Syntheses of Immobilized G Protein-Coupled Receptor

Laboratory of Clinical Investigation, Intramural Research Program, National Institute on Aging, NIH,. Baltimore, Maryland 21224. Opioid receptors are ...
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Anal. Chem. 2003, 75, 4480-4485

Syntheses of Immobilized G Protein-Coupled Receptor Chromatographic Stationary Phases: Characterization of Immobilized µ and K Opioid Receptors Farideh Beigi and Irving W. Wainer*

Laboratory of Clinical Investigation, Intramural Research Program, National Institute on Aging, NIH, Baltimore, Maryland 21224

Opioid receptors are members of the superfamily of G protein-coupled receptors (GPCRs). Liquid chromatographic stationary phases containing either the human µ or K opioid receptor subtypes have been developed to study the binding between the opioid receptors and their ligands. The receptors were obtained from chinese hamster ovary cells stably transfected with human µ or K receptor subtypes. The receptors were isolated through partial solubilization with sodium cholate detergent, and the partially purified receptor complex was immobilized in the phospholipid analogue monolayer of an immobilized artificial membrane liquid chromatographic stationary phase. The resulting phase was packed into a glass column (1.8 × 0.5 (i.d.) cm) and used in the on-line chromatographic determination of drug/ligand binding affinities to the immobilized opioid receptors. Preliminary on-line binding studies employing frontal chromatographic techniques were conducted with the known µ antagonist (naloxone) and a K agonist (U69593). The calculated dissociation constants (Kd) were 110 nM for naloxone and 84 nM for U69593. The results indicate that the immobilized receptors retained their ability to specifically bind ligands and were active for 1200 column volumes applied over two weeks. Zonal chromatographic experiments were also conducted, and competitive displacements of the marker ligands were observed. The results suggest that the immobilized opioid receptor stationary phases can be used to qualitatively assess the binding affinities of compounds to the immobilized receptors and represent the first example of the use of immobilized GPCRs in a chromatographic system. Opiates are a large class of pharmacologically active compounds that mediate various behaviors and effects, such as analgesia and euphoria.1,2 The activity of these compounds is a result of their binding to opioid receptors, which exist as the µ, * Corresponding author. Fax: 410-558-8409. Phone: (410)-558-8498. E.mail: [email protected]. (1) Jordan, B. A.; Cvejic S.; Devi, L. A. Neuropsychopharmacology 2000, 23, S4-S18. (2) Gether, U.; Kobilka, B. K. J. Biol. Chem. 1998, 273, 17979-17982.

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δ, and κ opioid receptor subtypes.3,4 The main endogenous ligands for the subtypes are known to be enkephalins, endorphins, and dynorphins.1,5 Opioid receptors are members of the superfamily of G proteincoupled receptors (GPCRs). GPCRs are characterized by the presence of seven transmembrane helices that are linked to the heterotrimeric G protein.5,6 These receptors function through a signaling pathway that is initiated via ligand binding to the receptor, followed by conformational changes that trigger the exchange of GDP for GTP and lead to the dissociation of the R-GTP subunit from the βγ subunit in the G protein complex.1,7,8 The GPCR family constitutes the largest class of cell surface receptors and a key target for the development of small-molecule drugs. Over 50% of marketed drugs are active at these receptors.9 In addition, it is estimated that in the mammalian genome ∼1000 genes encode for ∼10 000 GPCRs9 of which only about a few hundred are characterized. The rest are unknown orphan receptors whose activities have not been established. Since GPCRs are key targets for drug discovery, a number of high-throughput screens (HTS) have been developed. The predominate HTS approaches require the use of intact cells and are based upon the measurement of intracellular calcium or cAMP.10,11 The methods utilizing the measurement of intracellular calcium require the introduction of reporter genes into the target cells and measurements via colorimetric, chemiluminescence, or fluorescence spectrophotometry.10 The cAMP-based methods require pretreatment, incubations, lysis, and immunoassays.11 The cAMP levels are measured either by scintillation counting of incorporated radiolabels or with an ELISA-like assay. (3) Pleuvry, B. J. Br. J. Anesthesia 1991, 66, 370-380. (4) Conner, M.; Christie, McD. J. Clin. Exp. Pharm. Physiol. 1999, 26, 493499. (5) Wess, J. FASEB J. 1997, 11, 346-354. (6) Knapp, R. J.; Malatynska, E.; Collins, N.; Fang, L.; Wang, J. Y.; Hruby, V. J.; Roeske, W. R.; Yamamura, H. I. FASEB J. 1985, 9, 516-525. (7) Berntein, M. A.; Welch, S. P. Mol. Brain Res. 1998, 55, 237-242. (8) Wess, J. Pharmacol. Ther. 1998, 80, 231-264. (9) Marinissen, M. J.; Gutkind, J. S. Trends Pharmacol. Sci. 2001, 22, 386376. (10) Hamman, B. D.; Pollok, B. A.; Bennett, T.; Allen J.; Heim, R. J. Biomol. Screening 2002, 7, 45-55. (11) Chiulli, A. C.; Trompeter, K.; Palmer, M. J. Biomol. Screening 2000, 5, 239245. 10.1021/ac034385q Not subject to U.S. Copyright. Publ. 2003 Am. Chem. Soc.

