- Email: [email protected]
Multiplexing G protein-coupled receptors in microarrays: A radioligand-binding assay
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 365 (2007) 266–273 www.elsevier.com/locate/yabio
Multiplexing G protein-coupled receptors in microarrays: A radioligand-binding assay Bruce Posner a, Yulong Hong b,*, Eric Benvenuti a, Michael Potchoiba a, Dave Nettleton a, Li Lui b, Ann Ferrie b, Fang Lai b, Ye Fang b, Juan Miret c, Chris Wielis a, Brian Webb b,* a
b c
Pfizer, Groton, CT 06340, USA Corning, Corning, NY 14831, USA Pfizer, Cambridge, MA 02139, USA Received 19 December 2006 Available online 18 March 2007
Abstract Multiplexing of G protein-coupled receptors (GPCRs) in microarrays promises to increase the efficiency, reduce the costs, and improve the quality of high-throughput assays. However, this technology is still nascent and has not yet achieved the status of ‘‘high throughput’’ or laid claim to handling a large set of receptors. In addition, the technology has been demonstrated only when using fluorescent ligands to detect binding, limiting its application to a subset of GPCRs. To expand the impact of multiplexing on this receptor class, we have developed a radiometric approach to the microarray assay. In these studies, we considered two receptors in the a-adrenergic receptor family, a2A and a2C, and the 125I-labeled agonist clonidine. We demonstrate that microarrays of these receptors can be readily detected (signal/noise ratio 160) using a Typhoon 9210 PhosphorImager. In addition, biochemical characterization shows that ligand-binding profiles and selectivity are preserved with the selective antagonists BRL44408 and ARC239. Importantly, these microarrays use approximately 200- to 400-fold less membrane preparation required by conventional assay methods and allow two or more receptors to be assayed in an area equivalent to a standard well of a microtiter plate. The impact of this approach on screening in drug discovery is discussed. 2007 Elsevier Inc. All rights reserved. Keywords: GPCR; Microarrays; High-throughput screening; Radioligands; Adrenergic; Typhoon
G protein-coupled receptors (GPCRs)1 are seven transmembrane receptors that mediate many important physiological responses. They respond to a diverse range of stimuli, including small molecules, peptides, neurotransmitters, hormones, autocrine factors, odorants, and photons [1–3]. These receptors transduce the binding of an
*
Corresponding authors. Fax: +1 815 987 3949 (B. Webb). E-mail addresses: [email protected] (Y. Hong), brian.webb@ thermofisher.com (B. Webb). 1 Abbreviations used: GPCR, G protein-coupled receptor; CHO, Chinese hamster ovary; HEK293, human embryonic kidney 293; PBS, phosphatebuffered saline; GAPS, c-aminopropylsilane; PEI, polyethylenimine; MCID, Microcomputer Imaging Device; ROI, region of interest; MDC, molecular dynamic counts. 0003-2697/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2007.03.014
extracellular ligand to an intracellular signal through activation of heterotrimeric G proteins. This leads to activation of downstream effectors such as adenylyl cyclase, phospholipases, and ion channels. GPCRs constitute the largest superfamily of proteins with more than 800 members. Importantly, this protein class is a major focus of drug development efforts in the pharmaceutical industry ( 60% of current programs), and approximately 10% of its members account for 25% of the top 200 drugs on the market [4–6]. Because of the importance of GPCRs in generating new therapeutics, considerable efforts have been devoted to developing new GPCR assay technologies for rapid assessment of their interactions with small molecules. Most conventional methodologies used to study GPCR signaling involve
Multiplexing GPCRs in microarrays / B. Posner et al. / Anal. Biochem. 365 (2007) 266–273
monitoring either ligand binding (i.e., radioligand-binding assays) or various downstream signaling events using cellbased readouts (e.g., Ca2+-sensitive dyes, transcriptional reporters) [7–9]. Recently, a novel GPCR microarray technology was developed for screening multiple GPCRs in parallel using fluorescent ligand-binding assays [10,11]. The ability to screen multiple GPCR targets simultaneously in a single assay (i.e., multiplexing) would greatly improve the efficiency of compound screening and reduce the consumption of precious compound libraries. Potential applications of a multiplexed GPCR assay format include monitoring receptor selectivity and tracking potential drug safety issues. However, this technology has been demonstrated only when using fluorescent ligands to detect binding, limiting its application to the small subset of GPCR receptors for which fluorescent ligands are available [11–13]. The majority of GPCRs are studied using commercially or privately produced radiolabeled ligands that carry either 3 H or 125I isotopes. To expand the scope of GPCR microarray assays, we have developed an approach for detecting the binding of radioligand to microarrayed GPCR membrane preparations. The a2A and a2C subtypes of the adrenergic a2 family of receptors were selected as a model system for assay development and validation [14–16]. We demonstrate binding of 125I-labeled clonidine to microarrays of immobilized GPCR membrane preparations containing these receptors. Detection of the receptor-bound ligand was achieved using a PhosphorImager. Receptor selectivity and rank order potency are preserved in the microarray format as compared with conventional assay formats. Moreover, we demonstrate that the amount of receptor membrane required is considerably less than that with conventional methods ( 200–400-fold). These results further extend the utility of the GPCR microarray assay format in both industrial and academic settings. Materials and methods Compounds
267
was resuspended in cold phosphate-buffered saline (PBS) and stored at –80 C. The thawed cell pellet was resuspended in homogenization buffer (50 mM Tris–HCl [pH 7.2], 10 mM MgCl2, 1 mM EDTA, 10% sucrose) and homogenized with a Dounce homogenizer (20 strokes). The solution was spun at 1000 g for 5 min. The resulting supernatant was centrifuged at 25,000g for 20 min, and the pellet was resuspended in homogenization buffer and stored at –80 C. The specific activity and concentration of each membrane preparation were as follows: a2A, 0.8 pmol/mg, 9.3 mg/ml; a2C, 6 pmol/mg, 14.4 mg/ml. Fabrication of GPCR microarrays We previously described a method for fabrication of GPCR microarrays using standard DNA microarray contact quill printing technology [11]. Preliminary radiometric assay results indicated that the approximately 100 lm GPCR microarray spots produced by the conventional CMP3 pin are not sufficient for robust detection of radioligand binding using a Typhoon 9210 PhosphorImager (data not shown). To obtain better spatial resolution and detection of bound radioligand, we explored alternative printing technologies to fabricate larger spots for the GPCR microarrays. First, a quill pin with a larger pin diameter was used (CMP10B, Telechem International, Sunnyvale, CA, USA). Briefly, GPCR membrane preparations (2 mg/ml final protein concentration) were robotically transferred from a 384-well microplate reservoir and deposited in an array configuration onto glass slides coated with c-aminopropylsilane (GAPS) using a CMP10B quill pin on a Cartesian PixSys 5500 printer (Cartesian Technologies, Irvine, CA, USA) (for a schematic of the microarrays, see Fig. 1 later). The CMP10B pin produces GPCR microarray spots that are approximately 400 lm in diameter. To produce even larger microspots, arrays were fabricated using a Caliper Sciclone inL10 Workstation (Caliper Life Sciences, Hopkinton, MA, USA). With this approach, GPCR microarray spots of approximately 850 lm in diameter were produced. Printed GPCR microarrays were stored under nitrogen at 4 C.
