TRIFUNCTIONAL LIGANDS: A RADIOIODINATED HIGH AFFINITY ACYLATING ANTAGONIST FOR THE A1 ADENOSINE RECEPTOR.

NIH Public Access Author Manuscript Pharmacol Commun. Author manuscript; available in PMC 2014 November 03.

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Published in final edited form as: Pharmacol Commun. 1992 ; 1(2): 145–154.

TRIFUNCTIONAL LIGANDS: A RADIOIODINATED HIGH AFFINITY ACYLATING ANTAGONIST FOR THE A1 ADENOSINE RECEPTOR KENNETH A. JACOBSON, MARK E. OLAH*, and GARY L. STILES* Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892

Abstract


A new xanthine (adenosine antagonist) radioligand that binds covalently to A1-adenosine receptors was prepared and used as a receptor probe. BH-DITC-XAC was synthesized via a trifunctional aryl diisothiocyanate crosslinker. containing the p-hydroxyphenylpropionyl group for radioiodination. The xanthine competed against agonist or antagonist A1 receptor radioligands in bovine brain membranes with an IC50, of 40nM. 125I-BH-DITC-XAC, prepared directly by the chloramine T method and purified by HPLC. bound specifically to A1 receptors. This binding was inhibited in the presence of the adenosine agonists R-PIA, S-PIA. and NECA in a dose dependent manner and with the order of potency characteristic of bovine A1 receptors. Incubation of affinity purified bovine A1-receptors with 125I-BH-DITC-XAC (0.8 nM) for 2 hours resulted in the specific and clean labelling of a polypeptide band corresponding to MW 36,000, identical to that previously found for the A1 receptor.

Keywords xanthines; adenosine receptors; affinity labeling; radioiodination

INTRODUCTION NIH-PA Author Manuscript

The A1-adenosine receptor mediates a number of the depressant effects of the neuromodulator and local hormone adenosine (reviewed in Jacobson et al., 1992). The development of ligands, radioligands, photoaffinity probes and chemical affinity labels for the A1 receptor has permitted the rapid expansion of knowledge concerning its structure, function and regulation (Stiles et al., 1985, 1986b, 1987, 1990; Barrington et al., 1989). Recently, the receptor has been purified to homogeneity and its pharmacological properties studied (Olah et al., 1990). The existing affinity labels have various deficiencies (see Discussion section), principally in the percentage of covalent incorporation into the receptor protein and in specific activity.

© 1992 Harwood Academic Publishers GmbH Address all correspondence to: Kenneth A. Jacobson, Ph.D., Laboratory of Bioorganic Chemistry, Bldg.8A, Rm.B1A-17. NIDDK, Bethesda, MD 20892-1008. [Tel. (301) 496-9024] [Fax. (301) 402-0008]. *Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, NC 27710.

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A general trifunctional approach to systematic modification of the structure of chemical affinity labels for receptors has been explored to provide analogues with specific chemical, physical or spectroscopic properties (Boring et al., 1991). In order to produce a truly useful covalent probe for complete structural studies of the purified A1AR (as has been accomplished for other receptor systems (Dohlman et al., 1988) ) we have synthesized a high affinity A1AR selective acylating antagonist for covalent attachment to the receptor. This xanthine was synthesized via a tri-functional crosslinking reagent which contains a prosthetic group (p-hydroxy-phenylpropionyl group or Bolton-Hunter reagent, “BH”) for radiolabelling with 125I (Bolton and Hunter, 1973) and an isothiocyanate group for covalent attachment.

