Functional Characterization and Screening of G αi -Coupled GPCRs using AlphaScreen camp Detection Technology

Transcription

1 Functional Characterization and Screening of G αi -Coupled GPCRs using AlphaScreen camp Detection Technology Nathalie Bouchard, Erik Robitaille and Dean Wenham

2 1 Introduction G-protein coupled receptors (GPCRs) are a large family of cell surface transmembrane receptors that represent an extremely tractable class of drug targets. GPCRs are essentially membrane-associated proteins that induce the activation of G-proteins. GPCRs couple to three main families of G α subunits: G αi/o, G αs and G αq. The specificity of G α subunit coupling is dependent upon the GPCR and its cellular environment. G αs and G αi subunits act through the camp pathway by respectively activating or inhibiting adenylate cyclase, an enzyme catalyzing the conversion of ATP to camp. The measurement of camp has been used predominantly as a method to monitor G αs and G αi coupled receptor activation. Traditionally, radioimmunoassay/enzyme linked immunosorbent assays (RIA/ELISA) have been used, and more recently, homogeneous radiometric techniques such as scintillation proximity assays (Amersham Pharmacia Biotech, Bucks, UK) and Flashplates (, Boston, MA, USA) have been developed. In order to exclude the use of radioactivity from screening laboratories for the reasons of safety and economy, various nonradiometric platforms have been developed. AlphaScreen (, Boston, MA, USA) is a bead-based non-radioactive Amplified Luminescent Proximity Homogeneous Assay developed for high-throughput screening (HTS) applications. When a biological interaction brings the beads together, a cascade of chemical reactions acts to produce a greatly amplified signal. To demonstrate the application of the AlphaScreen-based camp detection technology for G αi coupled receptors, the G αi-coupled 5-hydroxytryptamine- 1A (5-HT 1A) serotonergic receptor has been chosen as a model system. In this study, the utility of the AlphaScreen-based camp Assay kit for the pharmacological characterization and the potential high-throughput screening of G αi-coupled receptors have been demonstrated. 2 Principles of AlphaScreen When a biological interaction brings the beads together, a cascade of chemical reactions acts to produce a greatly amplified signal. On laser excitation, a photosensitizer in the Donor bead converts ambient oxygen to a more excited singlet state. The singlet state oxygen molecules diffuse to react with a thioxene derivative in the Acceptor bead, generating chemiluminescence at 370 nm that further activates fluorophores contained in the same bead. The fluorophores subsequently emit light at nm. In the absence of a specific biological interaction, the singlet state oxygen molecules produced by the Donor bead go undetected without the close proximity of the Acceptor bead. As a result no signal is produced. The basis of this assay is the competition between endogenous camp and exogenously added biotincamp. The capture of camp is achieved by using a specific antibody conjugated to Acceptor beads. Signal generation only occurs if biotin-camp is captured since Donor beads are coated with streptavidin. Cells are stimulated to either increase or decrease intracellular camp levels followed by a combined cell lysis / detection step. 2