Published on Web 07/30/2003

Another approach to the discovery of new drugs directed at GPCRs is the screening of compounds for their binding affinity to the selected target. One possible route to the development of the required screens is the immobilization of the target GPCRs on liquid chromatographic supports and the use of the resulting stationary phases in frontal or zonal affinity chromatographic studies. This technique has been previously reported for the membrane-bound ligated ion channels, such as the nicotinic acetylcholine receptors,12 and transmembrane transporters, such as the Glut1 glucose transporter13 and P-glycoprotein,14 but has not been applied to GPCRs. Among the GPCRs, bovine rhodopsin receptor15 and a fluorlabeled β2-adrenergic receptor16 have been immobilized on gold or glass surfaces using antibodies or by streptavidin-biotin affinity intereractions. The resulting immobilized receptors were utilized in binding studies employing surface plasmon resonance techniques15 or to monitor agonist-induced conformational changes using real-time fluorescence microscopy.16 This project was designed to develop GPCR-based liquid chromatographic stationary phases for on-line determination of ligand-receptor binding interactions. The GPCRs chosen for the initial studies were the µ and κ subtypes of the opioid receptor obtained from cell lines expressing these receptors. The chromatographic backbone was the immobilized artificial membrane (IAM) stationary phase developed by Pigeon and Venkataram.17 The IAM support is a monolayer of phospholipid analogues, with functional headgroups, covalently coupled to silica beads. The specific feature of IAM beads is the hydrophobicity of the monolayers that allows immobilization of membrane proteins and receptors.18,19 In the following study, the membranes containing the expressed opioid receptors were solubilized, IAM support was added to the resulting solution, and the mixture was dialyzed. The removal of the detergent allowed the lipid monolayer of the IAM beads to incorporate into the reassembled membrane and lead to the immobilization of the receptor on the stationary phase. The resulting opioid-receptor stationary phase was utilized in frontal and zonal affinity chromatographic studies. The data from theses studies indicate that the immobilized receptors retained their ability to specifically bind known opioid receptor agonists and antagonists. EXPERIMENTAL SECTION Materials. The radioactive compounds, [3H]-naloxone, [3H]DAMGO (D-Ala2, N-Me-Phe4, Gly5-ol enkephalin), and [3H]-U69593 ((+)-(5R,7R,8β)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]benzeneacetamide), were purchased from Amersham (12) Zhang, Y.; Xiao, X.; Kellar, K.; Wainer, I. W. Anal. Biochem. 1998, 264, 22-25. (13) Yang, Q.; Lundahl, P. Biochemistry 1995, 34, 7289-7294. (14) Lu, L.; Leonessa, F.; Clarke, H.; Wainer, I. W. Mol. Pharmacol. 2001, 59, 62-68. (15) Bieri, C.; Ernst, O. P.; Heyse, S.; Hofmann, K. P.; Vogel, H. Nat. Biotechnol. 1999, 17, 1105-1108. (16) Neumann, L.; Wohland, T.; Whelan, R. J.; Zare, R. N.; Kobilka, B. K. ChemBioChem 2002, 3, 993-998. (17) Pigeon, C.; Venkataram, U. V. Anal. Biochem. 1989, 176, 36-47. (18) Moaddel, R.; Lu, L.; Baynham, M.; Wainer, I. W. J. Chromatogr., B 2002, 768, 41-53. (19) Moaddel, R.; Cloix, J.-F.; Ertem, G.; Wainer, I. W. Pharm. Res. 2002, 19, 104-107.