125
I-labeled clonidine (NEX253, iodoclonidine: 100 lCi [3.7MBq]; specific activity: 2200 Ci [81.4TBq]/mmol) was purchased from PerkinElmer. Unlabeled clonidine (cat. no. 0690), BRL14408 (cat. no. 1133), and ARC239 (cat. no. 0928) were purchased from Tocris. GPCR membrane preparations Cell lines expressing the a2A and a2C receptors were a gift from Hyunsuk (Tim) Min and Jiansu Zhang (Pfizer). The a2A receptor was expressed in Chinese hamster ovary (CHO) cells, and the a2C receptor was expressed in human embryonic kidney 293 (HEK293) cells. Membrane preparations were prepared from these cell lines using the following method. Cells were harvested with trypsin/versene solution and centrifuged for 5 to 6 min at 1000 rpm. The cell pellet
Microarray radioligand-binding assay Slides containing the printed GPCR microarrays were transferred from storage at 4 C to a humidified chamber and allowed to come to ambient temperature (20 min). A 10 ll spot of assay buffer (50 mM Tris [pH 7.5], 10 mM MgCl2, 1 mM EDTA) containing 125I-labeled clonidine (4 nM for most experiments except for radioligand titrations) and an unlabeled compound (if appropriate) was hand-pipetted onto each microarray subgrid contained on the slide. Microarrays were incubated for 1 h in a humidified chamber at room temperature. After the incubation period, the treated slides were washed manually with a stream of deionized water and dried with nitrogen. The slides were then placed in exposure cassettes for the
CHO
Multiplexing GPCRs in microarrays / B. Posner et al. / Anal. Biochem. 365 (2007) 266–273
HEK α2A α2C
268
Fig. 1. Schematic representation of GPCR microarrays printed on GAPS slide. A total of 16 microarrays were printed onto a GAPS slide in a 2 · 8 pattern. Each microarray contains three replicate membrane spots for HEK293, a2A, a2C, and CHO membrane preparations, as shown (a 4 · 3 array). Multiplexed binding assays were performed by incubating the microarrays with an assay solution containing the ligand 125I-labeled clonidine in the presence or absence of an unlabeled test compound. Thus, each slide allows the screening of 16 different compounds at a single dose or multiple doses of one or more compounds to be explored simultaneously against multiple receptors.
Typhoon 9210 PhosphorImager (GE Healthcare Biosciences, Piscataway, NJ, USA) and left for approximately 17 h in a lead/copper-lined exposure cabinet. Filter-binding assay A high-density (384-well) filter plate assay was used to determine equilibrium binding constants (Kd values) for 125 I-labeled clonidine and the a2A and a2C receptors (C. Jorgensen et al., manuscript in preparation). In preparation for the assay, polyethylenimine (PEI, Sigma–Aldrich, St. Louis, MO, USA) was added to 384-well Millipore Multiscreen FC filter plates to a final concentration of 0.3% in water with a Multidrop liquid dispenser (Thermo Electron, Franklin, MA, USA) and left for 2 to 6 h at room temperature before being stored at 4 C until ready for use. The binding assay was performed in a 384-well polypropylene plate (Matrix Technologies, Hudson, NH, USA). Using a Multidrop, 30 ll of assay buffer (50 mM Tris– HCl [pH 7.5], 10 mM MgCl2, 1 mM EDTA) was added to the 384-well polypropylene assay plate. In each experiment, membranes were prepared in assay buffer, and 30 ll of this membrane stock was added to each assay well (final assay concentrations: a2A, 8 lg/assay well; a2C,
4 lg/assay well). Radioligand (concentrations varied from 0.125 to 16 nM) was added to binding reactions in a volume of 30 ll of assay buffer. In assays designed to determine nonspecific binding, unlabeled clonidine (10 lM) was added with the radioligand. The binding assays for a2A and a2C were incubated at room temperature with gentle agitation for 2 and 1 h, respectively. Binding assays were transferred to 384-well filter plates (Millipore Multiscreen FC, pretreated with PEI as described above and rinsed with assay buffer). The filter plates were then washed four times with assay buffer. After the last wash, the plates were filtered dry and allowed to stand overnight on the benchtop behind appropriate shielding. Bottoms of plates were sealed with thin plate seals (Perkin Elmer Wallac, Downers Grove, IL, USA). Optiphase Supermix scintillation fluid (Perkin Elmer Wallac) was added to each well (15 ll/well) and incubated for 10 min. Tops of plates were sealed with thin film plate seals and read on a Trilux Betaplate counter (Perkin Elmer Wallac). Data for each assay were collected on a Trilux Betaplate counter and analyzed using GraphPad Prism 4.0 software (GraphPad, San Diego, CA, USA). Detection and quantitation using Typhoon instrument Binding of 125I-labeled clonidine to the adrenergic receptor microarray spots was detected and quantitated using a Typhoon 9210 PhosphorImager. Evaluation of GPCR autoradioluminographic images for 125I radioactivity was performed using a Microcomputer Imaging Device (MCID Elite, version 6.0, GE Healthcare Biosciences). Specific regions of interest (ROIs) were clearly delineated by the intensities of the photo-stimulated luminescence when displayed on the MCID Elite monitor, having a resolution of 1600 · 1200 pixels with 65,536 gray levels. The amount of radioactivity in each GPCR microspot was measured using a standardized ellipse sampling tool (8 · 8 pixels). The GPCR data were expressed as molecular dynamic counts (MDC)/mm2. The local background (amount of radioactivity bound nonspecifically to the GAPS surface) was calculated using an ellipse sampling tool positioned in the microarray area adjacent to the GPCR microspots. The signal/noise ratio values were calculated as follows: S/N = (mean signal – mean background) / standard deviation of background [17]. To optimize the assay performance, four different scanner settings were tested (10, 25, 50, and 100 lm). The scan setting is the unit area read incrementally during the processing of the image plate by the Typhoon 9210. The scan setting was found to have a marked effect on the detection sensitivity of the radioligand-bound arrays. Specifically, as the scan setting was increased (toward lower resolution), a significantly higher phosphorescence signal was observed from the radioligand-bound arrays. The maximum sensitivity was observed at the 100 lm setting. However, a significant ‘‘blooming’’ effect was also obtained at the 50and 100 lm settings. Blooming occurs when the radiation
Multiplexing GPCRs in microarrays / B. Posner et al. / Anal. Biochem. 365 (2007) 266–273
emitted by the scanned area strays into adjacent regions of the exposure screen, giving an artifactual ‘‘signal.’’ The 25lm setting proved to be the optimal detection setting to maximize sensitivity and minimize blooming, and it was used for all subsequent assays. Results Multiplexed GPCR binding assays using a microarray approach have been demonstrated recently [11]. GPCR membrane preparations microarrayed on flat glass substrates coated with GAPS retain proper receptor function and pharmacology, allowing parallel analysis of multiple GPCRs in a single assay. The methodologies used for fabrication and detection of GPCR microarrays, namely contact quill printing technology and fluorescent scanners, were ‘‘borrowed’’ from the DNA microarray industry [12]. However, because many important classes of GPCRs have nonpeptidic ligands, corresponding fluorescently labeled, small molecule ligands for these GPCRs are not available. To extend the utility of the GPCR microarray approach to these receptor classes, we developed a radioligand GPCR microarray assay. PhosphorImager technology was used to detect binding of a 125I-labeled ligand to printed GPCR microspots. Optimization of printing and assay conditions was performed using the a2C receptor. Preliminary experiments indicated that the conventional approximately 100 lm GPCR microarray spots used for fluorescent binding assays were not sufficient for robust detection of radioligand binding using a Typhoon 9210 PhosphorImager (data not shown). In an effort to address
a
b
c
+unlabeled clonidine
this issue, larger GPCR microarray spots were printed using a larger quill pin (CMP10B). A schematic of the GPCR microarray printing format is shown in Fig. 1. Adrenergic receptor a2C microarrays ( 400 lm spots) fabricated using this approach exhibited saturatable binding of 125I-labeled clonidine (for representative Typhoon 9210 microarray images and a binding isotherm for the a2C receptor, see Fig. 2). Printing larger microarray spots resulted in the immobilization of greater amounts of membrane per unit area of glass surface, providing improved signal for detection while maintaining good spatial resolution of the microarray spots. The dissociation constant obtained for a2C in the radioligand microarray assay was in good agreement with the value obtained using a conventional filter-binding assay (Table 1). The level of nonspecific binding to the a2C membrane microspots was determined by inclusion of excess unlabeled clonidine and found to be low (Fig. 2B). The dissociation constant for the adrenergic a2A receptor determined in the GPCR
Table 1 Thermodynamic dissociation constants for a2A and a2C and radiolabeled clonidine Receptor subtype
Reference Kda (nM)
Microarray Kdb (nM)
a2A a2C
1.4 ± 0.5 4.0 ± 2.5
2.1 ± 1.0 2.4 ± 1.3
a The reference Kd values are reported as the means and standard deviations of at least two measurements using the filter-binding assay, as described in Materials and methods. b The microarray Kd values are reported as the means and standard deviations of at least two measurements.