3 3 Materials and Methods Materials: The AlphaScreen camp Assay Kit (PerkinElmer Life Sciences, Boston, MA, USA; Cat. # M, assay points) was used in this study. Culture media, HBSS and HEPES were from Invitrogen Canada (Burlington, ON) and all other reagents were obtained from Sigma-Aldrich Canada Ltd (Oakville, ON). Cell Culture: CHO-h5-HT 1A transfected cells (B max=1.1pmol/mg protein) were maintained in culture with MEM media supplemented with 10% FBS and 2 mm glutamine and passaged using 1:3 1:20 dilutions. Cells were detached using PBS: EDTA, centrifuged and resuspended in stimulation buffer immediately before use at 0.6 million cells/ml (3000 cells/well). 2-Step Assay: 1. Stimulation step in 10 µl HBSS containing 5 mm HEPES ph 7.4, 0.1% BSA and 0.5 mm IBMX 2. Detection step with 15 µl of a mix of biotin-camp (10 nm final) + Donor beads (20 µg/ml final) in 5 mm HEPES ph 7.4, 0.1% BSA and 0.3% Tween-20, incubated 1 hr in the dark and read on AlphaQuest All assays were performed in white, opaque 384-well plates in a final volume of 25 µl. Standard curve: 5 µl Acceptor beads (15 µg/ml final) 5 µl camp (1 µm to 10 pm final) 30 min incubation at RT in the dark Detection Agonist stimulation: 5 µl cells + Acceptor beads (15 µg/ml final) 2.5 µl agonist 2.5 µl forskolin 30 min incubation at RT in the dark Detection Antagonist stimulation: 5 µl cells + Acceptor beads (15 µg/ml final) 2.5 µl antagonist (30 min incubation) 2.5 µl mix agonist + forskolin 30 min incubation at RT in the dark Detection Data analysis: Data were analyzed using GraphPad Prism software; the data points and vertical error bars on the graphs depict the mean ± SD of a representative experiment performed in triplicate. Each experiment was performed on at least three separate occasions. pk b values were calculated according to a derivation of the Cheng & Prusoff equation: K b = IC 50/(1+A/EC 50). pa 2 values were calculated using Schild Analysis of agonist dose-response curves performed in presence of varying concentrations of antagonist. Assay performance was calculated using the Z factor using the equation Z = 1- [ (3xSD stimulated + 3xSD control)/ mean stimulated-mean control ] 4 Results Forskolin stimulated camp in CHO-h5-HT 1A cells (pec 50 = 5.6 ± 0.06) where camp levels were increased from ± (basal) to 0.9 ± 0.28 pmol/well (100 µm forskolin) (slide 5) The full agonists 5-HT and R(+)8-OH-DPAT inhibited forskolin (10 µm) stimulated camp in a concentration dependent manner with pec 50= 7.8 ± 0.24 and 7.7 ± 0.23 respectively. Buspirone acted as a partial agonist (E max = 44.7 ± 13.4% relative to R(+)8-OH-DPAT) with lower potency (pec 50 = 6.8 ± 0.17) (slide 6) The 5-HT 1A selective antagonists spiperone (pk b = 8.9 ± 0.23), NAN-190 (pk b = 9.4 ± 0.28) and S(-)propranolol (pk b = 6.8 ± 0.25) all inhibited the R(+)8-OH-DPAT (100nM) response with the expected pharmacology. Clozapine (5-HT 6/7 selective) and metergoline (5-HT 1d/5-HT 2 selective) failed to inhibit the response (slide 7) Schild analysis of NAN-190 and spiperone yielded slopes close to unity and pa 2 values of 8.8 ± 0.23 and 8.4 ± 0.17 respectively (slide 8) In DMSO tolerance studies, the R(+)8-OH-DPAT response tolerated DMSO up to 2% (slide 9) In variability / precision studies, where Z values were measured, forskolin (10 µm) stimulation (Z = 0.77), R(+)8-OH-DPAT (100 nm) stimulation (Z = 0.54) and spiperone (1 µm) inhibition (Z = 0.34) all demonstrated a robustness suitable for screening (slide 10) 3

4 5 camp Standard Curve and Cell Number Optimization (A) Standard curve to camp used for interpolation of camp concentrations in the cell based assay (B) Forskolin concentration effect curves performed over a range of CHO-h5-HT 1A cells densities (C) Forskolin concentration effect curve for 3000 CHO-5-HT 1A cells/well converted to camp concentration using camp standard curve. 6 Agonist Pharmacological Profile Cells were stimulated with the 5-HT agonists 5-HT, R(+)8-OH-DPAT and buspirone, the antagonist spiperone and the dopamine receptor agonist, SKF38393 in the presence of 10 µm forskolin. pec 50 values are shown below. Agonists pec 50 Emax (%) Published values for pec 50 5HT 7.8 ± ± ± 0.2 R(+)8-OH-DPAT 7.7 ± ± 0.3 Buspirone 6.8 ± ± ± 0.3 Published pec50 values are means ± S.D of references 1 to 8. 4

5 7 Antagonist Pharmacological Profile CHO-5-HT 1A cells were pre-incubated for 30mins with the 5-HT 1A selective antagonists spiperone, NAN-190 and S(-) propranolol, the 5-HT 6/7 selective antagonist clozapine or the 5-HT 1/d/5-HT 2 selective antagonist metergoline before stimulation with 100 nm R(+)8-OH-DPAT and 10 µm forskolin. IC 50 and pk b values calculated from above and pa 2 values derived from slide 8 are shown in the table below. Antagonists IC 50 (nm) pk b pa 2 Published values for pk b Spiperone 7.9 ± ± ± NAN ± ± ± ± 0.2 (pa 2) S(-)Propanolol ± ± ± 0.1 Published pkb values are from references 1, 2, 5 and 7. 8 Schild Analysis Cells were pre-incubated for 30mins in the absence ( ) or in the presence of 10 nm ( ), 30 nm ( ), 100 nm ( ) and 300 nm ( ) NAN-190 (A) or spiperone (B) before performing a concentration effect curve to R(+)8-OH-DPAT. The inserts show Schild plots with a Schild slope of 1.0 ± 0.2 for NAN-190 and 0.95 ± 0.05 for spiperone and pa 2 values of 8.8 ± 0.2 and 8.4 ± 0.2 respectively. 5