Biosciences (Boston, MA). Egg phosphatidylcholine (PC) lipid was from Avanti Polar Lipids (Alabaster, AL). Fetal bovine serum (FBS) and 1× phosphate buffer saline (PBS) were from Bioscource International (Camarillo, CA). Ham’s F-12 nutrient media and Geniticin (G-418) were from Invitrogen Corp. (Carlsbad, CA). The enzyme inhibitors, phenylmethanesulfonyl floride (PMSF), benzamidine, leupeptin, and pepstatin A, and also all nonradioactive compounds and chemicals, naloxone, DAMGO, U69593, butorphanol, levorphanol, sodium cholate and igepal detergents, were from Sigma Aldrich (St. Louis, MO). Immobilized artificial membrane (IAM-PC) silica beads (12-µm particle size, 300-Å pore size) were from Regis Chemical Co. (Morton Grove, IL). Nitrocellulose dialysis tubing (cutoff at 10 000 Da) was from Pierce Chemical (Rockford, IL), and chromatographic glass columns HR 5/2 (i.d., mm/length, cm) were from Amersham Pharmacia Biotech (Uppsala, Sweden). Buffers. All the buffers were prepared at room temperature, the pH was adjusted to 7.4 using HCl (4 M), and the buffers were filtered (0.45 µm) and degassed prior to use. All the salts were of analytical grade. Homogenization buffer: MgCl2 (2 mM), PMSF (1 mM), benzamidine (1 mM), leupeptin (0.030 mM), pepstatin A (0.005 mM), and EDTA (1 mM) in 50 mM Trizma-hydrochloride (TrisHCl). Solubilization buffer: prepared from the homogenization buffer with the addition of 2% (w/v) sodium cholate detergent. Dialysis buffer: EDTA (1 mM), MgCl2 (2 mM), NaCl (150 mM), and PMSF (0.20 mM) in 50 mM Tris-HCl. Chromatographic running buffer: EDTA (1 mM) and MgCl2 (2 mM) in Tris-HCl (10 mM). Lysis buffer: NaCl (500 mM) supplemented with 2% Igepal and 2% sodium cholate detergents in Tris-HCl (10 mM). Cell Culture and Membrane Preparation. Chinese hamster ovary (CHO) cells stably transfected with the human µ receptor, hMOR, subtype20 were kindly provided by Dr. Jia Bei Wang (School of Pharmacy, University of Maryland, Baltimore, MD) and CHO cells stably transfected with the human κ receptor, hKOR, subtype,20 were kindly provided by Dr. Lawrence R. Toll (SRI International in Menlo Park, CA). The cells were cultured in plastic flasks in Ham’s F-12 media supplemented with 10% FBS and 0.25 mg/mL G-418, then harvested, and stored in PBS at 70 °C. The cells were cultured and harvested similarly in the Ham’s F-12 media supplemented with 10% FBS. For membrane preparation and immobilization, 108 cells were suspended in homogenization buffer and homogenized with a Versonic-100 sonicator (The Virtis Co. Inc., Gardiner, NY.) at setting 6 until a uniform suspension was achieved. For removal of large cell particles and organelles, the suspension was first centrifuged at 180g for 10 min, the pellet was discarded, and the supernatant was centrifuged at 55000g for 25 min. The resultant pellet was suspended in solubilization buffer overnight at 4 °C. The suspension was then centrifuged at 100000g for 35 min. Immobilization. The chromatographic support (180 mg, IAMPC) and PC-lipids (80 µM) were added to the supernatant containing the crude extracted receptor, and the resulting mixture was stirred at room temperature for 3 h, transferred into (5 cm (20) Xu, H.; Lu, Y.-F.; Rice, K. C.; Ananthan, S.; Rothman, R. B. Brain Res. Bull. 2001, 55, 507-511.

