TLR-Induced IL-12 and CCL2 Production by Myeloid Cells Is Dependent on Adenosine A3 Receptor-Mediated Signaling.

TLR-induced signaling potently activates cells of the innate immune system and is subject to regulation at different levels. Inflammatory conditions are associated with increased levels of extracellular adenosine, which can modulate TLR-induced production of cytokines through adenosine receptor-mediated signaling. There are four adenosine receptor subtypes that induce different signaling cascades. In this study, we demonstrate a pivotal contribution of adenosine A3 receptor (A3R)-mediated signaling to the TLR4-induced expression of IL-12 in different types of human myeloid APC. In dendritic cells, IL-12 and CCL2 responses as evoked by TLR2, 3, 4, 5, and 8, as well as IL-12 responses evoked by whole pathogens, were all reduced when A3R-mediated signaling was blocked. As a result, concomitant production of IFN-γ and IL-17 by T cells was significantly inhibited. We further show that selective inhibition of A3R-mediated signaling reduced TLR-induced phosphorylation of the transcription factor STAT1 at tyrosine 701. Next-generation sequencing revealed that A3R-mediated signaling controls the expression of metallothioneins, known inhibitors of STAT1 phosphorylation. Together our results reveal a novel regulatory layer of innate immune responses, with a central role for metallothioneins and autocrine/paracrine signaling via A3Rs.

A covalent antagonist for the human adenosine A2A receptor.

The structure of the human A2A adenosine receptor has been elucidated by X-ray crystallography with a high affinity non-xanthine antagonist, ZM241385, bound to it. This template molecule served as a starting point for the incorporation of reactive moieties that cause the ligand to covalently bind to the receptor. In particular, we incorporated a fluorosulfonyl moiety onto ZM241385, which yielded LUF7445 (4-((3-((7-amino-2-(furan-2-yl)-[1, 2, 4]triazolo[1,5-a][1, 3, 5]triazin-5-yl)amino)propyl)carbamoyl)benzene sulfonyl fluoride). In a radioligand binding assay, LUF7445 acted as a potent antagonist, with an apparent affinity for the hA2A receptor in the nanomolar range. Its apparent affinity increased with longer incubation time, suggesting an increasing level of covalent binding over time. An in silico A2A-structure-based docking model was used to study the binding mode of LUF7445. This led us to perform site-directed mutagenesis of the A2A receptor to probe and validate the target lysine amino acid K153 for covalent binding. Meanwhile, a functional assay combined with wash-out experiments was set up to investigate the efficacy of covalent binding of LUF7445. All these experiments led us to conclude LUF7445 is a valuable molecular tool for further investigating covalent interactions at this receptor. It may also serve as a prototype for a therapeutic approach in which a covalent antagonist may be needed to counteract prolonged and persistent presence of the endogenous ligand adenosine.

Scintillation proximity assay (SPA) as a new approach to determine a ligand's kinetic profile. A case in point for the adenosine A1 receptor.

Scintillation proximity assay (SPA) is a radio-isotopic technology format used to measure a wide range of biological interactions, including drug-target binding affinity studies. The assay is homogeneous in nature, as it relies on a "mix and measure" format. It does not involve a filtration step to separate bound from free ligand as is the case in a traditional receptor-binding assay. For G protein-coupled receptors (GPCRs), it has been shown that optimal binding kinetics, next to a high affinity of a ligand, can result in more desirable pharmacological profiles. However, traditional techniques to assess kinetic parameters tend to be cumbersome and laborious. We thus aimed to evaluate whether SPA can be an alternative platform for real-time receptor-binding kinetic measurements on GPCRs. To do so, we first validated the SPA technology for equilibrium binding studies on a prototypic class A GPCR, the human adenosine A1 receptor (hA1R). Differently to classic kinetic studies, the SPA technology allowed us to study binding kinetic processes almost real time, which is impossible in the filtration assay. To demonstrate the reliability of this technology for kinetic purposes, we performed the so-called competition association experiments. The association and dissociation rate constants (k on and k off) of unlabeled hA1R ligands were reliably and quickly determined and agreed very well with the same parameters from a traditional filtration assay performed simultaneously. In conclusion, SPA is a very promising technique to determine the kinetic profile of the drug-target interaction. Its robustness and potential for high-throughput may render this technology a preferred choice for further kinetic studies.

The adhesion G protein-coupled receptor G2 (ADGRG2/GPR64) constitutively activates SRE and NFκB and is involved in cell adhesion and migration.