B 40000
Relative Imager Units
A
30000
20000 -Cold Clonidine +Cold Clonidine
10000
0 0.0
2.5
5.0 125
d
269
7.5
10.0
12.5
15.0
17.5
I-Labeled Clonidine (nM)
Fig. 2. Binding of 125I-labeled clonidine to adrenergic a2C GPCR microarray. A membrane preparation containing a2C receptors was printed on a GAPS slide in a microarray format, as described in Materials and methods. (A) Saturation binding assays were performed using 125I-labeled clonidine. Representative images from a Typhoon 9210 PhosphorImager show binding of four concentrations of 125I-labeled clonidine to a2C microspots: 4 nM (a), 2 nM (b), 1 nM (c), and 0.5 nM (d). Nonspecific binding was determined by the addition of excess unlabeled clonidine (1 lM) in the reaction. Bound radioligand was quantitated using the Typhoon 9210 PhosphorImager and analyzed using GraphPad software. (B) Total and nonspecific binding of 125 I-labeled clonidine to a2 C microarrays expressed in relative imager units. This experiment was repeated three times with similar results. The error bars represent the standard deviation.
Multiplexing GPCRs in microarrays / B. Posner et al. / Anal. Biochem. 365 (2007) 266–273
microarray format was also similar to conventional assay data (Table 1). The observed amount of radioligand bound to each printed microarray spot was found to be dependent on the quality of the membrane preparation, the total protein concentration of the membrane preparation, and the amount of receptor contained in the membrane preparation (pmol of receptor/mg of membrane protein or Bmax). For the receptors considered here (Kd values for clonidine ranging from 2 to 4 nM and Bmax values P 0.8 pmol/mg), a membrane protein concentration of 2 mg/ml was optimal for array printing with the CMP10B quill pin. This concentration ensured that enough receptor is deposited on the glass surface for a robust microarray assay at the lowest concentrations of radioligand (0.1 nM). In principle, this protein concentration can be adjusted downward (e.g., if Bmax > 2 and/or the affinity for the radioligand is high [<1 nM]). Importantly, control microarray spots printed with the parental CHO and HEK293 membranes displayed significantly lower binding of 125I-labeled clonidine relative to the a2A and a2C spots (Fig. 3), demonstrating that the majority of the radioligand is bound to the overexpressed receptors. Further enhancements in the signal/noise ratio were obtained using the Caliper Sciclone nanodispenser to fabricate GPCR microarrays with even larger spots (Fig. 4). Although not specifically tested here, this printing approach should provide a robust assay for receptor preparations with expression levels below 0.8 pmol/mg of membrane protein. The Sciclone nanodispenser expels membrane onto the surface by physically pushing a defined volume (10 nl) of membrane sample out of a ceramic tip. This is in contrast to the quill pin approach, where capillary action pulls a fixed volume of membrane solution onto the glass surface. Depositing viscous membrane prepara-
α2C Bac kground
S/N: 160
175000 125000
S/N: 20
10000
0
CMP 10
Sciclone
125
Fig. 4. Comparison of I-labeled clonidine binding signal on a2C microarrays printed using contact printing (CMP10 pin) and noncontact dispensing (Sciclone). Microarrays of a2C membrane preparation were printed onto GAPS slides using either the CMP10 contact printing or the Sciclone nanodispenser. The 125I-labeled clonidine (4 nM) binding signal of the printed a2C membrane microspots and the local background signal (i.e., the amount of radioactivity bound nonspecifically to the GAPS surface) were quantified using the Typhoon 9210 PhosphorImager. The signal/noise (S/N) ratio was determined by comparing the signal intensity of the a2C membrane spot with the local background. The S/N ratios were 20 and 160 for the CMP10- and Sciclone-printed a2C microarrays, respectively.
tions often is difficult using quill pin devices (e.g., CMP10B). The Sciclone produces GPCR microspots with a diameter of approximately 850 lm. The signal/noise ratio was approximately eightfold higher on the GPCR membrane microspots produced by the Sciclone as compared with the CMP10B-printed microspots (Fig. 4). This improvement can be attributed to both a larger GPCR microarray spot size and a greater amount of membrane
Percentage of Total Signal
100 75 50 25 0
125 100 75 50 25
EK
0 H
H C
Cell line 125
225000
B 125
O
Percentage of Total Signal
A
275000
Relative Imager Units
270
Cell line
Fig. 3. Comparison of I-labeled clonidine binding signals for a2A and a2C membranes and control CHO and HEK293 parental cell line membranes in GPCR microarray format. GPCR microarrays containing a2A, a2C, CHO, and HEK293 membrane preparations printed with a CMP10 pin were incubated with 125I-labeled clonidine (16 nM), and the binding signal of each membrane microspot was quantitated using the Typhoon 9210 PhosphorImager. (A) Relative binding signals of a2A membrane and corresponding CHO parental cell line membrane microspots. (B) Relative binding signals of a2C membrane and corresponding HEK293 parental cell line membrane microspots.
Multiplexing GPCRs in microarrays / B. Posner et al. / Anal. Biochem. 365 (2007) 266–273
material deposited per unit area of GAPS-treated glass surface. Subsequent studies were conducted using Scicloneprinted GPCR microarrays. The a2A and a2C members of the adrenergic family display subtype selectivity for commercially available antagonists. Two of these antagonists, ARC239 and BRL44408, were used in competition assays on GPCR microarrays containing these a2 receptors. The goal was to determine whether the microarray format could be used to investigate receptor subtype selectivity and rank order potency for these two receptors simultaneously. Displacement of 125 I-labeled clonidine from a2A and a2C microarrays by increasing doses of BRL44408 and ARC239 was quantitated and analyzed. The resulting dose–response curves are shown in Fig. 5. The Ki values obtained for BRL44408 and ARC239 in the microarray format are in good agreement with published Ki values for these compounds (Table 2) [18]. Importantly, the anticipated rank order for these compounds against these two adrenergic receptor subtypes was observed. These results demonstrate the feasibility of parallel compound selectivity measurements of the a2A and a2C receptors using the radiometric GPCR microarray format.
Discussion The potential of multiplexed assays using a microarray approach has been realized during the past few years for DNA, and significant progress has been made for proteins [19,20]. However, the complexity of membrane-bound proteins has hindered the development of GPCR microarrays. The challenges have included maintaining bilayer fluidity, immobilizing protein and lipid to a surface, and maintaining the functional integrity of the receptor. Recently, the fabrication of GPCR microarrays was reported using contact pin printing technology [11]. Multiplexed GPCR binding assays can be performed using fluorescently labeled ligands; however, the number of ligands available for this
Table 2 Binding constants for BRL44408 and ARC239 for a2A and a2C using radiolabeled clonidine Ligand
Receptor subtype
Kia (nM)
Microarray Kib (nM)
BRL44408 BRL44408 ARC239 ARC239
a2A a2C a2A a2C
5.68 ± 1.86 150 ± 41 1330 ± 50 31.8 ± 1.3
25 ± 17 236 ± 50 382 ± 232 70 ± 42
a
See Ref. [18]. The microarray Ki values are reported as the means and standard deviations of at least two measurements. b
approach is limited, and some of these ligands exhibit high levels of nonspecific binding [11,13,21]. To expand the scope of the microarray approach, we have developed and validated a radiometric GPCR microarray assay. Robust detection of bound radioligand required larger GPCR microspots than the conventional GPCR microarray spots demonstrated previously [11]. The microarray spots were fabricated with either the CMP10 contact printing pin or the Sciclone nanodispenser, producing GPCR microspots with diameters of approximately 400 and 850 lm, respectively. Specific saturatable binding of 125 I-labeled clonidine to the subset of adrenergic receptors, the a2A and a2C subtypes, was demonstrated, and dissociation constants that compare favorably with those determined using a conventional filter-binding approach were obtained (Table 1). Detection of radioligand binding to the GPCR microarrays was accomplished using the Typhoon 9210 PhosphorImager. The signal/noise ratio was considerably higher for the microarray spots printed with the Sciclone nanodispenser (160 vs. 20 for the CMP10-printed microarrays), presumably due to the larger spot size and larger volume of membrane preparation printed with the Sciclone. The pharmacological fidelity of the immobilized receptors was further demonstrated using competitive displacement assays. Using the radiometric microarray assay, potency values for two receptor-selective reference
B 110
% Clonidine Bound
% Clonidine Bound
A
271
α2C α2A
80 50 20
120 α2A α2C
80
40
0 -10 -10
-9
-8
-7
-6
log [BRL44408] (M)
-5
-4
-9
-8
-7
-6
-5
-4
Log [ARC239] (M)
Fig. 5. GPCR microarray competition binding assays for adrenergic a2A and a2C receptors. Compound selectivity was determined for GCPR microarrays containing a2A and a2C receptors. 125I-labeled clonidine (4 nM) was incubated with the GPCR microarrays with half log dilutions of unlabeled BRL44408 (A) or ARC239 (B). Bound radioligand was quantitated using the Typhoon 9210 PhosphorImager, and the data were analyzed using GraphPad software.