MATERIALS AND METHODS Chemical Synthesis


300 MHz proton nuclear magnetic resonance spectra were measured on a Varian XL-300 FT-NMR spectrometer. Chemical shifts are expressed as ppm downfield from tetramethylsilane. Chemical ionization mass spectroscopy (CIMS) was carried out on a Finnigan 1015 mass spectrometer modified with EXTREL electronics. Accurate mass (using fast atom bombardment) was measured on a JEOL SX102 high resolution mass spectrometer. 4-[2-[[[2-[[3.5-Bis[(tert-butyloxycarbonyl)amino]benzoyl]amino]ethyl]amino]carbonyl]ethyl]-2-iodophenol, 6b—2[[3,5-Bis[(tertbutyloxycarbonyl)amino]benzoyl]amino]ethylamine (5, 15 mg, 38μmol, Boring et al., 1991) and compound 4b (15 mg, 38μmol, Michelot et al., 1980) were dissolved by sonication in 1 ml of dimethylformamide. After 1 h, 3 ml of ethyl acetate was added, and the mixture extracted successively with 0.1 NHCl and pH 7 phosphate buffer. The organic layer was dried and chromatographed on a silica thin layer plate (250μ) using chloroform:methanol:acetic acid 85/10/5, by volume, resulting in 12 mg of pure product (47% yield, Rf in same system = 0.66, compared to 0.60 for 6a). Accurate mass (669.1785 calc. for C28H37N4O7I) was +0.9 ppm.


4-[2-[[[2[[3,5-(Diisothiocyanato)benzoyl]amino]ehtyl]amino]-carbonyl]ethyl]-2iodophenol, 7b—Compound 6b was deprotected by dissolving in neat trifluoroacetic acid for 5 min, followed by evaporation and precipitation with ether to provide homogeneous 4[2-(((2-[(3,5-diamino)benzoyl]amino]ethyl]amino]carbonyl]-ethyl]-2-iodophenol (Rf in chloroform:methanol:acetic acid 85/10/5, by volume = 0.13). Accurate mass (469.0737 calc. for C18H21N4O3I) was +0.4 ppm. The diamine (10 mg, 21μmol) was dissolved in a mixture of chloroform (1 ml), dimethylformamide (0.5 ml) and saturated sodium bicarbonate (1 ml). The resultant solution was stirred and cooled in an ice bath and thiophosgene (60μ, 0.8 mmol) was added. After one hour, the organic layer was removed by pipette, washed with water, and treated with ether. A white precipitate was collected, yield 8 mg (69% yield), and was further purified by TLC. Accurate mass (469.0737 calc. for C20H17N4O3S2I) was found to be +0.4 ppm.

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Reaction of XAC, 1, with diisothiocyanate crosslinkers, 2—XAC (8-[2Aminoethyl[amino]carbonyl[methyl[oxyphenyl]]]]]-1,3-dipropyl--xanthine, 21 mg, 50 μmol, Research Biochemicals, Inc., Natick, MA) and compound 2 (>100 μmol) were suspended in 1 ml dimethylformamide and sonicated for several minutes. After stirring for 1 h, the volume of solvent was reduced under a stream of nitrogen with slight warming. Ether was added, and a precipitate formed. The product, 3, was recrystallized from dimethylformamide/ether to give yields in the 50–80% range. See also Boring et al. (1991). Radioiodination of BH-DITC-XAC BH-DITC-XAC, 3b, was prepared as described above and prior to radioiodination further purified by HPLC (retention time 17 min) on a Vydac C4 protein column, 1.0 × 25 cm, with a mobile phase consisting of 35% acetonitrile in water, containing 0.1% trifluoroacetic acid.


Purified BH-DITC-XAC (0.1 mg) was dissolved in dimethylformamide and then added to 0.4M sodium phosphate buffer pH 7.44 (1:10) to a total volume of 20 μl. To this was added 1.5 mCi of Na 125I (15 μl) followed by 10 μl of freshly prepared chloramine T (1 mg/ml) at room temperature. The reaction was stopped at 1 minute with 15 μl sodium metabisulfate (2mg/ml). This mixture was then separated by HPLC on a C18 μbondapak column using an isocratic elution with a mobile phase consisting of a mixture of 75% methanol and 25% 20 mM ammonium formate at pH 8.0. A single radioactive peak emerged at 22 min, and the starting material emerged at 17 min. The sample was stored in the elution buffer at −20° in the dark. Radioligand Binding Experiments Reagents: 125I-APNEA was synthesized as described previously (Stiles et al., 1985). Na 125I was purchased from Amersham (Arlington Heights, IL). Bovine brain was from a local abattoir. [3H]XAC was from NEN Research Products (Boston, MA). Electrophoresis reagents were from BioRad (Richmond, CA). Membrane Preparation: Bovine cerebral cortex membranes were prepared as previously described (Stiles et al., 1986a). Membranes were treated with adenosine deaminase (0.5 units/ml) for 20 min at 37° before radioligand binding assays.