Adhesion G protein-coupled receptors (ADGRs) are believed to be activated by auto-proteolytic cleavage of their very large extracellular N-terminal domains normally acting as a negative regulator of the intrinsically constitutively active seven transmembrane domain. ADGRG2 (or GPR64) which originally was described to be expressed in the epididymis and studied for its potential role in male fertility, is highly up-regulated in a number of carcinomas, including breast cancer. Here, we demonstrate that ADGRG2 is a functional receptor, which in transfected HEK293 cells signals with constitutive activity through the adhesion- and migration-related transcription factors serum response element (SRE) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) presumably via coupling to Gα12/13 and Gαq. However, activation of these two pathways appears to occur through distinct molecular activation mechanisms as auto-proteolytic cleavage is essential for SRE activation but not required for NFκB signaling. The overall activation mechanism for ADGRG2 is clearly distinct from the established ADGR activation mechanism as it requires the large extracellular N-terminal domain for proper intracellular signal transduction. Knockdown of ADGRG2 by siRNA in the highly motile breast cancer cell lines Hs578T and MDA-MB-231 resulted in a strong reduction in cell adhesion and subsequent cell migration which was associated with a selective reduction in RelB, an NFκB family member. It is concluded that the adhesion GPCR ADGRG2 is critically involved in the adhesion and migration of certain breast cancer cells through mechanisms including a non-canonical NFkB pathway and that ADGRG2 could be a target for treatment of certain types of cancer.

A yeast screening method to decipher the interaction between the adenosine A2B receptor and the C-terminus of different G protein α-subunits.

The expression of human G protein-coupled receptors (GPCRs) in Saccharomyces cerevisiae containing chimeric yeast/mammalian Gα subunits provides a useful tool for the study of GPCR activation. In this study, we used a one-GPCR-one-G protein yeast screening method in combination with molecular modeling and mutagenesis studies to decipher the interaction between GPCRs and the C-terminus of different α-subunits of G proteins. We chose the human adenosine A2B receptor (hA2BR) as a paradigm, a typical class A GPCR that shows promiscuous behavior in G protein coupling in this yeast system. The wild-type hA2BR and five mutant receptors were expressed in 8 yeast strains with different humanized G proteins, covering the four major classes: Gαi, Gαs, Gαq, and Gα12. Our experiments showed that a tyrosine residue (Y) at the C-terminus of the Gα subunit plays an important role in controlling the activation of GPCRs. Receptor residues R103(3.50) and I107(3.54) are vital too in G protein-coupling and the activation of the hA2BR, whereas L213(IL3) is more important in G protein inactivation. Substitution of S235(6.36) to alanine provided the most divergent G protein-coupling profile. Finally, L236(6.37) substitution decreased receptor activation in all G protein pathways, although to a different extent. In conclusion, our findings shed light on the selectivity of receptor/G protein coupling, which may help in further understanding GPCR signaling.

Functional selectivity of adenosine A1 receptor ligands?

The concept of functional selectivity offers great potential for the development of drugs that selectively activate a specific intracellular signaling pathway. During the last few years, it has become possible to systematically analyse compound libraries on G protein-coupled receptors (GPCRs) for this 'biased' form of signaling. We screened over 800 compounds targeting the class of adenosine A(1) receptors using a ß-arrestin-mediated signaling assay in U2OS cells as a G protein-independent readout for GPCR activation. A selection of compounds was further analysed in a G protein-mediated GTPγS assay. Additionally, receptor affinity of these compounds was determined in a radioligand binding assay with the agonist [(3)H]CCPA. Of all compounds tested, only LUF5589 9 might be considered as functionally selective for the G protein-dependent pathway, particularly in view of a likely overestimation of ß-arrestin signaling in the U2OS cells. Altogether, our study shows that functionally selective ligands for the adenosine A(1) receptor are rare, if existing at all. A thorough analysis of biased signaling on other GPCRs also reveals that only very few compounds can be considered functionally selective. This might indicate that the concept of functional selectivity is less common than speculated.

Three "hotspots" important for adenosine A(2B) receptor activation: a mutational analysis of transmembrane domains 4 and 5 and the second extracellular loop.