272
Multiplexing GPCRs in microarrays / B. Posner et al. / Anal. Biochem. 365 (2007) 266–273
compounds, BRL44408 and ARC239, and the a2 receptors were in reasonably good agreement with published literature values obtained using a conventional filter-binding assay (Fig. 5 and Table 2) [18]. Importantly, appropriate rank order of Ki values was observed for the two compounds considered, demonstrating the feasibility of running selectivity screens of closely related GPCR family members that are of interest for academic and pharmaceutical research. Although the assays described were performed manually using a slide format, transition to a more convenient microplate format should be possible. We recently demonstrated printing of nine GPCR microspots inside the wells of a prototype 96-well GAPS microplate with the Sciclone nanodispenser (data not shown). The smaller GPCR microspots produced by the CMP10B will allow at least twice this density inside the well, although the signal/noise ratio is reduced with these smaller GPCR microspots (Fig. 4). However, the requirement for intimate contact between the microarray assay surface and the PhosphorImager screen during assay detection necessitates the use of a microplate that allows removal of the glass bottom. Commercially available devices that convert slides into a microplate format (e.g., ProPlate from Grace Biolabs) may be suitable for this application and remain to be tested. The development of a microplate-based radioligand microarray assay should enable efficient coupling of multiplexed GPCR targets (i.e., multiple GPCRs within each well) with compound screening. Although the direct measurement of ligand binding using radiolabeled ligands remains the predominant highthroughput assay format for characterization of GPCR ligand affinity and selectivity, the recent demonstration of functional GPCR microarrays represents a new dimension in microarray assays [22]. The functional GPCR microarray assays measure the binding of the fluorescently labeled GTP analogue to G proteins that have been activated via agonist-stimulated activation of their cognate GPCR [23]. This approach enables the identification of agonists, antagonists, and inverse agonists, and it complements ligand-binding approaches described here and in other works. Thus, multiplexed GPCR microarray assays have now been demonstrated using both fluorescent and radiometric formats. Of course, both approaches have inherent advantages and disadvantages. For example, the abundance of commercially available radioligands must be weighed against the disposal issues associated with radioactivity. In conclusion, the ability to monitor compound selectivity against a collection of targets in a single assay holds great promise for improving the efficiency of assaying and characterizing the interactions of GPCRs with natural and synthesized ligands. Potential benefits of the multiplexed GPCR assay format include reagent savings (e.g., 200–400-fold less receptor membrane), reduced consumption of compound libraries, and time savings. For example, a single 96-well microtiter plate containing
a 4 · 4 GPCR array in each well represents 1536 assays across multiple receptors. With continued progress, the microarray assay format should have a significant impact on the efficiency of secondary screening (e.g., selectivity) and ultimately may find use in high-throughput screening. Acknowledgments We are grateful to Jeremy Everett, Eryl Sharp, Chris Williams, Anne Schmidt, and Bill Martin for their support of this work and their critical review of the manuscript. We also thank Brian Bush for his support of this work and Csilla Jorgensen for her help in setting up and executing the 384-well filter plate assay. References [1] A.G. Gilman, G proteins: Transducers of receptor-generated signals, Annu. Rev. Biochem. 56 (1987) 615–649. [2] J.R. Hepler, A.G. Gilman, G proteins, Trends Biochem. Sci. 17 (1992) 383–387. [3] C. Liebmann, G protein-coupled receptors and their signaling pathways: Classical therapeutical targets susceptible to novel therapeutic concepts, Curr. Pharm. Des. 10 (2004) 1937–1958. [4] A.L. Hopkins, C.R. Groom, The druggable genome, Nat. Rev. Drug Disc. 1 (2002) 727–730. [5] K. Lundstrom, The future of G protein-coupled receptors as targets in drug discovery, IDrugs 8 (2005) 909–913. [6] A. Wise, K. Gearing, S. Rees, Target validation of G-protein coupled receptors, Drug Disc. Today 7 (2002) 235–246. [7] W.P. Walters, M. Namchuk, Designing screens: How to make your hits a hit, Nat. Rev. Drug Disc. 2 (2003) 259–266. [8] P.A. Johnston, P.A. Johnston, Cellular platforms for HTS: Three case studies, Drug Disc. Today 7 (2002) 353–363. [9] C. Williams, A. Sewing, G-protein coupled receptor assays: To measure affinity or efficacy that is the question, Comb. Chem. High Throughput Screen 8 (2005) 285–292. [10] Y. Fang, J. Lahiri, L. Picard, G protein-coupled receptor microarrays for drug discovery, Drug Disc. Today 8 (2003) 755–761. [11] Y. Fang, A.G. Frutos, J. Lahiri, Membrane protein microarrays, J. Am. Chem. Soc. 124 (2002) 2394–2395. [12] Y. Fang, A.G. Frutos, B. Webb, Y. Hong, A. Ferrie, F. Lai, J. Lahiri, Membrane biochips, BioTechniques 33 (Suppl.) (2002) S62–S65. [13] R.J. Middleton, B. Kellam, Fluorophore-tagged GPCR ligands, Curr. Opin. Chem. Biol. 9 (2005) 517–525. [14] J.P. Hieble, W. Bondinell, R.R. Ruffolo Jr., a- and (-Adrenoceptors: From the gene to the clinic: I. Molecular biology and adrenoceptor subclassification, J. Med. Chem. 38 (1995) 3415–3444. [15] L. Hein, B.K. Kobilka, Neurotransmitter receptors: IV. Adrenergic receptor signal transduction and regulation, Neuropharmacology 34 (1995) 357–366. [16] D.B. Bylund, D.C. Eikenberg, J.P. Hieble, S.Z. Langer, R.J. Lefkowitz, K.P. Minneman, P.B. Molinoff, R.R. Ruffolo Jr., U. Trendelenburg, International Union of Pharmacology nomenclature of adrenoceptors, Pharmacol. Rev. 46 (1994) 121–136. [17] J. Zhang, T.D. Chung, K.R. Oldenburg, A simple statistical parameter for use in evaluation and validation of high throughput screening assays, J. Biomol. Screen 4 (1999) 67–73. [18] S. Uhlen, A.C. Porter, R.R. Neubig, The novel a-2 adrenergic radioligand [3 H]-MK912 is a-2C selective among human a-2A, a-2B, and a-2C adrenoceptors, J. Pharmacol. Exp. Ther. 271 (1994) 1558– 1565. [19] E. Phizicky, P.I. Bastiaens, H. Zhu, M. Snyder, S. Fields, Protein analysis on a proteomic scale, Nature 422 (2003) 208–215.
Multiplexing GPCRs in microarrays / B. Posner et al. / Anal. Biochem. 365 (2007) 266–273 [20] M. Schena, D. Shalon, R.W. Davis, P.O. Brown, Quantitative monitoring of gene expression patterns with a complementary DNA microarray, Science 270 (1995) 467–470. [21] J.C. McGrath, S. Arribas, C.J. Daly, Fluorescent ligands for the study of receptors, Trends Pharmacol. Sci. 17 (1996) 393– 399.
273
[22] Y. Hong, B.L. Webb, H. Su, E.J. Mozdy, Y. Fang, Q. Wu, L. Liu, J. Beck, A.M. Ferrie, S. Raghavan, J. Mauro, A. Carre, D. Mueller, F. Lai, B. Rasnow, M. Johnson, H. Min, J. Salon, J. Lahiri, Functional GPCR microarrays, J. Am. Chem. Soc. 127 (2005) 15350–15351. [23] T. Haga, G. Berstein (Eds.), G Protein-Coupled Receptors, CRC Press, Boca Raton, FL, 1999.