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length) nitrocellulose dialysis membrane, and placed in 1 L of dialysis buffer at 4 °C for 1 day. The dialysis step was repeated twice; each time fresh buffer was used. After dialysis, the mixture was centrifuged at 120g for 3 min, the supernatant was discarded, and the pellet of IAM support containing the immobilized receptor-bearing membrane was collected. The pellet was resuspended in 2 mL of running buffer, and the suspension was pumped through a HR 5/2 chromatographic glass column at a flow rate of 0.3 mL/min using a peristaltic pump. After the packing of the material as a gel bed, the end adaptors were assembled and the total gel-bed volume was 0.4 mL. Chromatographic Experiments. Chromatographic System. The chromatographic system consisted of an HPLC (10-AD) pump (Shimadzu Inc., Columbia, MD), a manually controlled FPLC injector (Amersham Biotechnology, Uppsala, Sweden) with either a small or a large sample loop, the packed immobilized receptor column, and an on-line radioactive flow detector (IN/US, Tampa, FL), all connected sequentially. In the zonal chromatographic studies, a 20-µL sample loop was used, and for the frontal chromatographic studies, sample volumes of 5-10 mL in the nanomolar range of concentration were applied continuously by a super loop (50 mL) until the elution profile showed a plateau region. Chromatographic Conditions. Column storage and the chromatographic experiments were carried out at room temperature. To eliminate the remaining detergent residues in the immobilizedmembrane and IAM beads, the column was equilibrated for at least 4-5 h prior to the initial use with the running buffer (see Buffer section) at 0.2 mL/min. Then the ligands were injected individually through the injector into the column and detected on-line by the radioflow detector in frontal and zonal chromatographic mode. Analysis of Frontal Chromatographic Data. The data were analyzed using the equation derived by Kasai et al.21 for the chromatographic determination of association and dissociation constants for water-soluble proteins. This equation was modified and adapted to membrane proteins22,23 where the number of binding sites and the dissociation constant can be determined by linear regression curve as in eq 1. In the above equation, Vi is the

1 1 1 ) + M Vi - Vmin PKa P

(1)

elution volume of the solute due to specific binding, Vmin is the elution volume of the solute at the saturation point (highest ligand concentration) of the receptor also corresponding to the nonspecific binding, P is the number of available binding sites, M is the concentration of the marker ligand and Ka and Kd are association and dissociation constants, respectively. Protein Extraction from Immobilized IAM Beads. After performance of all chromatographic experiments, the gel bed was removed from the glass column. A 100-µL aliquot of the stationary (21) Kasai, K.; Oda, Y.; Nishikata, M.; Ishii, S. J. Chromatogr. 1986, 376, 3347. (22) Brekkan, E.; Lundqvist, A.; Lundahl, P. Biochemistry 1996, 35, 1214112145. (23) Lundqvist, A.; Lundahl, P. J. Biochem. Biophys. Methods 2001, 49, 507521.