G protein-coupled receptors (GPCRs) are a major drug target and can be activated by a range of stimuli, from photons to proteins. Despite the progress made in the last decade in molecular and structural biology, their exact activation mechanism is still unknown. Here we describe new insights in specific regions essential in adenosine A(2B) receptor activation (A(2B)R), a typical class A GPCR. We applied unbiased random mutagenesis on the middle part of the human adenosine A(2B)R, consisting of transmembrane domains 4 and 5 (TM4 and TM5) linked by extracellular loop 2 (EL2), and subsequently screened in a medium-throughput manner for gain-of-function and constitutively active mutants. For that purpose, we used a genetically engineered yeast strain (Saccharomyces cerevisiae MMY24) with growth as a read-out parameter. From the random mutagenesis screen, 12 different mutant receptors were identified that form three distinct clusters; at the top of TM4, in a cysteine-rich region in EL2, and at the intracellular side of TM5. All mutant receptors show a vast increase in agonist potency and most also displayed a significant increase in constitutive activity. None of these residues are supposedly involved in ligand binding directly. As a consequence, it appears that disrupting the relatively "silent" configuration of the wild-type receptor in each of the three clusters readily causes spontaneous receptor activity.

Functional selectivity of adenosine receptor ligands.

Adenosine receptors are plasma membrane proteins that transduce an extracellular signal into the interior of the cell. Basically every mammalian cell expresses at least one of the four adenosine receptor subtypes. Recent insight in signal transduction cascades teaches us that the current classification of receptor ligands into agonists, antagonists, and inverse agonists relies very much on the experimental setup that was used. Upon activation of the receptors by the ubiquitous endogenous ligand adenosine they engage classical G protein-mediated pathways, resulting in production of second messengers and activation of kinases. Besides this well-described G protein-mediated signaling pathway, adenosine receptors activate scaffold proteins such as ß-arrestins. Using innovative and sensitive experimental tools, it has been possible to detect ligands that preferentially stimulate the ß-arrestin pathway over the G protein-mediated signal transduction route, or vice versa. This phenomenon is referred to as functional selectivity or biased signaling and implies that an antagonist for one pathway may be a full agonist for the other signaling route. Functional selectivity makes it necessary to redefine the functional properties of currently used adenosine receptor ligands and opens possibilities for new and more selective ligands. This review focuses on the current knowledge of functionally selective adenosine receptor ligands and on G protein-independent signaling of adenosine receptors through scaffold proteins.

Allosteric modulation of adenosine receptors.

Allosteric ligands for G protein-coupled receptors (GPCRs) may alter receptor conformations induced by an orthosteric ligand. These modulators can thus fine-tune classical pharmacological responses. In this review we will describe efforts to synthesize and characterize allosteric modulators for one particular GPCR subfamily, the adenosine receptors. There are four subtypes of these receptors: A(1), A(2A), A(2B) and A(3). Allosteric enhancers for the adenosine A(1) receptor may have anti-arrythmic and anti-lipolytic activity. They may also act as analgesics and neuroprotective agents. A(3) allosteric enhancers are thought to be beneficial in ischemic conditions or as antitumor agents. We will summarize recent developments regarding the medicinal chemistry of such compounds. Most data have been and are published about the adenosine A(1) and A(3) receptor, whereas limited or no information is available for the A(2A) and A(2B) receptor, respectively. Receptor mutation studies are also discussed, as they may shed light on the localization of the allosteric binding sites. This article is part of a Special Issue entitled: "Adenosine Receptors".

The endocannabinoid 2-arachidonylglycerol is a negative allosteric modulator of the human A3 adenosine receptor.

Studies of endogenous cannabinoid agonists, such as 2-arachidonylglycerol (2-AG), have revealed their potential to exert modulatory actions on other receptor systems in addition to their ability to activate cannabinoid receptors. This study investigated the effect of cannabinoid ligands on the human adenosine A(3) (hA(3)R) receptor. The endocannabinoid 2-AG was able to inhibit agonist ([125I]N(6)-(4-amino-3-iodobenzyl) adenosine-5'-(N-methyluronamide)--[125I] AB MECA) binding at the hA(3)R. This inhibition occurred over a narrow range of ligand concentration and was characterized by high Hill coefficients suggesting a non-competitive interaction. Furthermore, in the presence of 2-AG, the rate of [125I] AB MECA dissociation was increased, consistent with an action as a negative allosteric modulator of the hA(3)R. Moreover, by measuring intracellular cAMP levels, we demonstrate that 2-AG decreases both the potency of an agonist at the hA(3)R and the basal signalling of this receptor. Since the hA(3)R has been shown to be expressed in astrocytes and microglia, these findings may be particularly relevant in certain pathological states such as cerebral ischemia where levels of 2-AG and anandamide are raised.

