Structure-activity relationships of adenine and deazaadenine derivatives as ligands for adenine receptors, a new purinergic receptor family.

Adenine derivatives bearing substituents in the 2-, N(6)-, 7-, 8-, and/or 9-position and a series of deazapurines were synthesized and investigated in [(3)H]adenine binding studies at the adenine receptor in rat brain cortical membrane preparations (rAde1R). Steep structure-activity relationships were observed. Substitution in the 8-position (amino, dimethylamino, piperidinyl, piperazinyl) or in the 9-position (2-morpholinoethyl) with basic residues or introduction of polar substituents at the 6-amino function (hydroxy, amino, acetyl) represented the best modifications. Functional evaluation of selected adenine derivatives in adenylate cyclase assays at 1321N1 astrocytoma cells stably expressing the rAde1R showed that all compounds investigated were agonists or partial agonists. A subset of compounds was additionally investigated in binding studies at human embryonic kidney (HEK293) cells, which also express a high-affinity adenine binding site. Structure-affinity relationships at the human cell line were similar to those at the rAde1R, but not identical. In particular, N(6)-acetyladenine (25, K(i) rat: 2.85 microM; K(i) human: 0.515 microM) and 8-aminoadenine (33, K(i) rat: 6.51 microM; K(i) human: 0.0341 microM) were much more potent at the human as compared to the rat binding site. The new AdeR ligands may serve as lead structures and contribute to the elucidation of the functions of the adenine receptor family.

[(3)H]Adenine is a suitable radioligand for the labeling of G protein-coupled adenine receptors but shows high affinity to bacterial contaminations in buffer solutions.

[(3)H]Adenine has previously been used to label the newly discovered G protein-coupled murine adenine receptors. Recent reports have questioned the suitability of [(3)H]adenine for adenine receptor binding studies because of curious results, e.g. high specific binding even in the absence of mammalian protein. In this study, we showed that specific [(3)H]adenine binding to various mammalian membrane preparations increased linearly with protein concentration. Furthermore, we found that Tris-buffer solutions typically used for radioligand binding studies (50 mM, pH 7.4) that have not been freshly prepared but stored at 4 degrees C for some time may contain bacterial contaminations that exhibit high affinity binding for [(3)H]adenine. Specific binding is abolished by heating the contaminated buffer or filtering it through 0.2-mum filters. Three different, aerobic, gram-negative bacteria were isolated from a contaminated buffer solution and identified as Achromobacter xylosoxidans, A. denitrificans, and Acinetobacter lwoffii. A. xylosoxidans, a common bacterium that can cause nosocomial infections, showed a particularly high affinity for [(3)H]adenine in the low nanomolar range. Structure-activity relationships revealed that hypoxanthine also bound with high affinity to A. xylosoxidans, whereas other nucleobases (uracil, xanthine) and nucleosides (adenosine, uridine) did not. The nature of the labeled site in bacteria is not known, but preliminary results indicate that it may be a high-affinity purine transporter. We conclude that [(3)H]adenine is a well-suitable radioligand for adenine receptor binding studies but that bacterial contamination of the employed buffer solutions must be avoided.

Evidence for the functional expression and pharmacological characterization of adenine receptors in native cells and tissues.

An orphan G protein-coupled receptor from rat has recently been discovered to be activated by the nucleobase adenine (Proc Natl Acad Sci USA 99:8573-8578, 2002). In the present study, we show for the first time that the adenine receptor is expressed in membrane preparations of native tissues and cell lines in high density, including rat brain cortex, rat brain striatum, and the mouse neuroblastoma x rat glioma hybrid cell line NG108-15. Saturation analysis with [3H]adenine at rat brain cortical membranes exhibited a single high-affinity binding site with a KD value of 27.2 nM, and a binding capacity of 2.28 pmol/mg of protein. Kinetic studies revealed unusual binding kinetics of [3H]adenine with rapid association and slow dissociation. A series of compounds were investigated in [3H]adenine competition experiments at rat brain cortex. Only minor substitution of the adenine structure was tolerated, the most potent compounds of the present series being 2-fluoroadenine (Ki value of 620 nM), 8-thioadenine (Ki value of 2.77 microM), N6-methyladenine (Ki value of 3.64 microM), and 7-methyladenine (Ki value of 4.13 microM), all of which were partial agonists (40-60% intrinsic activity). Adenine dose dependently inhibited forskolin-stimulated adenylate cyclase in membrane preparations of NG108-15 cells as well as in intact cells, showing that the receptor is functional in NG108-15 cells. Reverse transcriptase-polymerase chain reaction experiments followed by sequencing indicate that the NG108-15 cells express the murine ortholog of the adenine receptor. Moreover, preliminary radioligand binding studies with [3H]adenine at membranes of human astrocytoma 1321N1 cells suggest that a human ortholog of the rat adenine receptor exists.

Novel amino acid derived natural products from the ascidian Atriolum robustum: identification and pharmacological characterization of a unique adenosine derivative.

Investigation of the methanolic extract of the Australian ascidian Atriolum robustum led to the isolation and characterization of five new amino acid derived structures (1-5). The structures were elucidated employing spectroscopic techniques (NMR, MS, UV, and IR). The absolute stereochemistry of 1 and 2 was established by chemical degradation, derivatization, and chiral GC-MS analysis. Structures 4 and 5 are complex nucleosides containing rare methylthioadenosine and methylsulfinyladenosine moieties, respectively. In radioligand binding studies the 5'-deoxy-5'-methylthioadenosine-2',3'-diester 4 exhibited affinity for A(1) and A(3) adenosine receptors with K(i) values below 10 microM. Its affinity was somewhat lower for A(2A) (K(i) = 17 microM) and much lower for A(2B) adenosine receptors. Analytical experiments using capillary electrophoresis showed that compound 4 was stable under the conditions of radioligand binding studies. Incubation with carboxylesterase resulted in slow hydrolysis of the adenosine derivative to 5'-deoxy-5'-methylthioadenosine (MTA), which was about 10-fold more potent at adenosine receptors than compound 4. Thus, the 2',3'-diester derivative 4 may act as a lipophilic prodrug of MTA in addition to its own adenosine receptor activity. GTP shift experiments indicated that the adenosine derivative was a partial agonist at A(1) adenosine receptors of rat brain cortical membranes. Compound 4 inhibited cAMP accumulation in Chinese hamster ovary (CHO) cell membranes recombinantly expressing the human A(3) adenosine receptor, thus indicating that the adenosine derivative also acted as a partial agonist at A(3)ARs. Homology models of the A(1) and the A(3) adenosine receptors in their putative active and inactive conformations were built and used for docking of the sterically demanding compound 4. It was found that this ligand fit well into the binding pockets of both receptor subtypes because of its highly flexible structure, although in somewhat different binding modes.