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phase was treated with a mixture of 2-propanol/water (75:25, v/v) and incubated for 2 h at 4 °C, the mixture was then centrifuged at 300g for 5 min, and the supernatant was collected and evaporated under vacuum using a rotary evaporator for 15 min at elevated temperature. The residue was resuspended in the running buffer and the protein content determined as described below. Protein Assay. The total amounts of solubilized membrane proteins and proteins stripped from the stationary phase were determined by bicinchoninic acid (BCA) protein assay (Pierce Biotechnology). The interference and compatibility of detergents and lipids were checked prior to final protein determinations. To eliminate detergent incompatibility with the protein assays, the solutions obtained from cell membranes or from the stationary phase were diluted 100-fold with the lysis buffer to a final concentration of 5 mM NaCl, 0.02% Igepal, and 0.02% sodium cholate detergents in 0.1 mM Tris-HCl at pH 7.4. A protein standard series was prepared with BSA in the concentration range of 0-20 µg/mL in the above-mentioned lysis buffer. The protein content was determined following the instructions provided in the Pierce BCA protein assay kit in which 2.5 mL of reagent A and 2.4 mL of reagent B were mixed with 0.1 mL of reagent C in a Falcon tube at 4 °C. Aliquots (100 µL) of samples and standards were added in triplicate to a 96-well plate, and 100 µL of the mixed BCA reagent was added to each well. The plate was incubated at 37 °C for 2 h, and the absorbance values were measured at λ ) 570 nm with a Bio-Rad (San Diego, CA) microplate reader. The amount of protein was calculated by using the Microsoft Excel program. RESULTS AND DISCUSSION Immobilization of Solubilized Receptors. In previous studies involving the immobilization of receptors and transporters on the IAM support, the solubilization buffers were composed of TrisHCl (50 mM, pH 7.4) containing 2% cholate (nicotinic acetylcholine receptors12) or 1.5% CHAPS (P-glycoprotein14) detergent. Dialysis of the mixture of the receptor/transporter containing buffer and IAM support removed the detergent and forced the receptor/ transporter into the support, producing viable immobilized receptor/transporter stationary phases. In this study, using the same methodology as above and dialysis of the receptor suspension containing cholate detergent did not produce any significant immobilization of the solubilized opioid receptors on the IAM support nor did the support display any observable specific opioid receptor binding activity. The results from a previous study of the isolation of GPCRs indicate that the detergents used in the solubilization and the dialysis processes removed lipids associated with the integrity of the receptor.24 Additional studies have demonstrated that the stability and integrity of solubilized GPCRs can be increased by the addition of lipids to the solution containing the solubilized receptors.25,26 On the basis of these results, phosphatidylcholine lipids (80 µM) were added to the supernatant containing the crude extracted receptor before the addition of the IAM stationary phase and the initiation of the dialysis. Using this approach, 20-30 µg (24) Lagane, B.; Gaibelet, G.; Meilhoc, E.; Masson, J.-M.; Cezanne, L.; Lopez, A. J. Biol. Chem. 2000, 275, 33197-33200. (25) Bidlack, J. M.; Abood, L. G. Life Sci. 1980, 27, 331-340. (26) Banerjee, P.; Joo, J. B.; Buse, J. T.; Dawson, G. Chem. Phys. Lipids 1995, 77, 65-78.

Table 1. Number of Binding Sites and Affinity Constants for the Opioid Receptor Ligands Obtained by Immobilized Opioid Receptor Chromatographic Matrixa ligand (opioid subtype)

binding sites (pmol)

dissociation const, Kd (nM)

naloxone (µ) U69593 (κ)

95 147

110 84

a The data are presented as the average of two determinations, N ) 2.

Figure 1. Frontal affinity chromatographic studies with an immobilized µ opioid receptor stationary phase: (A) frontal curves obtained using naloxone as the marker ligand in concentrations of (A) 0.08, (B) 30, (C) 80, (D) 160, and (E) 300 nM; (B) analysis of the frontal chromatographic data using eq 1, Y ) 0.0105x + 1.159; R 2 ) 0.99.

