware and software of a fluorometric imaging plate reader (FLIPR) to enable transfer of nanoliters of test compound directly to a 1536-well assay plate, and measure the resulting calcium response from all 1536 wells simultaneously. An intracellular calcium functional assay against the rat muscarinic acetylcholine receptor subtype 1 (rmAchR1) G-protein coupled receptor (GPCR) was miniaturized and executed on this modified instrument. In experiments measuring the activity of known muscarinic receptor agonists and antagonists, the miniaturized FLIPR assay gave EC(50) and IC(50) values and rank order potency comparable to the 384-well format assays. Calculated Z' factors for the miniaturized agonist and antagonist assays were, respectively, 0.56 +/- 0.21 and 0.53 +/- 0.22, which were slightly higher (Z'(agonist) = 0.55 +/- 0.33) and lower (Z'(antagonist) = 0.70 +/- 0.18) than the corresponding values in the 384-well assays. A mock agonist HTS campaign against the muscarinic receptor in miniaturized format was able to identify all wells spiked with the rmAchR1 agonist carbachol.

Pharmacological characterization of canine bradykinin receptors in prostatic culture and in isolated prostate

The objective of this study was to characterize pharmacologically bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg, BK) receptors in the canine prostate. Primary cultures of canine prostate stromal (PS) and epithelial cells (PE) were established and then characterized using cell-specific antibodies (actin, vimentin and cytokeratin). Cultured cells were assayed for BK receptors using fluorometric imaging plate reader assays. In addition, isolated strips of the canine prostate were studied for BK-induced isometric contraction. PS cells were labeled only with anti-actin and -vimentin antibodies, while the anti-cytokeratin antibodies labeled only the PE cells. In cultured prostate cells, the BK receptor 2 (B2)-preferring agonist BK induced mobilization of intracellular Ca(2+) in a concentration-dependent manner with potencies (log[EC(50)]mid R:PE, pEC(50)) of 8.72+/-0.12 in PS and 8.75+/-0.06 in PE cells. In contrast, the BK receptor 1 (B1)-selective agonist [des-Arg(9)]BK (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe) did not elicit any significant effect (pEC(50)<5) on Ca(2+) responses. BK agonism (10 nm) was inhibited by HOE-140 (D-arginyl-L-arginyl-L-prolyl-trans-4-hydroxy-L-prolylglycyl-3-(2-thienyl)-L-alanyl-L-seryl-D-1,2,3,4-tetrahhydro-3-isoquinolinecarbonyl-L-(2a,3b,7ab)-octahydro-1H-indole-2-carbonyl-L-arginine), a B2-selective antagonist, with a log[IC(50)] (pIC(50)) of 8.11+/-0.19 and 9.23+/-0.20 in PS and PE cells, respectively. [des-Arg(10)]HOE-140 (d-arginyl-l-arginlyl-l-prolyl-trans-4-hydroxy-l-prolylglycyl-3-(2-thienyl)-L-alanyl-L-seryl-D-1,2,3,4-tetrahydro-3-isoquinolinecarbonyl-L-(2a, 3b,7ab)-octahydro-1H-indole-2-carbonyl), a B1-selective antagonist, displayed weak antagonism with pIC(50) values of 4.87+/-0.23 and 6.38+/-0.16 in PS and PE cells, respectively. Isolated tissue strips of the canine prostate contracted to BK (10 microm) but not to [des-Arg(9)]BK (10 microm). BK-induced contractility was attenuated by HOE-140 (1 microm). In conclusion, canine prostates express functional B2 receptors, with no apparent B1 receptor subtypes.

Characterization of the mouse cold-menthol receptor TRPM8 and vanilloid receptor type-1 VR1 using a fluorometric imaging plate reader (FLIPR) assay.

1. TRPM8 (CMR1) is a Ca(2+)-permeable channel, which can be activated by low temperatures, menthol, eucalyptol and icilin. It belongs to the transient receptor potential (TRP) family, and therefore is related to vanilloid receptor type-1 (VR1, TRPV1). We tested whether substances which are structurally related to menthol, or which produce a cooling sensation, could activate TRPM8, and compared the responses of TRPM8 and VR1 to these ligands. 2. The effects of 70 odorants and menthol-related substances on recombinant mouse TRPM8 (mTRPM8), expressed in HEK293 cells, were examined using a FLIPR assay. In all, 10 substances (linalool, geraniol, hydroxycitronellal, WS-3, WS-23, FrescolatMGA, FrescolatML, PMD38, CoolactP and Cooling Agent 10) were found to be agonists. 3. The EC(50) values of the agonists defined their relative potencies: icilin (0.2+/-0.1 microM)>FrescolatML (3.3+/-1.5 microM) > WS-3 (3.7+/-1.7 microM) >(-)menthol (4.1+/-1.3 microM) >frescolatMAG (4.8+/-1.1 microM) > cooling agent 10 (6+/-2.2 microM) >(+)menthol (14.4+/-1.3 microM) > PMD38 (31+/-1.1 microM) > WS-23 (44+/-7.3 microM) > Coolact P (66+/-20 microM) > geraniol (5.9+/-1.6 mM) > linalool (6.7+/-2.0 mM) > eucalyptol (7.7+/-2.0 mM) > hydroxycitronellal (19.6+/-2.2 mM). 4. Known VR1 antagonists (BCTC, thio-BCTC and capsazepine) were also able to block the response of TRPM8 to menthol (IC(50): 0.8+/-1.0, 3.5+/-1.1 and 18+/-1.1 microM, respectively). 5. The Ca(2+) response of hVR1-transfected HEK293 cells to the endogenous VR1 agonist N-arachidonoyl-dopamine was potentiated by low pH. In contrast, menthol- and icilin-activated TRPM8 currents were suppressed by low pH. 6. In conclusion, in the present study, we identified 10 new agonists and three antagonists of TRPM8. We found that, in contrast to VR1, TRPM8 is inhibited rather than potentiated by protons.