Radioligand Binding Assay: Membranes (40 μg of protein, 150 μl) were incubated for 1 h at 37° in a total volume of 250 μl, containing 50 μl of radioligand at the indicated concentration and 50 μl of competing ligand. Bound and free radioligand were separated by addition of 4 ml of 50 mM TRIS/10 mM MgCl2/1 mM EDTA, pH 8.26 at 5° (buffer A) with 0.02% CHAPS, followed by vacuum filtration on glass filters with additional washes totaling 12 ml of buffer. Filters were counted in a γ counter at an efficiency of 75%. Nonspecific binding was defined with 10−5 M R-PIA. Saturation and competition binding data were analyzed using computer modeling programs as previously described (DeLean et al., 1978, 1982).


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Receptor Purification and Labeling


A1AR was purified from bovine brain cortex as previously described (Olah et al., 1990). The receptor preparation contained A1AR at a specific activity of approximately 6,000 pmol/mg. Receptor labeling was carried out by incubating approximately 0.5 pmol of receptor with 125I-BH-DITC-XAC (0.8 nM) in a buffer containing 0.1% CHAPS, 50 mM TRIS, 1 mM EDTA, 125 mM NaCl, 400 mM KCl, pH 7.4. Incubation proceeded for 2 hours at 25°C in a shaking water bath. An aliquot of the receptor preparation was added to SDS buffer (10% SDS, 10% glycerol, 25 mM TRIS-HCl, pH 6.8, 5% β-mercaptoethanol) prior to SDSPAGE. Samples were then subjected to electrophoresis in 11% polyacrylamide gels as described by Laemmli (1970). Dried gels were then exposed to Kodak XAR-5 film at −80°C.

RESULTS

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Figure 1 illustrates the application of the trifunctional strategy (Boring et al., 1991) to derivatives of the xanthine amine congener (XAC), 1. XAC may be treated with a chemically reactive symmetrical cross-linking reagent, 2, resulting in a xanthine isothiocyanate derivative, 3, for irreversible reaction with the receptor protein. The 5position of the isothiocyanate-bearing ring is reserved as an attachment site for a reporter group X (radioactive or non-radioactive), and we have shown that such substitution is tolerated in A1 receptor binding (Boring et al., 1991). The simplest analog, in which X = H, 3a, m-DITC-XAC, is a potent affinity label for A1 receptors (Stiles and Jacobson, 1988), and higher molecular weight derivatives, in which group X is elongated, are also irreversible inhibitors of the receptor. Compound 3b, BH-DITC-XAC, is a trifunctional xanthine that contains a prosthetic group (Bolton and Hunter, 1973) for radioiodination (BH) attached at the 5-position via a spacer chain. The 3-(4-hydroxyphenyl)propionyl group was introduced via an N-hydroxysuccinimide ester precursor 4a (Michelot et al., 1980), as shown in Figure 2. Compound 3b was effectively radiolabeled using the sodium iodide/chloramine T method, and the product 125I-BH-DITC-XAC, 3c, was purified by reverse phase HPLC. The synthesis of nonradioactively labeled BH-DITC-XAC, 3c, as a standard was carried out by an analogous route beginning with the iodinated Bolton-Hunter reagent, 4b. Since radioiodinated 4b is readily available, this method of preparing 3c is also potentially adaptable for its radiochemical synthesis. BH-DITC-XAC competed for specific [125I]APNEA or [3H]XAC binding (A1AR selective agonist and antagonist radioligands, respectively) in bovine cerebral cortex membranes as shown in Figure 3. The IC50 for inhibition of binding was 40±7 nM (n = 3). Since BHDITC-XAC is an irreversible ligand, and true equilibrium conditions are never achieved, the IC50 value represents only the apparent IC50 under the specific conditions described. The true potency of an irreversible ligand or radioligand will increase with the time of incubation. We next assessed the pharmacological characteristics of 125I-labeled BH-DITC-XAC. The A1AR ligands, R-PIA, S-PIA and NECA compete for binding of 125I-BH-DITC-XAC with the appropriate order of potency expected for interaction with the A1AR (Figure 4). As


