Identification of Regions of the -1 Receptor Ligand Binding Site Using a Novel Photoprobe

Receptors, once considered a class of opioid receptors, are now regarded as a unique class of receptors that contain binding sites for a wide range of ligands, including the drug 1-N(2 ,6 -dimethylmorpholino)3-(4-t-butylpropylamine) (fenpropimorph), a yeast sterol isomerase inhibitor. Because fenpropimorph has high-binding affinity to the -1 receptor, we have synthesized a series of fenpropimorph-like derivatives with varying phenyl ring substituents and have characterized their binding affinities to the -1 receptor. In addition, we have synthesized a carrier-free, radioiodinated fenpropimorph-like photoaffinity label, 1-N-(2 ,6 -dimethyl-morpholino)-3-(4-azido3-[I]iodo-phenyl)propane ([I]IAF), which covalently derivatized the -1 receptor (25.3 kDa) in both the rat liver and guinea pig liver membranes and the -2 receptor (18 kDa) in rat liver membranes with high specificity. Furthermore, after cleaving the specific [I]IAF-photolabeled -1 receptor in guinea pig and rat liver membranes and the pure guinea pig -1 receptor with EndoLys-C and cyanogen bromide, the [I]IAF label was identified both in a peptide containing steroid binding domain-like I (SBDLI) (amino acids 91–109) and in a peptide containing steroid binding domain-like II (SBDLII) (amino acids 176–194). Because a single population of binding sites (R 0.992) for [I]IAF interaction with the -1 receptor was identified by ( )-[H]pentazocine competitive binding with nonradioactive [I]IAF, it was concluded that SBDLI (amino acids 91–109) and SBDLII (amino acids 176–194) comprises, at least in part, regions of the -1 receptor ligand binding site(s). The receptor is a unique receptor that, for the past 30 years, has been persistently enigmatic. receptors were initially proposed to be subtypes of opioid receptors based on work performed by Martin and colleagues (1976) to study the actions of antipsychotic drugs. In these experiments, they proposed the existence of a -opioid receptor based on the psychomimetic effects of SKF-10047, which could not be explained by or -opioid receptors (Martin et al., 1976). This hypothesis, however, was later refuted when the receptor was shown to be insensitive to naloxone, a common opioid receptor antagonist (Iwamoto, 1981; Su, 1982; Vaupel, 1983). As a result, receptors were reclassified as unique nonopioid and nonphencyclidine binding sites present in the central nervous system and peripheral organs that are distinct from other known neurotransmitter or hormone receptors (Quirion et al., 1992). To date, two subtypes of the receptor have been identified, the -1 and -2 receptors, which are distinguishable by their pharmacology, function, and molecular mass. The -1 receptor was first cloned from guinea pig liver in 1996 (Hanner et al., 1996) and subsequently from other sources, including human placental choriocarcinoma cells (Kekuda et al., 1996), human brain (Prasad et al., 1998), rat brain (Seth et al., 1998; Mei and Pasternak, 2001), and mouse brain (Pan et al., 1998). The -2 receptor, however, has yet to be cloned. The -1 receptor has 223 amino acids and shares 30% identity and 67% similarity with a yeast sterol C8–C7 isomerase, which is involved in cholesterol synthesis (Moebius et al., 1997). Unlike this yeast sterol isomerase, however, the -1 receptor does not have any sterol isomerase activity (Hanner et al., 1996), and it shares no sequence homology with any known mammalian proteins, including the mammalian C8–C7 sterol isomerase. This work was supported by the National Institutes of Health grant R01MH065503 (to A.E.R.). Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.107.038307. ABBREVIATIONS: SKF-10047, N-allyl-normetazocine; IACoc, 3-iodo-4-azido cocaine; IAF, 1-N-(2 ,6 -dimethyl-morpholino)-3-(4-azido-3-iodophenyl) propane; SBDLI, sterol binding domain-like I; SBDLII, sterol binding domain-like II; TMD, transmembrane domain; CNBr, cyanogen bromide; PAGE, polyacrylamide gel electrophoresis; DTG, ditolylguanidine; MBP, maltose binding protein; THF, tetrahydrofuran; TLC, thin-layer chromatography; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. 0026-895X/07/7204-921–933$20.00 MOLECULAR PHARMACOLOGY Vol. 72, No. 4 Copyright © 2007 The American Society for Pharmacology and Experimental Therapeutics 38307/3255037 Mol Pharmacol 72:921–933, 2007 Printed in U.S.A. 921 at A PE T Jornals on M ay 4, 2017 m oharm .aspeurnals.org D ow nladed from In mammalian systems, the -1 receptor is ubiquitously expressed in the central nervous system and peripheral organs of the endocrine and immune systems. Because of its broad distribution among tissues, it is speculated that the -1 receptor is able to mediate different cellular events such as modulation of voltage-gated K channels (Wilke et al., 1999), calcium release (Hayashi and Su, 2001), regulation of lipid compartmentalization on the endoplasmic reticulum (Hayashi and Su, 2003), regulation of cocaine effects (McCracken et al., 1999a,b), neuroprotective effects (Lysko et al., 1992), increase in extracellular acetylcholine levels (Matsuno et al., 1993), and inhibition of proliferative responses to mitogens (Paul et al., 1994). -1 Receptor knockout mice are viable and fertile, showing no overt constitutive phenotype (Langa et al., 2003). Langa and colleagues did find, however, that when the knockout mice were injected with the ligand ( )SKF-10047, there was abrogation of the hypermotility response, suggesting a role for -1 receptors in psychostimulant actions, which is further supported by the fact that methamphetamine also binds to the -1 receptor (Nguyen et al., 2005). A unique trait of this receptor is that it has high binding affinity for an assorted array of naturally occurring compounds such as steroids and neuropeptides, but it has yet to be determined which, if any, of these compounds are the endogenous ligands for the -1 receptor. Pharmacological studies have indicated that this receptor binds to a wide range of compounds including opiates, antipsychotics, antidepressants, antihistamines, phencyclidine-like compounds, -adrenergic receptor ligands, serotonergic compounds, cocaine and cocaine analogs, neurosteroids, and neuropeptides. Previous work from our laboratory showed that a cocainebased radioiodinated photoaffinity label [I]3-iodo-4-azido cocaine ([I]IACoc) is a high-affinity ligand for the -1 receptor (Kahoun and Ruoho, 1992) and specifically identified steroid binding domain-like II (amino acids 176–194) as part of the guinea pig -1 receptor binding site for cocaine (Chen et al., 2007). In this article, we report the synthesis and characterization of several fenpropimorph-like ligands that bind to -1 and -2 receptors as determined in guinea pig liver membranes and rat liver membranes, respectively. The synthesis of the carrier-free, radioiodinated fenpropimorph-like photoaffinity label, 1-N-(2 ,6 -dimethyl-morpholino)-3-(4-azido-3-[I]iodo-phenyl)propane ([I]IAF), which covalently derivatizes both the -1 and -2 receptors with high specificity, is reported. In addition, -1 receptor binding site peptides that