Multiplexing G protein-coupled receptors in microarrays: A radioligand-binding assay Bruce Posner a, Yulong Hong b,*, Eric Benvenuti a, Michael Potchoiba a, Dave Nettleton a, Li Lui b, Ann Ferrie b, Fang Lai b, Ye Fang b, Juan Miret c, Chris Wielis a, Brian Webb b,* a
b c
Pfizer, Groton, CT 06340, USA Corning, Corning, NY 14831, USA Pfizer, Cambridge, MA 02139, USA Received 19 December 2006 Available online 18 March 2007
Abstract Multiplexing of G protein-coupled receptors (GPCRs) in microarrays promises to increase the efficiency, reduce the costs, and improve the quality of high-throughput assays. However, this technology is still nascent and has not yet achieved the status of ‘‘high throughput’’ or laid claim to handling a large set of receptors. In addition, the technology has been demonstrated only when using fluorescent ligands to detect binding, limiting its application to a subset of GPCRs. To expand the impact of multiplexing on this receptor class, we have developed a radiometric approach to the microarray assay. In these studies, we considered two receptors in the a-adrenergic receptor family, a2A and a2C, and the 125I-labeled agonist clonidine. We demonstrate that microarrays of these receptors can be readily detected (signal/noise ratio 160) using a Typhoon 9210 PhosphorImager. In addition, biochemical characterization shows that ligand-binding profiles and selectivity are preserved with the selective antagonists BRL44408 and ARC239. Importantly, these microarrays use approximately 200- to 400-fold less membrane preparation required by conventional assay methods and allow two or more receptors to be assayed in an area equivalent to a standard well of a microtiter plate. The impact of this approach on screening in drug discovery is discussed. 2007 Elsevier Inc. All rights reserved. Keywords: GPCR; Microarrays; High-throughput screening; Radioligands; Adrenergic; Typhoon
G protein-coupled receptors (GPCRs)1 are seven transmembrane receptors that mediate many important physiological responses. They respond to a diverse range of stimuli, including small molecules, peptides, neurotransmitters, hormones, autocrine factors, odorants, and photons [1–3]. These receptors transduce the binding of an
*
Corresponding authors. Fax: +1 815 987 3949 (B. Webb). E-mail addresses: [email protected] (Y. Hong), brian.webb@ thermofisher.com (B. Webb). 1 Abbreviations used: GPCR, G protein-coupled receptor; CHO, Chinese hamster ovary; HEK293, human embryonic kidney 293; PBS, phosphatebuffered saline; GAPS, c-aminopropylsilane; PEI, polyethylenimine; MCID, Microcomputer Imaging Device; ROI, region of interest; MDC, molecular dynamic counts. 0003-2697/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2007.03.014
extracellular ligand to an intracellular signal through activation of heterotrimeric G proteins. This leads to activation of downstream effectors such as adenylyl cyclase, phospholipases, and ion channels. GPCRs constitute the largest superfamily of proteins with more than 800 members. Importantly, this protein class is a major focus of drug development efforts in the pharmaceutical industry ( 60% of current programs), and approximately 10% of its members account for 25% of the top 200 drugs on the market [4–6]. Because of the importance of GPCRs in generating new therapeutics, considerable efforts have been devoted to developing new GPCR assay technologies for rapid assessment of their interactions with small molecules. Most conventional methodologies used to study GPCR signaling involve
Multiplexing GPCRs in microarrays / B. Posner et al. / Anal. Biochem. 365 (2007) 266–273
monitoring either ligand binding (i.e., radioligand-binding assays) or various downstream signaling events using cellbased readouts (e.g., Ca2+-sensitive dyes, transcriptional reporters) [7–9]. Recently, a novel GPCR microarray technology was developed for screening multiple GPCRs in parallel using fluorescent ligand-binding assays [10,11]. The ability to screen multiple GPCR targets simultaneously in a single assay (i.e., multiplexing) would greatly improve the efficiency of compound screening and reduce the consumption of precious compound libraries. Potential applications of a multiplexed GPCR assay format include monitoring receptor selectivity and tracking potential drug safety issues. However, this technology has been demonstrated only when using fluorescent ligands to detect binding, limiting its application to the small subset of GPCR receptors for which fluorescent ligands are available [11–13]. The majority of GPCRs are studied using commercially or privately produced radiolabeled ligands that carry either 3 H or 125I isotopes. To expand the scope of GPCR microarray assays, we have developed an approach for detecting the binding of radioligand to microarrayed GPCR membrane preparations. The a2A and a2C subtypes of the adrenergic a2 family of receptors were selected as a model system for assay development and validation [14–16]. We demonstrate binding of 125I-labeled clonidine to microarrays of immobilized GPCR membrane preparations containing these receptors. Detection of the receptor-bound ligand was achieved using a PhosphorImager. Receptor selectivity and rank order potency are preserved in the microarray format as compared with conventional assay formats. Moreover, we demonstrate that the amount of receptor membrane required is considerably less than that with conventional methods ( 200–400-fold). These results further extend the utility of the GPCR microarray assay format in both industrial and academic settings. Materials and methods Compounds
267
was resuspended in cold phosphate-buffered saline (PBS) and stored at –80 C. The thawed cell pellet was resuspended in homogenization buffer (50 mM Tris–HCl [pH 7.2], 10 mM MgCl2, 1 mM EDTA, 10% sucrose) and homogenized with a Dounce homogenizer (20 strokes). The solution was spun at 1000 g for 5 min. The resulting supernatant was centrifuged at 25,000g for 20 min, and the pellet was resuspended in homogenization buffer and stored at –80 C. The specific activity and concentration of each membrane preparation were as follows: a2A, 0.8 pmol/mg, 9.3 mg/ml; a2C, 6 pmol/mg, 14.4 mg/ml. Fabrication of GPCR microarrays We previously described a method for fabrication of GPCR microarrays using standard DNA microarray contact quill printing technology [11]. Preliminary radiometric assay results indicated that the approximately 100 lm GPCR microarray spots produced by the conventional CMP3 pin are not sufficient for robust detection of radioligand binding using a Typhoon 9210 PhosphorImager (data not shown). To obtain better spatial resolution and detection of bound radioligand, we explored alternative printing technologies to fabricate larger spots for the GPCR microarrays. First, a quill pin with a larger pin diameter was used (CMP10B, Telechem International, Sunnyvale, CA, USA). Briefly, GPCR membrane preparations (2 mg/ml final protein concentration) were robotically transferred from a 384-well microplate reservoir and deposited in an array configuration onto glass slides coated with c-aminopropylsilane (GAPS) using a CMP10B quill pin on a Cartesian PixSys 5500 printer (Cartesian Technologies, Irvine, CA, USA) (for a schematic of the microarrays, see Fig. 1 later). The CMP10B pin produces GPCR microarray spots that are approximately 400 lm in diameter. To produce even larger microspots, arrays were fabricated using a Caliper Sciclone inL10 Workstation (Caliper Life Sciences, Hopkinton, MA, USA). With this approach, GPCR microarray spots of approximately 850 lm in diameter were produced. Printed GPCR microarrays were stored under nitrogen at 4 C.