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described above, the binding conditions do not of necessity achieve equilibrium in nature but the IC50 values for R-PIA= 1.9±0.61 nM, S-PIA = 3.8± 1.7 nM and NECA 79±38 nM are similar to those that have been described for competitive radioligand binding assays (Stiles et al, 1985). This suggests that 125I-BH-DITC-XAC indeed does interact with the A1AR. In order to demonstrate that 125I-BH-DITC-XAC can irreversibly label the A1AR, purified preparations of A1AR were incubated with the radioprobe and then subjected to SDS-PAGE. A single labeled polypeptide migrating within Mr 36,000 is shown to be labeled with 125IBH-DITC-XAC (Figure 5). Covalent incorporation under these conditions represent roughly 20–25% of the specifically bound radioligand. As a control, the same procedure was carried out in the presence of 5 mM theophylline, and this band disappeared. These data taken together suggest that this new compound (BH-DITC-XAC) can bind to the A1AR with all the appropriate pharmacology and selectivity and can be radioiodinated to high specific activity. This radioprobe can then be utilized to label covalently the A1AR as documented by SDS-PAGE/autoradiography.


DISCUSSION The A1-selective agonist APNEA, N6-(4-aminophenyiethyl)adenosine (Stiles et al., 1985) and the A1-selective antagonist PAPA-XAC, the p-aminophenylacetyl derivative of 8-[2aminoethyl[amino]carbonyl]methyl-(oxyphenyl)]]]]-1,3-dipropylxanthine (Stiles and Jacobson, 1987), have been radioiodinated and crosslinked photochemically to the A1 adenosine receptor glycoprotein indicating a molecular weight in the range of 36–40,000 daltons. The covalent attachment of radioiodinated APNEA and PAPA-XAC to A1 receptors using the crosslinker SANPAH has a low efficiency of incorporation, in the range of 0.1 to 3%. The corresponding azido photoaffinity probes (Barrington et al., 1989) incorporate with much higher efficiency but still only in the range of 5–20% in membranes and with lower efficiencies in solubilized preparations. Tritiated covalent affinity probes that have much higher efficiency of incorporation have been reported, but they display lower specific activity (Stiles and Jacobson, 1988; Dickinson et al., 1985; Pitha et al., 1980; Regan et al., 1984).


Several years ago we synthesized an isomeric pair of high affinity acylating A1AR antagonists [3H]DITC-XAC (para- or meta-isothiocyanate, 3a) and demonstrated affinity labeling of the A1AR with high efficacy (Stiles and Jacobson, 1988). The nonradiolabeled m-DITC-XAC (1b, X = H) at a concentration of 500 nM covalently incorporated into the A1AR with an efficiency greater than 90%, as determined by its ability to block subsequent radioligand binding (Stiles and Jacob-son, 1988). A tritiated form of m-DITC-XAC was able to label the A1AR in membranes from bovine brain, although high nonspecific binding was observed and fluororadiography was required to allow detection. This complicated method of detection was necessitated by the low energy of tritium and the low specific activity of tritiated precursors for DITC-XAC relative to radioiodinated compounds. Each of the abovementioned radioligands, photoaffinity and affinity probes have their own intrinsic deficiencies.