were specifically derivatized by [I]IAF upon photolysis have been identified both for the membrane-bound -1 receptor and the pure guinea pig -1 receptor (Ramachandran et al., 2007) using cleavage strategies with EndoLys-C and cyanogen bromide. Materials and Methods General. Melting points were determined with a Thomas-Hoover capillary melting point apparatus and are reported uncorrected. NMR spectra were recorded on a Varian 300 spectrometer with the free-base form of the compounds except where noted (Varian, Palo Alto, CA). Spectra were obtained in CDCl3 with tetramethylsilane as an internal standard. Chemicals were obtained from Aldrich Chemical Co. (Milwaukee, WI). Elemental analysis was performed by the Research Institute of Petroleum Industry (Tehran, Iran). Frozen rat and guinea pig livers were obtained from Pel-Freez (Rogers, AR). The synthesis of 1-N-(2 ,6 -dimethyl-morpholino)-3-(4-fluoro-3phenyl propionamide (2) and 1-N-(2 ,6 -dimethyl-morpholino)-3-(4fluorophenyl-propylamine) (3) is outlined in Scheme 1 and that of the remainder of the compounds (6-11) is outlined in Scheme 2. 1-N-(2 ,6 -Dimethyl-morpholino)-3-(4-fluoro-3-phenyl propionamide) (2). To a solution of dicyclohexylcarbodiimide (5) (4.5 g, 22 mmol) in CH2Cl2 (120 ml), a mixture of 3-(4-fluoro-3-phenyl) propionic acid (1) (3.4 g, 20 mmol) in CH2Cl2 (80 ml) was added. After 10 min of stirring at room temperature, 2,6-dimethylmorpholine (22 mmol, 2.5 g) was added drop-wise. The reaction mixture was stirred at room temperature until the starting acid disappeared (3 h) (TLC, hexane/EtOAc, 9:1). The solid was removed by filtration, and the solution was washed with H2O (2 100 ml), 10% NaHCO3 (2 100 ml) before drying with MgSO4. The solvent was evaporated by rotary evaporator, and the crude product was purified by column chromatography (silica gel, n-hexane/EtOAc, 9:1) as an oil with 90% yield (4.77 g, 18 mmol). H NMR: 7.18 (m, 2 H), 6.97 (m, 2 H), 6.05 (S, 1 H, NH), 3.84 (m, 2 H), 3.43 (d, J 8.1 Hz, 4H), 2.83 (t, 2 H), 2.51 (t, 2 H), 1.21 (d, J 8.0 Hz, 6 H). C: 173.6, 139.6, 135.0, 129.7 (2 C), 115.9 (2 C), 71.1 (2 C), 57.8 (2 C), 34.5, 31.6, 18.4. Anal. Calcd. for C15H20FNO2: C, 67.90; H, 7.55; N, 5.28%. Found: C, 68.00; H, 5.67; N, 6.10%. 1-N-(2 ,6 -Dimethyl-morpholino)-3-(4-fluorophenyl-propylamine) (3). In a double-necked round-bottomed flask equipped with septum and condenser, a solution of 1-N-(2 ,6 -dimethylmorpholino)-3-(4-fluoro-3-phenyl propionamide) (2) (2.65 g, 10 mmol) was added drop-wise to a stirred solution of LiAlH4 (0.74 g, 20 mmol) in anhydrous tetrahydrofuran (THF) (20 ml) under argon. TLC indicated that the reaction was almost completed after 15 min at room temperature. The reaction was driven to completion by brief refluxing (15 min), and the reaction mixture was cooled to room temperature followed by the dilution with 100 ml of THF. The excess LiAlH4 was destroyed by drop-wise addition of water (3 ml), 15% aqueous NaOH (3 ml), and finally water (10 ml). The reaction mixture was stirred for 30 min at room temperature, and the solids were removed by filtration. The filtrate was dried with MgSO4 and evaporated by a rotary evaporator to give pure product as yellow oil in 95% yields (2.38 g, 9.5 mmol). H NMR: 7.12 (m, 2 H), 6.95 (m, 2 H), 3.84 (m, 2 H), 3.33 (d, J 8.1 Hz, 4H), 2.38 (t, 2 H), 2.55 (m, 2 H), 1.72 (t, 2 H), 1.21 (d, J 8.0 Hz, 6 H). C: 160.6, 135.6, 130.0 (2 C), 115.7 (2 C), 71.0 (2 C), 57.8 (2 C), 34.5, 34.6, 32.2 18.6. Anal. Calcd. for C15H22FNO: C, 71.68; H, 8.82; N, 5.57%. Found: C, 71.59; H, 8.90; N, 5.70%. 