Differential expression of adenosine A3 receptors controls adenosine A2A receptor-mediated inhibition of TLR responses in microglia.

Microglia activation is a prominent feature in many neuroinflammatory disorders. Unrestrained activation can generate a chronic inflammatory environment that might lead to neurodegeneration and autoimmunity. Extracellular adenosine modulates cellular activation through adenosine receptor (ADORA)-mediated signaling. There are four ADORA subtypes that can either increase (A(2A) and A(2B) receptors) or decrease (A(1) and A(3) receptors) intracellular cyclic AMP levels. The expression pattern of the subtypes thus orchestrates the cellular response to extracellular adenosine. We have investigated the expression of ADORA subtypes in unstimulated and TLR-activated primary rhesus monkey microglia. Activation induced an up-regulation of A(2A) and a down-regulation of A(3) receptor (A(3)R) levels. The altered ADORA-expression pattern sensitized microglia to A(2A) receptor (A(2A)R)-mediated inhibition of subsequent TLR-induced cytokine responses. By using combinations of subtype-specific agonists and antagonists, we revealed that in unstimulated microglia, A(2A)R-mediated inhibitory signaling was effectively counteracted by A(3)R-mediated signaling. In activated microglia, the decrease in A(3)R-mediated signaling sensitized them to A(2A)R-mediated inhibitory signaling. We report a differential, activation state-specific expression of ADORA in microglia and uncover a role for A(3)R as dynamically regulated suppressors of A(2A)R-mediated inhibition of TLR-induced responses. This would suggest exploration of combinations of A(2A)R agonists and A(3)R antagonists to dampen microglial activation during chronic neuroinflammatory conditions.

Allosteric modulation of adenosine receptors.

Allosteric modulators for adenosine receptors may have potential therapeutic advantage over orthosteric ligands. Allosteric enhancers at the adenosine A(1) receptor have been linked to antiarrhythmic and antilipolytic activity. They may also have therapeutic potential as analgesics and neuroprotective agents. A(3) allosteric enhancers are postulated to be useful against ischemic conditions or as antitumor agents. In this review, we address recent developments regarding the medicinal chemistry of such compounds. Most efforts have been and are directed toward adenosine A(1) and A(3) receptors, whereas limited or no information is available for A(2A) and A(2B) receptors. We also discuss some findings, mostly receptor mutation studies, regarding localization of the allosteric binding sites on the receptors.

Synthesis and evaluation of homodimeric GnRHR antagonists having a rigid bis-propargylated benzene core.

The fact that GPCRs might function in a dimeric fashion is currently well accepted. For GnRHR, a GPCR that regulates gonadotropin release, there is evidence that the receptor also functions as a dimer. We here describe the design and synthesis of a set of dimeric GnRHR antagonists in order to understand the interaction of dimeric ligands to the receptor and to address the question whether GnRHR dimerization is a prerequisite for signalling. Biological evaluation of the compounds shows no discrimination between monomeric and dimeric-ligands in respect to binding affinities, however, the dimeric ligands appear to have different functional properties.

ZM241385, DPCPX, MRS1706 are inverse agonists with different relative intrinsic efficacies on constitutively active mutants of the human adenosine A2B receptor.

The human adenosine A(2B) receptor belongs to class A G protein-coupled receptors (GPCRs). In our previous work, constitutively active mutant (CAM) human adenosine A(2B) receptors were identified from a random mutation bank. In the current study, three known A(2B) receptor antagonists, 4-{2-[7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-yl-amino]ethyl}phenol (ZM241385), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and N-(4-acetylphenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]acetamide (MRS1706) were tested on wild-type and nine CAM A(2B) receptors with different levels of constitutive activity in a yeast growth assay. All three compounds turned out to be inverse agonists for the adenosine A(2B) receptor because they were able to fully reverse the basal activity of four low-level constitutively active A(2B) receptor mutants and to partially reverse the basal activity of three medium-level constitutively active A(2B) receptor mutants. We also discovered two highly constitutively active mutants whose basal activity could not be reversed by any of the three compounds. A two-state receptor model was used to explain the experimental observations; fitting these yielded the following relative intrinsic efficacies for the three inverse agonists ZM241385, DPCPX, and MRS1706: 0.14 +/- 0.03, 0.35 +/- 0.03, and 0.31 +/- 0.02, respectively. Moreover, varying L, the ratio of active versus inactive receptors in this model, from 0.11 for mutant F84L to 999 for two highly constitutively active mutants yielded simulated dose-response curves that mimicked the experimental curves. This study is the first description of inverse agonists for the human adenosine A(2B) receptor. Moreover, the use of receptor mutants with varying levels of constitutive activity enabled us to determine the relative intrinsic efficacy of these inverse agonists.