125
I-labeled clonidine (NEX253, iodoclonidine: 100 lCi [3.7MBq]; specific activity: 2200 Ci [81.4TBq]/mmol) was purchased from PerkinElmer. Unlabeled clonidine (cat. no. 0690), BRL14408 (cat. no. 1133), and ARC239 (cat. no. 0928) were purchased from Tocris. GPCR membrane preparations Cell lines expressing the a2A and a2C receptors were a gift from Hyunsuk (Tim) Min and Jiansu Zhang (Pfizer). The a2A receptor was expressed in Chinese hamster ovary (CHO) cells, and the a2C receptor was expressed in human embryonic kidney 293 (HEK293) cells. Membrane preparations were prepared from these cell lines using the following method. Cells were harvested with trypsin/versene solution and centrifuged for 5 to 6 min at 1000 rpm. The cell pellet
Microarray radioligand-binding assay Slides containing the printed GPCR microarrays were transferred from storage at 4 C to a humidified chamber and allowed to come to ambient temperature (20 min). A 10 ll spot of assay buffer (50 mM Tris [pH 7.5], 10 mM MgCl2, 1 mM EDTA) containing 125I-labeled clonidine (4 nM for most experiments except for radioligand titrations) and an unlabeled compound (if appropriate) was hand-pipetted onto each microarray subgrid contained on the slide. Microarrays were incubated for 1 h in a humidified chamber at room temperature. After the incubation period, the treated slides were washed manually with a stream of deionized water and dried with nitrogen. The slides were then placed in exposure cassettes for the
CHO
Multiplexing GPCRs in microarrays / B. Posner et al. / Anal. Biochem. 365 (2007) 266–273
HEK α2A α2C
268
Fig. 1. Schematic representation of GPCR microarrays printed on GAPS slide. A total of 16 microarrays were printed onto a GAPS slide in a 2 · 8 pattern. Each microarray contains three replicate membrane spots for HEK293, a2A, a2C, and CHO membrane preparations, as shown (a 4 · 3 array). Multiplexed binding assays were performed by incubating the microarrays with an assay solution containing the ligand 125I-labeled clonidine in the presence or absence of an unlabeled test compound. Thus, each slide allows the screening of 16 different compounds at a single dose or multiple doses of one or more compounds to be explored simultaneously against multiple receptors.
Typhoon 9210 PhosphorImager (GE Healthcare Biosciences, Piscataway, NJ, USA) and left for approximately 17 h in a lead/copper-lined exposure cabinet. Filter-binding assay A high-density (384-well) filter plate assay was used to determine equilibrium binding constants (Kd values) for 125 I-labeled clonidine and the a2A and a2C receptors (C. Jorgensen et al., manuscript in preparation). In preparation for the assay, polyethylenimine (PEI, Sigma–Aldrich, St. Louis, MO, USA) was added to 384-well Millipore Multiscreen FC filter plates to a final concentration of 0.3% in water with a Multidrop liquid dispenser (Thermo Electron, Franklin, MA, USA) and left for 2 to 6 h at room temperature before being stored at 4 C until ready for use. The binding assay was performed in a 384-well polypropylene plate (Matrix Technologies, Hudson, NH, USA). Using a Multidrop, 30 ll of assay buffer (50 mM Tris– HCl [pH 7.5], 10 mM MgCl2, 1 mM EDTA) was added to the 384-well polypropylene assay plate. In each experiment, membranes were prepared in assay buffer, and 30 ll of this membrane stock was added to each assay well (final assay concentrations: a2A, 8 lg/assay well; a2C,
4 lg/assay well). Radioligand (concentrations varied from 0.125 to 16 nM) was added to binding reactions in a volume of 30 ll of assay buffer. In assays designed to determine nonspecific binding, unlabeled clonidine (10 lM) was added with the radioligand. The binding assays for a2A and a2C were incubated at room temperature with gentle agitation for 2 and 1 h, respectively. Binding assays were transferred to 384-well filter plates (Millipore Multiscreen FC, pretreated with PEI as described above and rinsed with assay buffer). The filter plates were then washed four times with assay buffer. After the last wash, the plates were filtered dry and allowed to stand overnight on the benchtop behind appropriate shielding. Bottoms of plates were sealed with thin plate seals (Perkin Elmer Wallac, Downers Grove, IL, USA). Optiphase Supermix scintillation fluid (Perkin Elmer Wallac) was added to each well (15 ll/well) and incubated for 10 min. Tops of plates were sealed with thin film plate seals and read on a Trilux Betaplate counter (Perkin Elmer Wallac). Data for each assay were collected on a Trilux Betaplate counter and analyzed using GraphPad Prism 4.0 software (GraphPad, San Diego, CA, USA). Detection and quantitation using Typhoon instrument Binding of 125I-labeled clonidine to the adrenergic receptor microarray spots was detected and quantitated using a Typhoon 9210 PhosphorImager. Evaluation of GPCR autoradioluminographic images for 125I radioactivity was performed using a Microcomputer Imaging Device (MCID Elite, version 6.0, GE Healthcare Biosciences). Specific regions of interest (ROIs) were clearly delineated by the intensities of the photo-stimulated luminescence when displayed on the MCID Elite monitor, having a resolution of 1600 · 1200 pixels with 65,536 gray levels. The amount of radioactivity in each GPCR microspot was measured using a standardized ellipse sampling tool (8 · 8 pixels). The GPCR data were expressed as molecular dynamic counts (MDC)/mm2. The local background (amount of radioactivity bound nonspecifically to the GAPS surface) was calculated using an ellipse sampling tool positioned in the microarray area adjacent to the GPCR microspots. The signal/noise ratio values were calculated as follows: S/N = (mean signal – mean background) / standard deviation of background [17]. To optimize the assay performance, four different scanner settings were tested (10, 25, 50, and 100 lm). The scan setting is the unit area read incrementally during the processing of the image plate by the Typhoon 9210. The scan setting was found to have a marked effect on the detection sensitivity of the radioligand-bound arrays. Specifically, as the scan setting was increased (toward lower resolution), a significantly higher phosphorescence signal was observed from the radioligand-bound arrays. The maximum sensitivity was observed at the 100 lm setting. However, a significant ‘‘blooming’’ effect was also obtained at the 50and 100 lm settings. Blooming occurs when the radiation
Multiplexing GPCRs in microarrays / B. Posner et al. / Anal. Biochem. 365 (2007) 266–273
emitted by the scanned area strays into adjacent regions of the exposure screen, giving an artifactual ‘‘signal.’’ The 25lm setting proved to be the optimal detection setting to maximize sensitivity and minimize blooming, and it was used for all subsequent assays. Results Multiplexed GPCR binding assays using a microarray approach have been demonstrated recently [11]. GPCR membrane preparations microarrayed on flat glass substrates coated with GAPS retain proper receptor function and pharmacology, allowing parallel analysis of multiple GPCRs in a single assay. The methodologies used for fabrication and detection of GPCR microarrays, namely contact quill printing technology and fluorescent scanners, were ‘‘borrowed’’ from the DNA microarray industry [12]. However, because many important classes of GPCRs have nonpeptidic ligands, corresponding fluorescently labeled, small molecule ligands for these GPCRs are not available. To extend the utility of the GPCR microarray approach to these receptor classes, we developed a radioligand GPCR microarray assay. PhosphorImager technology was used to detect binding of a 125I-labeled ligand to printed GPCR microspots. Optimization of printing and assay conditions was performed using the a2C receptor. Preliminary experiments indicated that the conventional approximately 100 lm GPCR microarray spots used for fluorescent binding assays were not sufficient for robust detection of radioligand binding using a Typhoon 9210 PhosphorImager (data not shown). In an effort to address
a
b
c
+unlabeled clonidine
this issue, larger GPCR microarray spots were printed using a larger quill pin (CMP10B). A schematic of the GPCR microarray printing format is shown in Fig. 1. Adrenergic receptor a2C microarrays ( 400 lm spots) fabricated using this approach exhibited saturatable binding of 125I-labeled clonidine (for representative Typhoon 9210 microarray images and a binding isotherm for the a2C receptor, see Fig. 2). Printing larger microarray spots resulted in the immobilization of greater amounts of membrane per unit area of glass surface, providing improved signal for detection while maintaining good spatial resolution of the microarray spots. The dissociation constant obtained for a2C in the radioligand microarray assay was in good agreement with the value obtained using a conventional filter-binding assay (Table 1). The level of nonspecific binding to the a2C membrane microspots was determined by inclusion of excess unlabeled clonidine and found to be low (Fig. 2B). The dissociation constant for the adrenergic a2A receptor determined in the GPCR
Table 1 Thermodynamic dissociation constants for a2A and a2C and radiolabeled clonidine Receptor subtype
Reference Kda (nM)
Microarray Kdb (nM)
a2A a2C
1.4 ± 0.5 4.0 ± 2.5
2.1 ± 1.0 2.4 ± 1.3
a The reference Kd values are reported as the means and standard deviations of at least two measurements using the filter-binding assay, as described in Materials and methods. b The microarray Kd values are reported as the means and standard deviations of at least two measurements.