1-N-(2 ,6 -Dimethyl-morpholino)-3-phenyl propane (6). A mixture of 1-bromo-3-phenyl propane (4) (3 mmol, 0.59 g) and 2,6dimethylmorpholine (5) (3.6 mmol, 0.41 g) was refluxed overnight, and the reaction mixture was cooled and purified by column chromatography (silica gel, n-hexane/EtOAc, 4:1) in yellow oil in 98% yield (0.6 g, 2.94 mmol). H NMR: 7.21–7.03 (m, 5 H), 3.67 (m, 2 H), 2.65 (m, 4 H), 2.31 (t, 2 H,), 1.77 (m, 2 H), 1.69 (t, 2 H), 1.18 (d, 6 H). C NMR, 128.46, 126.60, 126.03, 124.12, 73.23, 56.42, 52.25, 39.89, 26.55, 19.31. Anal. Calcd. for C15H23NO: C, 77.21; H, 9.93; N, 6.00%. Found: C, 77.48; H, 10.03; N, 5.78%. 1-N-(2 ,6 -Dimethyl-morpholino)-3-(4-nitrophenyl) propane and 1-N-(2 ,6 -dimethyl-morpholino)-3-(2-nitrophenyl) propane (7). In a round-bottomed flask, a mixture of 1-N-(2 ,6 -dimethyl-morpholino)-3-phenyl propane (6) (3 mmol, 0.69 g), Bi(NO3)3 5H2O (1.5 mmol, 0.73 g), and trifluoroacetic anhydride (3 mmol, 0.42 ml) was stirred at room temperature under solvent-free conditions Scheme 1. Schematic diagram for the synthesis of compounds 2 and 3. 922 Pal et al. at A PE T Jornals on M ay 4, 2017 m oharm .aspeurnals.org D ow nladed from (Hajipour et al., 2003, 2005a,b; Hajipour and Ruoho, 2005a,b). After approximately 5 min, the starting material, 1-N-(2 ,6 -dimethyl-morpholino)-3-phenyl propane (6), was consumed (determined by TLC developed in n-Hex/EtOAc, 4:1). The product was isolated by precipitation in CH2Cl2 (20 ml). The solvent was evaporated by a rotary evaporator, and the red residue was purified by column chromatography (silica gel, n-hexane/EtOAc, 4:1). The final product (yellow oil) consisted of a mixture of 2and 4-nitrobenzene isomers in a ratio of 9:1 (the isomers were not separated). The yield of the reaction was 90% (2.7 mmol, 0.75 g). H NMR: 7.14–8.23 (m, 4 H), 3.67 (m, 2 H), 2. 75 (m, 0.4 H), 2.71 (m, 3.6 H), 2.33 (m, 2 H), 1.79 (m, 2 H), 1.69 (m, 2 H), 1.16 (d, 0.6 H), 1.13 (d, 5.4 H). C NMR, 147.25, 144.12, 134.15, 130.25 128.46, 126.60, 0.126.03, 125.38, 124.12, 73.23, 59.70, 59.40, 58.09, 56.42, 52.25, 39.89, 28.66, 27.78, 26.55, 19.31. Anal. Calcd. for C15H22N2O3: C, 64.73; H, 7.97; N, 10.06%. Found: C, 64.52; H, 8.13; N, 9.87%. 1-N-(2 ,6 -Dimethyl-morpholino)-3-(4-aminophenyl) propane (8). The mixture of 1-N-(2 ,6 -dimethyl-morpholine)-3-(4-nitrobenzene) propane and 1-N-(2 ,6 -dimethyl-morpholine)-3-(2-nitrobenzene) propane (7) (3 mmol, 0.83 g) was reduced to the amino derivatives using Pd/C (10%, 0.20 g) in methanol and H2 gas overnight. The reaction mixture was filtered to give 1-N-(2 ,6 -dimethylmorpholino)-3-(aminophenyl) propane (8) as a mixture of 2and 4-aminobenzene isomers in 96% yield (2.88 mmol, 0.71 g). The 1-N(2 ,6 -dimethyl-morpholine)-3-(4-aminobenzene) propane isomer was separated from 1-N-(2 ,6 -dimethyl-morpholine)-3-(2-aminobenzene) propane by column chromatography (silica gel, n-hexane/EtOAc, 4:1) (90%, 0.64 g).Yellow oil. H NMR: 7.13 (d, J 8.4, 2 H), 6.63 (d, J 8.4, 2 H), 4.80 (br, 2 H, NH2), 3.75 (m, 2 H), 2.74 (m, 4 H), 2.62 (t, 2 H,), 2.22 (m, 2 H), 1.85 (t, 2 H), 1.18 (d, 6 H). C NMR, 129.93, 126.24, 118.46, 115.24, 71.73, 59.69, 58.43, 32.84, 26.46, 19.30. Anal. Calcd. for C15H24N2O: C, 72.54; H, 9.74; N, 12.28%. Found: C, 72.68; H, 9.86; N, 12.68%. 1-N-(2 ,6 -Dimethyl-morpholino)-3-(4-amino-3-iodo-phenyl) propane (9). In a mortar, a mixture of 1-N-(2 ,6 -dimethyl-morpholino)-3-(4-aminophenyl) propane (8) (3 mmol, 0.74 g), Me4N ICl2 (3.6 mmol, 1.0 g) was ground with a pestle under solid-state conditions for 30 min to afford 1-N-(2 ,6 -dimethyl-morpholino)-3-(4-amino-3-iodo-phenyl) propane (9). The reaction mixture was dissolved in a mixture of water and ethyl acetate (100 ml, 1:1). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (2 25 ml). The combined ethyl acetate solution was dried (MgSO4), decolorized with activated charcoal (2 g), and filtrated. The solvent was evaporated with a rotary evaporator to afford 1-N-(2 ,6 dimethyl-morpholino)-3-(4-amino-3-iodo-phenyl) propane (9) as a yellowish oil, 90% yield (2.7 mmol, 1.01 g). The crude product was used for the next step without purification. 1-N-(2 ,6 -Dimethyl-morpholino)-3-(4-azido-3-iodo-phenyl) propane (10). In a round-bottomed flask, 1-N-(2 ,6 -dimethylmorpholino)-3-(4-amino-3-iodo-phenyl) propane (9) (3 mmol, 1.12 g) was dissolved in 20% HCl (v/v) and allowed to stand for 5 min at 0°C. NaNO2 (3.6 mmol, 0.21 g, in 5 ml of H20) was then added, and the reaction mixture was stirred at room temperature for 30 min. An aqueous solution of NaN3 (3.6 mmol, 0.23 g, in 5 ml of H20) was added drop-wise to the reaction mixture. The reaction was stirred at room temperature in the dark for 30 min and then extracted with ethyl acetate (3 30 ml). The combined ethyl acetate extracts were dried with MgSO4, and the solvent was evaporated using a rotary evaporator to afford an orange oil. The crude product was purified by column chromatography (silica gel, initially toluene/Et2NH, 20:1, followed by toluene/Et2NH, 4:1). Yield, 95%, (2.85 mmol, 1.14 g), Yellowish oil. H NMR: 8.40 (d, J 1.8, 1 H), 8.20 (dd, J 1.8, 8.4 1 H), 7.2 (d, J 8.4, 1 H), 3.75 (m, 2 H), 2.74 (m, 4 H), 2.62 (t, 2 H,), 2.22 (m, 2 H), 1.85 (t, 2 H), 1.18 (d, 6 H). C NMR, 158.90, 139.10, 135.25, 130.20, 128.10, 127.40, 71.61, 59.73, 58.43, 32.84, 26.46, 19.30. Anal. Calcd. for C15H22IN4O: C, 44.90; H, 5.53; N, 13.96%. Found: C, 45.11; H, 5.79; N, 13.74%. 1-N-(2 ,6 -Dimethyl-morpholino)-3-(4-azidophenyl) propane (11). This reaction was performed as described above using 1-N(2 ,6 -dimethyl-morpholino)-3-(4-aminophenyl) propane (8) (3 mmol, 0.74 g) as the starting material. The crude product was purified by column chromatography as described above (silica gel, first toluene/ Et2NH, 20:1, and then toluene/Et2NH, 4:1) to yield 1-N-(2 ,6 -dimethyl-morpholino)-3-(4-azidophenyl) propane (11) in 96% yield (0.26 g, 2.88 mmol), m.p. 128–131. H NMR, 7.92 (d, J 8.4, 2 H), 7.23(d, J 8.4, 2 H), 3.72 (m, 2 H), 2.74 (m, 4 H), 2.62 (t, 2 H,), 2.22 (m, 2 H), 1.85 (t, 2 H), 1.18 (d, 6 H). C NMR, 142.13, 135.25, 130.20, 128.40, 71.61, 59.73, 58.43, 32.84, 26.46, 19.30. Anal. Calcd. for C15H23N4O: C, 65.43; H, 8.42; N, 20.35%. Found: C, 64.29; H, 8.59; N, 20.54%. Scheme 2. Schematic diagram for the synthesis of compounds 6, 7, 8, 9, 10, and 11. Mapping of -1 Receptor Ligand Binding Site 923 at A PE T Jornals on M ay 4, 2017 m oharm .aspeurnals.org D ow nladed from Radiosynthesis of [I]IAF. Radioactive Na[I] (5 mCi) in 14 l of 0.1 M NaOH was neutralized by adding 14 l of 0.1 M HCl and then diluted with 50 l of 0.5 M sodium acetate buffer, pH 5.6. 1-N-(2 ,6 -dimethyl-morpholino)-3-(4-amino-phenyl) propane (10 l of 2.5 mg/ml in 0.5 M sodium acetate buffer, pH 5.6) was then added to the reaction. Iodination was initiated by adding 30 l of Chloramine-T (1 mg/ml in water) and continued for 15 min at room temperature. The reaction was terminated by the addition of 100 l of Na2S2O5 (5 mg/ml in water). The pH of the reaction was adjusted to approximately 9 by adding 20 l of 1 M NaOH. The reaction mixture was then extracted three times with 1 ml of ethyl acetate. The pooled extracts were evaporated under N2 to approximately 50 l and streaked on a 0.25-mm thick silica gel (60/254) plate (10 20 cm), which was developed with a solvent system of toluene/diethylamine (4:1 v/v). 