A cell-based microarrayed compound screening format for identifying agonists of G-protein-coupled receptors

The identification of agonist and antagonist leads for G-protein-coupled receptors (GPCRs) is of critical importance to the pharmaceutical and biotechnology industries. We report on the utilization of a novel, high-density, well-less screening platform known as microarrayed compound screening (μARCS) that tests 8640 compounds in the footprint of a standard microtiter plate for the identification of novel agonists for a specific G-protein-coupled receptor. Although receptors coupled to the Gα q protein can readily be assessed by fluorescence-based Ca 2+ release measurements, many GPCRs that are coupled to Gα s or Gα i/o proteins are not amenable to functional evaluation in such a high-throughput manner. In this study, the human dopamine D 4.4 receptor, which normally couples through the Gα i/o protein to inhibit adenylate cyclase and to reduce levels of intracellular cAMP, was coupled to intracellular Ca 2+ release by stably coexpressing this receptor with a chimeric G αqo5 protein in HEK-293 cells. In μARCS format, the cells expressing D 4.4 receptor and Gα qo5 protein were preloaded with fluo-4, cast into a 1% agarose gel, placed above the compound sheets, and imaged successively using a ViewLux charge-coupled device imaging system. Dopamine and other agonists evoked an increase in fluorescence response that appeared as bright spots in a time- and concentration-dependent manner. Utilizing this technology, a library of 260,000 compounds was rapidly screened and led to the identification of several novel agonists. These agonists were further characterized using a fluorometric imaging plate reader assay. Excellent confirmation rates coupled with enhanced efficiency and throughput enable μARCS to serve as an alternative platform for the screening and identification of novel GPCR agonists.

Comparative analysis of human and rat S1P 5 (edg8): differential expression profiles and sensitivities to antagonists

Five guanine nucleotide-binding protein-coupled receptors (S1P 1–5) for the lysophospholipid mediator sphingosine 1-phosphate (S1P) have thus far been described. Whereas tissue distribution and functional properties of the human S1P 1–4 genes are well characterized, only limited functional and expression data are available for S1P 5, todate. Northern blot analysis indicated that human S1P 5 (hS1P 5) is an alternatively spliced gene, with a 5.4-kb transcript that is predominantly expressed in peripheral tissues, and a 2.4-kb transcript expressed in brain, spleen, and peripheral blood leucocytes. In contrast, rat S1P 5 (rS1P 5) was exclusively detected in brain and skin. Expression of hS1P 5 and rS1P 5 in mammalian CHO-K1 or HEK293 cells conferred onto the cells the ability to mobilize intracellular calcium as determined by a functional Fluorometric Imaging Plate Reader assay, when challenged with S1P and dihydro S1P, respectively. Applying a lipid library with 200 bioactive lipids in a functional Fluorometric Imaging Plate Reader assay did not reveal additional agonists. However, both receptors exhibited differential sensitivity towards the S1P- and lysophosphatidic acid-receptor antagonist, suramin: rS1P 5-mediated intracellular calcium mobilization was partly inhibited by suramin ( ic 50: 5800 μM), whereas hS1P 5 was completely antagonized ( ic 50: 130 μM). Both receptors were sensitive towards inhibition with the related drug (8,8′-(carbonylbis(imino-3,1-phenylene))bis(1,3,5-naphthalenetrisulfonic acid)) but ic 50 values differed significantly (340 μM for hS1P 5, 4000 μM for rS1P 5). In addition, rS1P 5 displayed antiproliferative effects in transfected CHO-K1 and HEK293 cells in contrast to hS1P 5. Taken together, our data imply that differences between hS1P 5 and rS1P 5 will be an important point to be considered in the development of selective receptor antagonists.

Identification of nonpeptidic urotensin II receptor antagonists by virtual screening based on a pharmacophore model derived from structure-activity relationships and nuclear magnetic resonance studies on urotensin II.

The vasoactive cyclic 11-amino acid peptide urotensin II (U-II) has recently been discovered as the endogenous ligand of the orphan G-protein-coupled receptor GPR14. As U-II might be involved in the regulation of cardiovascular homeostasis and pathology, a nonpeptidic GPR14/U-II antagonist is of considerable basic and therapeutic interest. We have performed structure-activity relationship studies on U-II by investigating 25 peptide analogues to mobilize intracellular calcium in GPR14-transfected CHO cells, demonstrating that only the side chains of the residues Trp-7, Lys-8, and Tyr-9 are required for receptor recognition and activation. The solution structure of U-II derived by nuclear magnetic resonance has served as a structural template for a three-dimensional three point pharmacophore query for the virtual screening of the Aventis compound repository for nonpeptidic U-II receptor antagonists. Highly active lead compounds of six different scaffold classes could be identified, antagonizing the biological activity of U-II in vitro. The most potent compound identified by the virtual screening approach, 1-(3-carbamimidoyl-benzyl)-4-methyl-1H-indole-2-carboxylic acid (naphthalen-1-ylmethyl)amide, reveals an IC(50) of 400 nM in a functional fluorometric imaging plate reader assay and constitutes a promising lead.