B 40000
Relative Imager Units
A
30000
20000 -Cold Clonidine +Cold Clonidine
10000
0 0.0
2.5
5.0 125
d
269
7.5
10.0
12.5
15.0
17.5
I-Labeled Clonidine (nM)
Fig. 2. Binding of 125I-labeled clonidine to adrenergic a2C GPCR microarray. A membrane preparation containing a2C receptors was printed on a GAPS slide in a microarray format, as described in Materials and methods. (A) Saturation binding assays were performed using 125I-labeled clonidine. Representative images from a Typhoon 9210 PhosphorImager show binding of four concentrations of 125I-labeled clonidine to a2C microspots: 4 nM (a), 2 nM (b), 1 nM (c), and 0.5 nM (d). Nonspecific binding was determined by the addition of excess unlabeled clonidine (1 lM) in the reaction. Bound radioligand was quantitated using the Typhoon 9210 PhosphorImager and analyzed using GraphPad software. (B) Total and nonspecific binding of 125 I-labeled clonidine to a2 C microarrays expressed in relative imager units. This experiment was repeated three times with similar results. The error bars represent the standard deviation.
Multiplexing GPCRs in microarrays / B. Posner et al. / Anal. Biochem. 365 (2007) 266–273
microarray format was also similar to conventional assay data (Table 1). The observed amount of radioligand bound to each printed microarray spot was found to be dependent on the quality of the membrane preparation, the total protein concentration of the membrane preparation, and the amount of receptor contained in the membrane preparation (pmol of receptor/mg of membrane protein or Bmax). For the receptors considered here (Kd values for clonidine ranging from 2 to 4 nM and Bmax values P 0.8 pmol/mg), a membrane protein concentration of 2 mg/ml was optimal for array printing with the CMP10B quill pin. This concentration ensured that enough receptor is deposited on the glass surface for a robust microarray assay at the lowest concentrations of radioligand (0.1 nM). In principle, this protein concentration can be adjusted downward (e.g., if Bmax > 2 and/or the affinity for the radioligand is high [<1 nM]). Importantly, control microarray spots printed with the parental CHO and HEK293 membranes displayed significantly lower binding of 125I-labeled clonidine relative to the a2A and a2C spots (Fig. 3), demonstrating that the majority of the radioligand is bound to the overexpressed receptors. Further enhancements in the signal/noise ratio were obtained using the Caliper Sciclone nanodispenser to fabricate GPCR microarrays with even larger spots (Fig. 4). Although not specifically tested here, this printing approach should provide a robust assay for receptor preparations with expression levels below 0.8 pmol/mg of membrane protein. The Sciclone nanodispenser expels membrane onto the surface by physically pushing a defined volume (10 nl) of membrane sample out of a ceramic tip. This is in contrast to the quill pin approach, where capillary action pulls a fixed volume of membrane solution onto the glass surface. Depositing viscous membrane prepara-
α2C Bac kground
S/N: 160
175000 125000
S/N: 20
10000
0
CMP 10
Sciclone
125
Fig. 4. Comparison of I-labeled clonidine binding signal on a2C microarrays printed using contact printing (CMP10 pin) and noncontact dispensing (Sciclone). Microarrays of a2C membrane preparation were printed onto GAPS slides using either the CMP10 contact printing or the Sciclone nanodispenser. The 125I-labeled clonidine (4 nM) binding signal of the printed a2C membrane microspots and the local background signal (i.e., the amount of radioactivity bound nonspecifically to the GAPS surface) were quantified using the Typhoon 9210 PhosphorImager. The signal/noise (S/N) ratio was determined by comparing the signal intensity of the a2C membrane spot with the local background. The S/N ratios were 20 and 160 for the CMP10- and Sciclone-printed a2C microarrays, respectively.
tions often is difficult using quill pin devices (e.g., CMP10B). The Sciclone produces GPCR microspots with a diameter of approximately 850 lm. The signal/noise ratio was approximately eightfold higher on the GPCR membrane microspots produced by the Sciclone as compared with the CMP10B-printed microspots (Fig. 4). This improvement can be attributed to both a larger GPCR microarray spot size and a greater amount of membrane
Percentage of Total Signal
100 75 50 25 0
125 100 75 50 25
EK
0 H
H C
Cell line 125
225000
B 125
O
Percentage of Total Signal
A
275000
Relative Imager Units
270
Cell line
Fig. 3. Comparison of I-labeled clonidine binding signals for a2A and a2C membranes and control CHO and HEK293 parental cell line membranes in GPCR microarray format. GPCR microarrays containing a2A, a2C, CHO, and HEK293 membrane preparations printed with a CMP10 pin were incubated with 125I-labeled clonidine (16 nM), and the binding signal of each membrane microspot was quantitated using the Typhoon 9210 PhosphorImager. (A) Relative binding signals of a2A membrane and corresponding CHO parental cell line membrane microspots. (B) Relative binding signals of a2C membrane and corresponding HEK293 parental cell line membrane microspots.
Multiplexing GPCRs in microarrays / B. Posner et al. / Anal. Biochem. 365 (2007) 266–273
material deposited per unit area of GAPS-treated glass surface. Subsequent studies were conducted using Scicloneprinted GPCR microarrays. The a2A and a2C members of the adrenergic family display subtype selectivity for commercially available antagonists. Two of these antagonists, ARC239 and BRL44408, were used in competition assays on GPCR microarrays containing these a2 receptors. The goal was to determine whether the microarray format could be used to investigate receptor subtype selectivity and rank order potency for these two receptors simultaneously. Displacement of 125 I-labeled clonidine from a2A and a2C microarrays by increasing doses of BRL44408 and ARC239 was quantitated and analyzed. The resulting dose–response curves are shown in Fig. 5. The Ki values obtained for BRL44408 and ARC239 in the microarray format are in good agreement with published Ki values for these compounds (Table 2) [18]. Importantly, the anticipated rank order for these compounds against these two adrenergic receptor subtypes was observed. These results demonstrate the feasibility of parallel compound selectivity measurements of the a2A and a2C receptors using the radiometric GPCR microarray format.
Discussion The potential of multiplexed assays using a microarray approach has been realized during the past few years for DNA, and significant progress has been made for proteins [19,20]. However, the complexity of membrane-bound proteins has hindered the development of GPCR microarrays. The challenges have included maintaining bilayer fluidity, immobilizing protein and lipid to a surface, and maintaining the functional integrity of the receptor. Recently, the fabrication of GPCR microarrays was reported using contact pin printing technology [11]. Multiplexed GPCR binding assays can be performed using fluorescently labeled ligands; however, the number of ligands available for this
Table 2 Binding constants for BRL44408 and ARC239 for a2A and a2C using radiolabeled clonidine Ligand
Receptor subtype
Kia (nM)
Microarray Kib (nM)
BRL44408 BRL44408 ARC239 ARC239
a2A a2C a2A a2C
5.68 ± 1.86 150 ± 41 1330 ± 50 31.8 ± 1.3
25 ± 17 236 ± 50 382 ± 232 70 ± 42
a
See Ref. [18]. The microarray Ki values are reported as the means and standard deviations of at least two measurements. b
approach is limited, and some of these ligands exhibit high levels of nonspecific binding [11,13,21]. To expand the scope of the microarray approach, we have developed and validated a radiometric GPCR microarray assay. Robust detection of bound radioligand required larger GPCR microspots than the conventional GPCR microarray spots demonstrated previously [11]. The microarray spots were fabricated with either the CMP10 contact printing pin or the Sciclone nanodispenser, producing GPCR microspots with diameters of approximately 400 and 850 lm, respectively. Specific saturatable binding of 125 I-labeled clonidine to the subset of adrenergic receptors, the a2A and a2C subtypes, was demonstrated, and dissociation constants that compare favorably with those determined using a conventional filter-binding approach were obtained (Table 1). Detection of radioligand binding to the GPCR microarrays was accomplished using the Typhoon 9210 PhosphorImager. The signal/noise ratio was considerably higher for the microarray spots printed with the Sciclone nanodispenser (160 vs. 20 for the CMP10-printed microarrays), presumably due to the larger spot size and larger volume of membrane preparation printed with the Sciclone. The pharmacological fidelity of the immobilized receptors was further demonstrated using competitive displacement assays. Using the radiometric microarray assay, potency values for two receptor-selective reference
B 110
% Clonidine Bound
% Clonidine Bound
A
271
α2C α2A
80 50 20
120 α2A α2C
80
40
0 -10 -10
-9
-8
-7
-6
log [BRL44408] (M)
-5
-4
-9
-8
-7
-6
-5
-4
Log [ARC239] (M)
Fig. 5. GPCR microarray competition binding assays for adrenergic a2A and a2C receptors. Compound selectivity was determined for GCPR microarrays containing a2A and a2C receptors. 125I-labeled clonidine (4 nM) was incubated with the GPCR microarrays with half log dilutions of unlabeled BRL44408 (A) or ARC239 (B). Bound radioligand was quantitated using the Typhoon 9210 PhosphorImager, and the data were analyzed using GraphPad software.