1-N-(2 ,6 -Dimethyl-morpholino)-3-(4-amino-3-[I]iodophenyl)propane was detected by autoradiography using X-Omat film (Rf 0.63) (Eastman Kodak, Rochester, NY), and the corresponding silica gel was extracted three times with 1 ml of ethyl acetate and stored at 20°C (yield, 1.5 mCi, 30%). The ethyl acetate extract of 1-N-(2 ,6 -imethyl-morpholino)-3-(4amino-3-[I]iodo-phenyl)propane was evaporated to dryness under an N2 stream. H2SO4 (50 l of 3%) was added to the tube, vortexed, and kept on ice for approximately 15 min. To this reaction mixture, 10 l of 1 M sodium nitrite was added and maintained on ice in the dark for 30 min. Sodium azide (50 l of 1 M) was added, and the reaction was allowed to proceed on ice in the dark for 30 min. The reaction was terminated by the addition of 0.5 ml of 10% sodium bicarbonate and then extracted three times with 1 ml of ethyl acetate. The extracts were pooled and back-extracted with 0.5 ml of water. The extract was then reduced to approximately 50 l under an N2 stream and streaked on a 0.25-mm thick silica gel (60/254) plate (10 20 cm), which was developed in toluene/diethylamine (20:1). The major radioactive reaction product, [I]IAF, migrated with an Rf value of 0.81. The material was detected by autoradiography, and the corresponding silica gel was extracted three times with ethyl acetate and stored at 20°C overnight. The ethyl acetate extract was centrifuged the next day at 24,000 rpm for 10 min to remove dissolved silica (appeared as a solid white precipitate). The radioactive product comigrated with authentic nonradioactive 1-N(2 ,6 -imethyl-morpholino)-3-(4-azido-3-iodo-phenyl) propane prepared as described above. The overall yield for the synthesis of [I]IAF varied between 18 and 20%. Preparation of Rat Liver and Guinea Pig Liver Membranes. Minced frozen rat livers or guinea pig livers (65 g) were thawed in 100 ml of homogenization buffer (10 mM phosphate buffer, pH 7.4, containing 0.32 M sucrose, 1 M MgSO4, 0.5 M EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, 1 g/ml pepstatin A, and 10 g/ml p-toluenesulfonyl-L-arginine methyl ester) and then homogenized on ice with a homogenizer (setting 6, four bursts of 10-s each; Polytron; Kinematica, Basel, Switzerland) followed by a glass homogenizer (Teflon pestle by six slow passes at 3000 rpm). The homogenized tissues were then centrifuged at 17,000g for 10 min. The supernatants were recentrifuged at 100,000g for 1 h. The microsomal pellets were resuspended in homogenization buffer, snapfrozen with dry ice/ethanol, and stored at 80°C at a final protein concentration of 20 mg/ml. Preparation of Pure Guinea Pig -1 Receptor. Pure guinea pig -1 receptor was prepared as reported previously (Ramachandran et al., 2007). In brief, the maltose binding protein (MBP)-guinea pig -1 receptor fusion protein with a Factor Xa cleavage site linking the MBP and -1 receptor and a six-histidine tag on the C terminus of -1 receptor was expressed in Escherichia coli. The E. coli biomass was collected by centrifugation at 3000 rpm for 30 min, sonicated for 15 min at 4°C, centrifuged at 100,000g for 1 h at 4°C and the pellet resuspended, and extracted for 3 h with stirring at 4°C in 20 mM Tris, pH 8.0, 0.2 M NaCl, 1 mM 2-mercaptoethanol, and 1 mM EDTA supplemented with a protease inhibitor cocktail and Triton X-100. The extract was centrifuged at 100,000g for 1 h at 4°C. The supernatant containing the fusion protein was