6 9 DMSO Tolerance Studies (A) Acceptor beads and camp dilutions were incubated with increasing DMSO concentrations ( %; giving final concentrations of 0-5%) followed by the detection step. (B) In agonist stimulation experiments, stimulation was performed in presence of 0-5 % DMSO. 10 Variability / Precision Analysis Cells were stimulated with buffer ( ), 10 µm forskolin ( ), 10 µm forskolin nm R(+) 8-OH-DPAT ( ) and 10 µm forskolin nm R(+)8-OH-DPAT +1 µm spiperone ( ) in the presence of 1% DMSO. The data represents a total of 48 wells for each condition performed over 2 separate plates. Z data are represented in table below. Fsk / Fsk + R Fsk + R(+)8-OH-DPAT / Fsk + Basal / Fsk (+)8-OH-DPAT R(+)8-OH-DPAT + spiperone Day 1, plate Day 1, plate Day 2, plate Mean ± S.D ± ± ±

7 11 Conclusion The AlphaScreen camp Assay kit has been used to characterize the G αi-coupled h5-ht 1A receptor expressed in CHO cells and further, validated as a platform that can be used for the high-throughput screening of G αi-coupled receptors. The AlphaScreen camp Assay Kit demonstrated excellent utility, where standard agonists and antagonists demonstrated the expected pharmacology. The use of functional inhibition curves and Schild analysis for the determination of antagonist affinities yielded affinity constants consistent with published data. These data demonstrate the use of this detection platform for compound characterization such as that used in hit-to-lead seeking programs. Key to the success of a screening platform is its resistance to organic solvents such as DMSO as well as its precision in being able to distinguish active compounds from those with no activity. Here we have demonstrated the suitability of the AlphaScreen camp Assay Kit for screening where DMSO tolerance up to 1% was observed and assay precision as calculated by Z was 0.5 for G αi-coupled agonist stimulation and 0.3 for antagonist inhibition of this response. In view of the complexity of the types biomolecular interactions occurring in this system as well as the fact that the precision analysis was performed in the presence of 1% DMSO, the AlphaScreen camp Assay Kit represents a highly robust system for the detection of agonists and antagonists at G αi-coupled receptors. Thus in conclusion, the AlphaScreen homogeneous, non-radioactive camp detection platform demonstrates multifaceted flexibility for studying G αi-coupled receptors where primary screening and quantitative pharmacological studies can be performed with the same platform. 12 References 1. Schoeffter P. et al., Inhibition of camp accumulation via recombinant human serotonin 5-HT1A receptors: considerations on receptor effector coupling across systems, Neuropharmacolgy 36 (4/5), , Dumuis A. et al., Pharmacology of 5-HT1A receptors which inhibit camp production in hippocampal and cortical neurons in primary culture, Mol Pharmacol 33 (2), , Schoeffter P. et al., Centrally acting hypotensive agents with affinity for 5-HT1A binding sites inhibit forskolin-stimulated adenylate cyclase activity in calf hippocampus, Br J Pharmacol 95 (3), , Shenker A. et al., Pharmacology characterization of two 5-HT receptors coupled to adenylate cyclase in guinea pig hippocampal membranes, Mol Pharmacol 31 (4), , Pauwels P.J. et al., Activity of serotonin receptor agonists, partial agonists and antagonists at cloned human 5-HT1A receptors that are negatively coupled to adenylate cyclase in permanently transfected HeLa cells, Biochem Pharmacol 45 (2), , De Vry J. et al., Characterisation of the aminomethylchroman derivative BAY x 3702 as a highly potent 5-HT1A receptor agonist, J Pharmacol Exp Ther 284 (3), , De Vivo M. et al., Characterization of the 5-HT1A receptor-mediated inhibition of forskolin-stimulated adenylate cyclase activity in guinea pig and rat hippocampal membranes, J Pharmacol Exp Ther 238 (1), , Fargin A. et al., Effector coupling mechanisms of the cloned 5-HT1A receptor, J Biol Chem 264 (25), ,

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