of proteins from the solubilized membranes was immobilized per milligram of support, as determined by BCA protein assay. The resulting stationary phases were packed into HR 5/2 columns, and the presence and activities of the immobilized opioid receptors were determined using frontal and zonal chromatography. The columns prepared in this manner had a bed volume of 0.4 mL, and when the mobile-phase flow rate was 0.2 mL/min, the columns retained activity for 1200 column volumes. Characterization of the Immobilized µ Opioid Receptor. The activity and specificity of the immobilized µ opioid receptor subtype was investigated using naloxone as the marker ligand. In each experiment, 0.05 nM of [3H]-naloxone was supplemented with nonlabeled naloxone and 5-10 mL of the sample was injected continuously onto the column until a front and a plateau was observed. Increasing the ligand concentrations shifted the curve to the left, indicative of specific binding between the ligand and the immobilized receptor, Figure 1A. After each run, the column was washed at the same flow rate for a minimum of 1 h or until the control sample (0.05 nM) was displaced back to its original position.

The relationship between ligand concentrations and the corresponding elution volumes obtained at half-heights of the chromatographic plateaus was investigated using eq 1, Figure 1B. The equation for the fitted linear regression line was Y ) 0.0105x + 1.159; R2 ) 0.99. The results of the analysis indicated that the number of active binding sites (Bmax) for naloxone on the immobilized receptor column was 95 pmol and the calculated dissociation constant (Kd) was 110 nM, Table 1. In a previous study, Xu et al20 in the presence of [35S]-GTP-γ-S used the CHO-hMOR cell line and determined the binding effect of naloxone (µ receptor antagonist) on the membrane of the agonist-stimulated receptor. The apparent functional inhibition constant (Ki) for this interaction was 2.0 nM, as calculated using the Cheng and Pursoff equation. While there is a 55-fold difference between their Ki and the chromatographic Kd values, the results indicate that the immobilization of the CHO-hMOR membranes did not qualitatively alter the binding on the µ receptor subtype. The quantitative difference between the two values may simply reflect an artifact of the chromatographic approach. The immobilization of a receptor onto a solid support can alter the observed Kd values through the restriction of the conformational freedom of the receptor, alteration of the kinetics associated with the association and dissociation of the receptor-ligand complexes, or both. This has been observed in previous studies with the nicotinic acetylcholine receptor12 and P-glycoprotein14 stationary phases. The difference may also reflect the fact that the calculated Ki value is a relative value derived using functional data (IC50 and ED50 values); thus, a 55-fold difference may be well within the experimental errors associated with both experiments. The possibility that immobilization affects binding affinities on the µ opioid receptor was also suggested by frontal displacement studies using naloxone and the enkephalin peptide DAMGO. The apparent functional Ki of DAMGO in the rat CHO-rMOR cell line was 8 nM, roughly equivalent to that of naloxone for human CHOhMOR.20 However, when DAMGO was used as the displacer, mobile-phase concentrations of up to 30 µM had no effect on the retention volume of [3H]-naloxone; in this instance, the marker ligand concentration was 80 pM. When the situation was reversed and [3H] DAMGO was the marker and naloxone was the displacer, a 20 nM concentration of naloxone displaced the [3H] DAMGO peak by 20 µL and the 60 nM concentration produced a 60-µL displacement. The results suggest that there was a decrease in DAMGO affinity produced by the immobilization itself or by the ionic detergent used in the immobilization, since these detergents have been shown to decrease the opioid receptor binding affinities of Analytical Chemistry, Vol. 75, No. 17, September 1, 2003

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Table 2. Difference in Displaced Volumes of Marker Ligands, Naloxone (1.5 nM) on µ and or U69593 (0.05 nM) on K Opioid Subtypes, to the Displacer Compounds As Analyzed by Zonal(µ) and Frontal(K) Affinity Chromatography, Respectivelya displacer compd (receptor)

concn

displaced vol, ∆V (µL)

naltrexone(µ) levorphanol(µ) methadone(µ) norbinaltorphimine(κ) nobinaltorphimine(κ)

0.2 mM 0.15 mM 0.15 mM 20 nM 40 nM

300 680 740 560 750

a The data are presented as the average of two determinations, N ) 2.