then purified on an amylose column and cleaved with Factor Xa at room temperature for 36 to 48 h. The -1 receptor with a six-histidine tag at the C terminus was collected on an Ni column and eluted using 0.25 M imidazole in wash buffer (50 mM sodium phosphate, pH 8.0, and 0.3 M NaCl) containing 1% Triton X-100. The protein that was eluted from the Ni column was further purified by incubating with an agarose resin coupled with anti-Maltose binding protein antibody (binding capacity of approximately 0.4 mg for MBP per milliliter) at 4°C for 18 to 24 h. After incubation was complete, the mixture was centrifuged at 4000 rpm at room temperature, and the supernatant contained pure guinea pig -1 receptor containing a sixhistidine tag at the C terminus. Receptor Binding Assays. The binding affinities of the newly synthesized compounds for the -1 and -2 receptors were determined using competitive binding assays as described previously (Matsumoto et al., 1995; Nguyen et al., 2005). Assays for -1 receptors were performed using ( )-[H]pentazocine (10 nM) in guinea pig liver membranes (25 g/well) incubated at 30°C for 1 h with several Fig. 1. A, structures of the different fenpropimorph derivatives used in competitive binding assays. B, inhibition of ( )-[H]pentazocine binding by fenpropimorph derivatives in guinea pig liver membranes. Competitive binding curves against 10 nM ( )-[H]pentazocine were generated for the fenpropimorph derivatives, and data points were fit to a one-site nonlinear regression curve. Haloperidol (5 M) was used to determine nonspecific binding. The KD values for compounds were calculated using GraphPad Prism software (version 4.0c) and are listed in Table 1. Compound 2 was found not to bind to the -1 receptor (KD 100 M). Compounds 3 and 11 had KD values of 84 21.9 and 72.3 14.9 nM, respectively. Compounds 6, 7, and 8 had low affinities for -1 receptor and had KD values of 301 61.7, 242 47, and 1500 390 nM, respectively. IAF (compound 10) had moderate affinity to the -1 receptor with a KD value of 194 27.5 nM. 924 Pal et al. at A PE T Jornals on M ay 4, 2017 m oharm .aspeurnals.org D ow nladed from concentrations of competing ligands showed in Fig. 1A. After incubation, the guinea pig liver membranes were harvested on a 0.5% polyethylenimine-treated Whatman GF/B filters using a Brandel cell harvester (Brandel, Gaithersburg, MD). The assay for determining the -2 binding property of IAF was performed using rat liver membranes (25 g/well) and 30 nM [H]ditolylguanidine (DTG) in the presence of ( )-pentazocine (100 nM). Haloperidol (5 M) was used to determine nonspecific binding for both -1 and -2 receptor binding assays. Radioactivity on the filters was detected by liquid scintillation spectrometry using NEN formula 989 as scintillation cocktail (PerkinElmer Life and Analytical Sciences, Waltham, MA). The values were fit to a nonlinear regression curve using the software GraphPad Prism version 4.0c (GraphPad Software Inc., San Diego, CA), and reported dissociation equilibrium constants, KD, were calculated using the Cheng-Prusoff equation (Cheng and Prusoff, 1973). Photoaffinity Labeling of the Rat and Guinea Pig Liver Membrane -1 Receptor. Rat or guinea pig liver membranes (200 g/tube) were suspended in a final volume of 0.1 ml of incubation buffer (50 mM Tris-HCl, pH 7.4) in the presence or absence of the protecting drugs and incubated at 37°C for 20 min. [I]IACoc (Kahoun and Ruoho, 1992; Chen et al., 2007) or [I]IAF was added to the membrane suspensions to a final concentration of 1 nM (1% ethyl acetate, final