Mining a chemical database for fragment co-occurrence: discovery of "chemical clichés".

Nowadays millions of different compounds are known, their structures stored in electronic databases. Analysis of these data could yield valuable insights into the laws of chemistry and the habits of chemists. We have therefore explored the public database of the National Cancer Institute (>250,000 compounds) by pattern searching. We split the molecules of this database into fragments to find out which fragments exist, how frequent they are, and whether the occurrence of one fragment in a molecule is related to the occurrence of another, nonoverlapping fragment. It turns out that some fragments and combinations of fragments are so frequent that they can be called "chemical clichés". We believe that the fragment data can give insight into the chemical space explored so far by synthesis. The lists of fragments and their (co-)occurrences can help create novel chemical compounds by (i) systematically listing the most popular and therefore most easily used substituents and ring systems for synthesizing new compounds, (ii) being an easily accessible repository for rarer fragments suitable for lead compound optimization, and (iii) pointing out some of the yet unexplored parts of chemical space.

Allosteric modulators affect the internalization of human adenosine A1 receptors.

To study the effect of allosteric modulators on the internalization of human adenosine A(1) receptors, the receptor was equipped with a C-terminal yellow fluorescent protein tag. The introduction of this tag did not affect the radioligand binding properties of the receptor. CHO cells stably expressing this receptor were subjected during 16 h to varying concentrations of the agonist N(6)-cyclopentyladenosine (CPA) in the absence or presence of 10 microM of the allosteric enhancer PD 81,723 ((2-amino-4,5-dimethyl-3-thienyl)-[3-(trifluoromethyl)phenyl]methanone) or the allosteric inhibitor SCH-202676 (N-(2,3-diphenyl-1,2,4-thiadiazol-5(2H)-ylidene)methanamine). CPA itself was able to internalize 25% and 40% of the receptors at a concentration of 400 nM or 4 muM, respectively. Addition of either PD 81,723 or SCH-202676 alone had no effect on internalization. However, with PD 81,723 a slight amount of internalization was obtained already at 40 nM of CPA and at 400 nM CPA 59% of the receptors internalized. SCH-202676 on the other hand effectively prevented CPA-induced internalization of the receptor.

A "locked-on," constitutively active mutant of the adenosine A1 receptor.

We studied the wild-type human adenosine A1 receptor and three mutant receptors, in which the glycine at position 14 had been changed into an alanine, a leucine, or a threonine residue. All receptors were characterized in radioligand binding experiments, the wild-type and the Gly14Thr mutant receptor in greater detail. Both receptors were allosterically modulated by sodium ions and PD81,723 (2-amino-4,5-dimethyl-3-thienyl-[3(trifluoromethyl)-phenyl]methanone), although in a different way. All mutant receptors appeared to be spontaneously or "constitutively" active in a [35S]GTPgammaS binding assay, the first demonstration of the existence of such CAM (constitutively active mutant) receptors for the adenosine A1 receptor. The Gly14Thr mutant receptor was also constitutively active in another functional assay, i.e., the inhibition of forskolin-induced cAMP production in intact cells. Importantly, this mutant displayed a peculiar "locked-on" phenotype, i.e., neither agonist nor inverse agonist was capable of modulating the basal activity in both the GTPgammaS and the cAMP assay, unlike the wild-type and the two other mutant receptors.

Allosteric modulation and constitutive activity of fusion proteins between the adenosine A1 receptor and different 351Cys-mutated Gi alpha-subunits.