some enkephalin peptides.27 The data may also reflect the fact that the chromatographic process has identified multiple binding sites on the µ opioid receptor. The results suggest that DAMGO may share a binding site with naloxone, which is a low-affinity site for DAMGO and a high-affinity site for naloxone. In addition, DAMGO may bind at a second, high-affinity site, reflected by the apparent Ki value. The previous membrane binding studies with the CHO-hMOR cell line did not establish a Bmax for the binding of naloxone to the µ receptor subtype. However, a saturation binding study of 6β-iodo-3,14-dihydroxy-17-cyclopropylmethyl-4,5R-epoxymorphinan indicated that for this drug the number of active binding sites were 863 fmol/mg of proteins. In this study, it was determined that the stationary phase contained 20-30 µg of proteins/mg of gel matrix from the solubilized membranes and the calculated Bmax for naloxone was 95 pmol for the whole column bed. Thus, the apparent number of active binding sites per mg of protein was 20 pmol. There is a ∼30-fold difference in the number of available binding sites between the two methods, which could be a reflection of the fact that two different ligands and two different experimental approaches were used to generate the data. However, the difference also raises the question of whether the amount of immobilized protein can be adequately measured and whether this determination has any relevance to the characterization of the immobilized receptor stationary phase. The results of the frontal affinity studies indicate that when 108 CHO-hMOR cells were used, the resulting stationary phase contained 95 pmol of active naloxone binding sites. The number of active binding sites and not the estimated amount of immobilized protein should be the parameter used in the optimization of this and other GPCRbased stationary phases. The ability of the immobilized µ opioid receptor stationary phase to identify competitive binding was investigated using zonal chromatography with [3H]-naloxone (1.5 nM) as the marker ligand and known specific agonists and antagonists as the displacers. The addition of the competitive antagonist naltrexone (200 nM) or the competitive agonists levorphanol (150 nM) or methadone (150 nM) to the mobile phase reduced the chromatographic retentions of the marker ligand, Table 2and Figure 2. To our knowledge, the absolute or relative Kd values for the binding of these compounds to the human µ opioid receptor (27) Hulme, E. C. Receptor Biochemistry A Practical Approach; Oxford University Press: New York, 1990; Chapter 4.

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Figure 2. Zonal displacement chromatographic studies with an immobilized µ opioid receptor stationary phase using 1.5 nM [3H]naloxone as the marker ligand and (A) no displacer in the mobile phase, (B) 200 nM naltrexone added to the mobile phase, (C) 150 nM levorpahnol added to the mobile phase, and (D) 150 nM methadone added to the mobile phase.

subtype have not been reported. Indeed, a major pharmacology text ranks opioid receptor agonists and antagonists using pluses (+) for agonists and minuses (-) for antagonists to reflect their functional activity.28 In this system, the rating for naltrexone is (- - -) and the ratings for levorphanol and methadone are (+ + +). These data are neither inconsistent with nor comparable to the chromatographic data. In previous studies with the nicotinic acetylcholine receptor12 and P-glycoprotein14 stationary phases, the chromatographic data could be compared to Kd values obtained using standard membrane binding assays. The differences between the data were shown to be quantitative, not qualitative, and could be related by simple linear regression analyses. Since these data are not available for the µ opioid receptor subtype used in this study, this correlation cannot be established at this time. Characterization of the Immobilized K Opioid Receptor. The activity and specificity of the immobilized κ opioid receptor subtype were investigated using the κ agonist U69493 as the marker ligand. In these experiments, 0.08 nM [3H]-U69593 was supplemented with nonlabeled U69493 to produce ligand concentrations of 0.08, 40, 150, and 300 nM. In the same way as for the µ subtype, increasing the ligand concentrations shifted the marker curve to the left, indicative of specific binding between the ligand and the immobilized receptor, Figure 3. After each run, the column was washed at the same flow rate for a minimum of 1 h or until the control sample (0.08 nM) was displaced back to its original position. The relationship between ligand concentrations and the corresponding elution volumes obtained at half-heights of the chromatographic plateaus was investigated using eq 1. This equation was fitted to the linear regression curve Y ) 0.0068x + 0.5715; R2 ) 0.89. The results of the analysis indicated that the number of active binding sites for U69493 on the immobilized receptor was 147 pmol and the calculated dissociation constant (Kd) was 84 nM, Table 1. (28) Hardman, J. G., Limbird, L. E., Goodman Gilman, A., Eds. The pharmacological Basis of Therapeutics, 10th ed.; McGraw-Hill: New York, 2001; Chapter 23.