272
Multiplexing GPCRs in microarrays / B. Posner et al. / Anal. Biochem. 365 (2007) 266–273
compounds, BRL44408 and ARC239, and the a2 receptors were in reasonably good agreement with published literature values obtained using a conventional filter-binding assay (Fig. 5 and Table 2) [18]. Importantly, appropriate rank order of Ki values was observed for the two compounds considered, demonstrating the feasibility of running selectivity screens of closely related GPCR family members that are of interest for academic and pharmaceutical research. Although the assays described were performed manually using a slide format, transition to a more convenient microplate format should be possible. We recently demonstrated printing of nine GPCR microspots inside the wells of a prototype 96-well GAPS microplate with the Sciclone nanodispenser (data not shown). The smaller GPCR microspots produced by the CMP10B will allow at least twice this density inside the well, although the signal/noise ratio is reduced with these smaller GPCR microspots (Fig. 4). However, the requirement for intimate contact between the microarray assay surface and the PhosphorImager screen during assay detection necessitates the use of a microplate that allows removal of the glass bottom. Commercially available devices that convert slides into a microplate format (e.g., ProPlate from Grace Biolabs) may be suitable for this application and remain to be tested. The development of a microplate-based radioligand microarray assay should enable efficient coupling of multiplexed GPCR targets (i.e., multiple GPCRs within each well) with compound screening. Although the direct measurement of ligand binding using radiolabeled ligands remains the predominant highthroughput assay format for characterization of GPCR ligand affinity and selectivity, the recent demonstration of functional GPCR microarrays represents a new dimension in microarray assays [22]. The functional GPCR microarray assays measure the binding of the fluorescently labeled GTP analogue to G proteins that have been activated via agonist-stimulated activation of their cognate GPCR [23]. This approach enables the identification of agonists, antagonists, and inverse agonists, and it complements ligand-binding approaches described here and in other works. Thus, multiplexed GPCR microarray assays have now been demonstrated using both fluorescent and radiometric formats. Of course, both approaches have inherent advantages and disadvantages. For example, the abundance of commercially available radioligands must be weighed against the disposal issues associated with radioactivity. In conclusion, the ability to monitor compound selectivity against a collection of targets in a single assay holds great promise for improving the efficiency of assaying and characterizing the interactions of GPCRs with natural and synthesized ligands. Potential benefits of the multiplexed GPCR assay format include reagent savings (e.g., 200–400-fold less receptor membrane), reduced consumption of compound libraries, and time savings. For example, a single 96-well microtiter plate containing
a 4 · 4 GPCR array in each well represents 1536 assays across multiple receptors. With continued progress, the microarray assay format should have a significant impact on the efficiency of secondary screening (e.g., selectivity) and ultimately may find use in high-throughput screening. Acknowledgments We are grateful to Jeremy Everett, Eryl Sharp, Chris Williams, Anne Schmidt, and Bill Martin for their support of this work and their critical review of the manuscript. We also thank Brian Bush for his support of this work and Csilla Jorgensen for her help in setting up and executing the 384-well filter plate assay. References [1] A.G. Gilman, G proteins: Transducers of receptor-generated signals, Annu. Rev. Biochem. 56 (1987) 615–649. [2] J.R. Hepler, A.G. Gilman, G proteins, Trends Biochem. Sci. 17 (1992) 383–387. [3] C. Liebmann, G protein-coupled receptors and their signaling pathways: Classical therapeutical targets susceptible to novel therapeutic concepts, Curr. Pharm. Des. 10 (2004) 1937–1958. [4] A.L. Hopkins, C.R. Groom, The druggable genome, Nat. Rev. Drug Disc. 1 (2002) 727–730. [5] K. Lundstrom, The future of G protein-coupled receptors as targets in drug discovery, IDrugs 8 (2005) 909–913. [6] A. Wise, K. Gearing, S. Rees, Target validation of G-protein coupled receptors, Drug Disc. Today 7 (2002) 235–246. [7] W.P. Walters, M. Namchuk, Designing screens: How to make your hits a hit, Nat. Rev. Drug Disc. 2 (2003) 259–266. [8] P.A. Johnston, P.A. Johnston, Cellular platforms for HTS: Three case studies, Drug Disc. Today 7 (2002) 353–363. [9] C. Williams, A. Sewing, G-protein coupled receptor assays: To measure affinity or efficacy that is the question, Comb. Chem. High Throughput Screen 8 (2005) 285–292. [10] Y. Fang, J. Lahiri, L. Picard, G protein-coupled receptor microarrays for drug discovery, Drug Disc. Today 8 (2003) 755–761. [11] Y. Fang, A.G. Frutos, J. Lahiri, Membrane protein microarrays, J. Am. Chem. Soc. 124 (2002) 2394–2395. [12] Y. Fang, A.G. Frutos, B. Webb, Y. Hong, A. Ferrie, F. Lai, J. Lahiri, Membrane biochips, BioTechniques 33 (Suppl.) (2002) S62–S65. [13] R.J. Middleton, B. Kellam, Fluorophore-tagged GPCR ligands, Curr. Opin. Chem. Biol. 9 (2005) 517–525. [14] J.P. Hieble, W. Bondinell, R.R. Ruffolo Jr., a- and (-Adrenoceptors: From the gene to the clinic: I. Molecular biology and adrenoceptor subclassification, J. Med. Chem. 38 (1995) 3415–3444. [15] L. Hein, B.K. Kobilka, Neurotransmitter receptors: IV. Adrenergic receptor signal transduction and regulation, Neuropharmacology 34 (1995) 357–366. [16] D.B. Bylund, D.C. Eikenberg, J.P. Hieble, S.Z. Langer, R.J. Lefkowitz, K.P. Minneman, P.B. Molinoff, R.R. Ruffolo Jr., U. Trendelenburg, International Union of Pharmacology nomenclature of adrenoceptors, Pharmacol. Rev. 46 (1994) 121–136. [17] J. Zhang, T.D. Chung, K.R. Oldenburg, A simple statistical parameter for use in evaluation and validation of high throughput screening assays, J. Biomol. Screen 4 (1999) 67–73. [18] S. Uhlen, A.C. Porter, R.R. Neubig, The novel a-2 adrenergic radioligand [3 H]-MK912 is a-2C selective among human a-2A, a-2B, and a-2C adrenoceptors, J. Pharmacol. Exp. Ther. 271 (1994) 1558– 1565. [19] E. Phizicky, P.I. Bastiaens, H. Zhu, M. Snyder, S. Fields, Protein analysis on a proteomic scale, Nature 422 (2003) 208–215.
Multiplexing GPCRs in microarrays / B. Posner et al. / Anal. Biochem. 365 (2007) 266–273 [20] M. Schena, D. Shalon, R.W. Davis, P.O. Brown, Quantitative monitoring of gene expression patterns with a complementary DNA microarray, Science 270 (1995) 467–470. [21] J.C. McGrath, S. Arribas, C.J. Daly, Fluorescent ligands for the study of receptors, Trends Pharmacol. Sci. 17 (1996) 393– 399.
273
[22] Y. Hong, B.L. Webb, H. Su, E.J. Mozdy, Y. Fang, Q. Wu, L. Liu, J. Beck, A.M. Ferrie, S. Raghavan, J. Mauro, A. Carre, D. Mueller, F. Lai, B. Rasnow, M. Johnson, H. Min, J. Salon, J. Lahiri, Functional GPCR microarrays, J. Am. Chem. Soc. 127 (2005) 15350–15351. [23] T. Haga, G. Berstein (Eds.), G Protein-Coupled Receptors, CRC Press, Boca Raton, FL, 1999.