We studied fusion proteins between the human adenosine A1 receptor and different 351Cys-mutated G(i1) alpha-subunits (A1-Gialpha) with respect to two important concepts in receptor pharmacology, i.e. allosteric modulation and constitutive activity/inverse agonism. The aim of our study was twofold. We first analysed whether such fusion products are still subject to allosteric modulation, and, secondly, we investigated the potential utility of the fusion proteins to study constitutive receptor activity. We determined the pharmacological profile of nine different A1-Gialpha fusion proteins in radioligand binding studies. In addition, we performed [35S]GTPgammaS binding experiments to study receptor and G protein activation of selected A1-Gialpha fusion proteins. Compared to unfused adenosine A1 receptors, the affinity of N6-cyclopentyladenosine (CPA) at wild-type A1-Gialpha fusion proteins (351Cys) increased more than eightfold, while the affinity of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) did not change significantly. Furthermore, we showed that the allosteric enhancer of agonist binding, PD81,723 (2-amino-4,5-dimethyl-3-thienyl-[3-(trifluoromethyl)-phenyl]methanone), elicited similar effects on ligand binding; i.e. CPA binding to the A1-Gialpha fusion proteins was enhanced, whereas the affinity of DPCPX was hardly affected. Moreover, sodium ions were unable to decrease agonist binding to the majority of the A1-Gialpha fusion proteins, presumably because they exhibit their effect through uncoupling of the R-G complex. From [35S]GTPgammaS binding experiments, we learned that all the A1-Gialpha fusion proteins tested had a higher basal receptor activity than the unfused adenosine A1 receptor, thereby providing improved conditions to observe inverse agonism. Moreover, the maximal CPA-induced stimulation of basal [35S]GTPgammaS binding was increased for the five A1-Gialpha fusion proteins tested, whereas the inhibition induced by 8-cyclopentyltheophylline (CPT) was more pronounced at 351Cys, 351Ile, and 351Val A1-Gialpha fusion proteins. Thus, the maximal receptor (de)activation depended on the amino acid at position 351 of the Gi alpha-subunit. In conclusion, A1-Gialpha fusion proteins, especially with 351Cys and 351Ile, can be used as research tools to investigate inverse agonism, due to their increased readout window in [35S]GTPgammaS binding experiments.

New, non-adenosine, high-potency agonists for the human adenosine A2B receptor with an improved selectivity profile compared to the reference agonist N-ethylcarboxamidoadenosine.

The adenosine A(2B) receptor is the least well characterized of the four known adenosine receptor subtypes because of the absence of potent, selective agonists. Here, we present five non-adenosine agonists. Among them, 2-amino-4-(4-hydroxyphenyl)-6-(1H-imidazol-2-ylmethylsulfanyl)pyridine-3,5-dicarbonitrile, 17, LUF5834, is a high-efficacy partial agonist with EC(50) = 12 nM and 45-fold selectivity over the adenosine A(3) receptor but lacking selectivity versus the A(1) and A(2A) subtypes. Compound 18, LUF5835, the 3-hydroxyphenyl analogue, is a full agonist with EC(50) = 10 nM.

Synthesis and biological evaluation of disubstituted N6-cyclopentyladenine analogues: the search for a neutral antagonist with high affinity for the adenosine A1 receptor.

Novel 3,8- and 8,9-disubstituted N(6)-cyclopentyladenine derivatives were synthesised in moderate overall yield from 6-chloropurine. The derivatives were made in an attempt to find a new neutral antagonist with high affinity for adenosine A(1) receptors. N(6)-Cyclopentyl-9-methyladenine (N-0840) was used as a lead compound. Binding affinities of the new analogues were determined for human adenosine A(1) and A(3) receptors. Their intrinsic activity was assessed in [35S]GTPgammaS binding experiments. Elongation of the 9-methyl of N-0840 to a 9-propyl substituent was very well tolerated. A 9-benzyl group, on the other hand, caused a decrease in adenosine A(1) receptor affinity. Next, the 8-position was examined in detail, and affinity was increased with appropriate substitution. Most derivatives were A(1)-selective and 20 of the new compounds (6-9, 15-21, 23-26, 28, 31, 33, 35, and 36) had higher adenosine A(1) receptor affinity than the reference substance, N-0840. Compound 31 (N(6)-cyclopentyl-8-(N-methylisopropylamino)-9-methyladenine, LUF 5608) had the highest adenosine A(1) receptor affinity, 7.7 nM. In the [35S]GTPgammaS binding experiments, derivatives 5, 14, 22, 23, 25, 26, 33 and 34 did not significantly change basal [35S]GTPgammaS binding, thus behaving as neutral antagonists. Moreover, four of these compounds (23, 25, 26, and 33) displayed a 4- to 10-fold increased adenosine A(1) receptor affinity (75-206 nM) compared to N-0840 (852 nM). In summary, we synthesised a range of N-0840 analogues with higher affinity for adenosine A(1) receptors. In addition, four new derivatives, LUF 5666 (23), LUF 5668 (25), LUF 5669 (26) and LUF 5674 (33), behaved as neutral antagonists when tested in [35S]GTPgammaS binding studies. Thus, these compounds have improved characteristics as neutral adenosine A(1) receptor antagonists.