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This paper describes the synthesis, radiolabeling and characterization of a new iodinated affinity probe for the A1AR using a trifunctional approach to ligand design (Boring et al., 1991). The extension of chains at the 5-position of the isothiocyanate ring of m-DITC-XAC offers new flexibility in the design of irreversible antagonist probes for adenosine receptors. Our previous study of trifunctional xanthines (Boring et al., 1991) indicated a tolerance for sterically large groups at the 5-position of m-DITC-XAC (group X in Figure 1) in binding to A1 receptors. Groups attached at this position, which were designed to serve as reporter groups, included prosthetic groups for radiolabeling, fluorescent dyes, a spin label probe, and groups for chemical and photochemical cross-linking. Furthermore, we have utilized an amide linkage rather than a thiourea linkage at the 5-position, based on structure activity patterns noted previously (Boring et al., 1991), to maximize receptor affinity. Perhaps this methodology will serve as a model for developing ligands for other receptors in which a non-essential (relatively insensitive in receptor binding) amino group occurs (Michelot et al., 1980). The symmetrical crosslinking intermediate 2b, which bears two isothiocyanate groups in the 3 and 5 positions, when present as an excess reagent may be coupled to nucleophiles in general.


The advantages of the affinity ligand 125I-BH-DITC-XAC in comparison to previously reported adenosine receptor probes are that it can directly incorporate into the A1AR with high efficiency and specific activity (approximately 2200 Ci/mmol). Previous affinity ligands for the A1AR have either been nonradioactively labelled or contained tritium (Stiles and Jacobson, 1988) at considerably lower specific activity. This makes detection difficult and sensitivity in detecting small amounts of receptor is low.


This new compound interacts with the A1AR with high affinity and with the appropriate pharmacological order of potency. The reaction is nonreversible and covalent as documented by our ability to detect the receptor using SDS/PAGE autoradiography (Figure 5). The compound labeled the same 36,000 dalton receptor as the prototypic photoaffinity probe [125I]PAPA-XAC-SANPAH (Stiles and Jacobson, 1987). It should be noted that this new affinity probe will be most useful in studying the purified receptor but not the membrane bound receptor, since it contains a highly reactive chemical group which may label other membrane-bound proteins nonspecifically. The greatest utility for this new affinity probe will be in delineating the detailed structure of the A1AR, enabling the binding domain of the A1AR to be probed following covalent incorporation, and digestion of the receptor enzymatically or chemically followed by purification and sequencing of the peptide fragment. This type of work in association with the overall sequence information from the cloning of the cDNA for the A1AR (Libert et al., 1991) should provide important new information on the detailed structure of the A1AR.

Acknowledgments M.E.O. was supported by a N.I.H. Postdoctoral Fellowship (1F32-GM-I3713-02) from the National Institute of General Medical Sciences. G.L.S. is supported by NHLBI SCOR grant (P50HL17670) in ischemic disease, in part by NHLBI grant (ROIHL35134) and Supplement and a Grant-in-Aid (880662) from the American Heart Association and 3M Riker.


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FIGURE 1.

Synthesis of trifunctional XAC derivatives that act as irreversible antagonists at A1 adenosine receptors.

NIH-PA Author Manuscript Pharmacol Commun. Author manuscript; available in PMC 2014 November 03.

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FIGURE 2.

Synthesis of non-radioactive xanthine standards and intermediates.


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FIGURE 3.

Inhibition of binding of [125I]APNEA to bovine cerebral cortical A1-adenosine receptors by the trifunctional xanthine BH-DITC-XAC, 3b, a substrate for radioiodination. The binding was carried out at 37°C for 60 min. [125I]APNEA was present at a concentration of 0.5 nM in 0.05M TRIS at pH 8.26. 10mM MgCl2, 1 mM EDTA, at 5°C. Nonspecific binding was defined with 10−5 M R-PIA.


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FIGURE 4.

Inhibition of binding of 125I-BH-DITC-XAC to bovine cerebral cortical A1-adenosine receptors by adenosine agonists: R-PIA (○), S-PIA (□), and NECA (△). The binding was carried out at 25°C for 90 min. 125I-BH-DITC-XAC was present at a concentration of 0.8 nM in 0.05 M TRIS buffer at pH 7.4. Nonspecific binding was defined with 10−5 M R-PIA.


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FIGURE 5.

Affinity labeling of A1-adenosine receptors purified from bovine brain. Membranes were incubated with the antagonist affinity probe 125I-BH-DITC-XAC (0.8 nM) at pH 7.4 for two hours in the absence and presence of 5 mM theophylline. The labeled receptor preparation was then subjected to SDS-PAGE and autoradiography. Molecular weight standards are shown on the left.


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