Figure 3. Frontal affinity chromatographic studies with an immobilized κ opioid receptor stationary phase using U69493 as the marker ligand in concentrations of (A) 0.08, (B) 40, (C) 150, and (D) 300 nM.

The Kd for the binding interaction between U69493 and the human κ receptor subtype, expressed in the CHO-hKOR, has not been reported, and the results cannot be directly compared with in vitro data. However, a Kd value has been reported for the binding of U69493 to κ opioid receptors isolated from frog brain tissue, Kd ) 6.8 nM.29 Although the data cannot be directly compared, the 12-fold difference between the two values indicates that the results obtained on the immobilized κ opioid receptor stationary phase are reasonable. The ability of the immobilized κ opioid receptor stationary phase to identify competitive binding was investigated using [3H]U69593 (0.08 nM) as the marker ligand with a known specific antagonist used as the displacer in frontal affinity chromatography. The addition to the mobile phase of 20 and 40 nM concentrations of the competitive antagonist norbinaltrophimine displaced the marker ligand by 560 and 750 µL, respectively, Table 2. However, similar concentrations of the competitive agonists butorphanol and dynorphins B or the competitive antagonist naloxone did not displace the marker ligand (data not shown). CONCLUSION This work reports the immobilization of G protein-coupled receptors on a liquid chromatographic stationary phase and their (29) Newman, L. C.; Sands, S. S.; Wallace, D. R.; Stevens, C. W. J. Pharmacol. Exp. Ther. 2002, 301, 364-370. (30) Milligan, G.; Bond, R. A. Trends Phys. Sci. 1997, 18, 468-474.

use in on-line affinity and displacement chromatographic studies. The results of these studies indicate that the µ and the κ opioid receptor stationary phases retained their abilities to bind known agonists and antagonists of the respective receptor subtypes. The data suggest that the opioid receptor stationary phases could be used to assess the affinity of a compound for these receptors, although it is not clear whether this would be strictly a qualitative assessment or whether a quantitative measure of specific binding affinity, i.e., Kd values, could be obtained. However, to our knowledge, no comparable method exists for the on-line ranking of potential opioid ligands using liquid chromatographic techniques and the results of these studies are a positive step toward the establishment of such a screen. One of the aims of this project was to capture and immobilize both the receptor and its related G protein into the stationary matrix. Our preliminary results demonstrate that the GPCR has been immobilized; however, the presence of the G protein in the immobilized membrane-stationary phase complex has not been established. This will be the subject of further studies with this system as will the optimization of the solubilization and immobilization processes. The experimental approach described in the paper can be adapted to other GPCRs including known and orphan receptors, and also, ligands can be detected by detection methods such as mass spectrometry and all other standard chromatographic detection systems. Even though on-line affinity chromatography using the resulting immobilized GPCR stationary phases would not necessarily produce functional data, the approach can be used to identify ligands for known and unknown receptors, thus aid in “deorphanizing” a receptor. The method may also be useful in the identification of agonists, inverse agonist,30 antagonists, and noncompetitive antagonists that bind to the immobilized receptors, classifying the receptors in a specific family and subsequently obtaining first-generation drugs for therapeutic purposes. Experiments confirming these hypotheses are currently underway and will be reported elsewhere.

Received for review April 14, 2003. Accepted July 1, 2003. AC034385Q

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