concentration) and again incubated for 15 min at 37°C. The tubes containing membrane suspensions were then placed in an ice-cold water bath in the photoreactor, and the membrane suspensions were diluted 10-fold with ice-cold incubation buffer containing 20 mM -mercaptoethanol immediately before photolysis. Activation of the photoaffinity labels was accomplished by exposure to a highpressure AH-6 mercury lamp (Advanced Radiation Corporation, Santa Clara, CA) from a distance of 10 cm for 6 s. After photolysis, membrane suspensions were centrifuged at 100,000g for 1 h at 4°C, and the pellets were resuspended in 0.1 ml of incubation buffer. Proteins were separated by 16.5% SDS-tricine/PAGE and visualized by PhosphorImager (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK). Photoaffinity Labeling of the Pure Guinea Pig -1 Receptor. The pure guinea pig -1 receptor (10 g/tube) in 100 l of incubation buffer containing 20 mM -mercaptoethanol was incubated at 37°C for 20 min in the presence or absence of the protecting -1 ligands. [I]IACoc or [I]IAF was then added to each tube to a final concentration of 1 nM (1% ethyl acetate, final concentration) and again incubated for 15 min at 37°C as described above. The tubes were then placed in the ice-cold water bath of the photoreactor and photolysed as described above for 6 s. After photolysis, 5 SDS-PAGE sample buffer (25 l/tube) was added before separation by 16.5% SDS-Tricine/PAGE and finally visualized by PhosphorImaging. EndoLys C Digestion of the Photolabeled -1 Receptor. Before the enzymatic cleavage with the EndoLys C, the -1 receptor was photolabeled separately (10 g of pure protein in 100 l of incubation buffer per tube or 1 mg of rat and guinea pig liver membrane separately in 1 ml of incubation buffer per tube) in the presence and absence of the protector (10 M haloperidol) using both [I]IACoc (1 nM) and [I]IAF (1 nM) as described above. Photolabeled samples were purified by electrophoresis on a 15% SDSpolyacrylamide gel and visualized by wet gel (unstained) autoradiography. The autoradiogram was used as a template to excise the specifically labeled and the protected -1 receptor from the gel with a razor blade. The gel slices were minced and placed into a 1.5-ml microcentrifuge tube containing 1 ml of water. The tubes were maintained overnight at 4°C, and the gel slurries were transferred to spin columns (Bio-Rad Laboratories, Hercules, CA). The eluted -1 receptor in the supernatant extract was collected by centrifugation at 1600g for 5 min. In general, more than 80% of the photolabeled -1 receptor was recovered in the aqueous supernatant (as measured by radioactivity). The eluted materials were concentrated to 100 l by lyophilization and incubated with 0.25 g of EndoLys C (Promega, Madison, WI) per tube overnight at room temperature. The enzymatic digestions were terminated by adding 5 SDS-PAGE sample buffer and separated by 16.5% SDS-Tricine/PAGE. The peptides were again identified by wet-gel autoradiography, excised, and eluted with water as described above. The eluted peptides were concentrated (to approximately 30 l) and subjected to cyanogen bromide (CNBr) digestion separately. CNBr Digestion of the -1 Receptor Peptides. The concentrated peptides were mixed with 70 l of CNBr solution (0.15 M in 70% formic acid solution) for 15 to 18 h at room temperature by tumbling. The reaction mixtures were then diluted with 1 ml of water, lyophilized, and separated by 16.5% SDS-Tricine/PAGE. Gels were stained-destained, dried, and the radiolabeled peptides were visualized by PhosphorImaging.