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G protein-coupled receptors (GPCRs) form the most frequently exploited drug target family, moreover they are often found mutated in cancer. Here we used an aggregated dataset of mutations found in cancer patient samples derived from the Genomic Data Commons and compared it to the natural human variance as exemplified by data from the 1000 Genomes project. While the location of these mutations across the protein domains did not differ significantly in the two datasets, a mutation enrichment was observed in cancer patients among conserved residues in GPCRs such as the “DRY” motif. We subsequently created a ranking of high scoring GPCRs, using a multi-objective approach (Pareto Front Ranking). The validity of our approach was confirmed by re-discovery of established cancer targets such as the LPA and mGlu receptor families, and we identified novel GPCRs that had not been directly linked to cancer before such as the P2Y Receptor 10 (P2RY10). As a proof of concept, we projected the structurally investigated mutations in the crystal structure of the C-C Chemokine (CCR) 5 receptor, one of the high-ranking GPCRs previously linked to cancer. Several positions were pinpointed that relate to either structural integrity or endogenous and synthetic ligand binding, providing a rationale to their mechanism of influence in cancer. In conclusion, this study identifies a list of GPCRs that are prioritized for experimental follow up characterization to elucidate their role in cancer. The computational approach here described can be adapted to investigate the roles in cancer of any protein family.Author summaryDespite cancer being one of the most studied diseases due to its high mortality rate, one underexplored aspect is the association of certain protein families with tumor pathogenicity. We focused here on the G protein-coupled receptors family for three reasons. Firstly, it has been shown that this is the second most mutated class of proteins in cancer following kinases. Secondly, this family has been extensively studied resulting in a wide availability of experimental data for these proteins. Finally, more than 30 % of the drugs currently in the market target its members. For these receptors, we explored the mutational landscape across cancer patients compared to healthy individuals. Our findings show the existence of cancer-related alteration patterns that occur at conserved positions. Additionally, we computationally ranked these G protein-coupled receptors on their importance in the pathogenesis of cancer based on multiple objectives. The result is a list of recommendations on where to focus next. These results suggest that there is room for repurposing existing therapies for cancer treatment while also highlighting the risk of potential interactions between cancer treatments and common drugs. All in all, we present a window of opportunity for new targeting strategies in oncology for G protein-coupled receptors.

Kv11.1 (hERG) channels play a critical role in repolarization of cardiomyocytes during the cardiac action potential (AP). Drug mediated Kv11.1 blockade results in AP prolongation, which poses an increased risk of sudden cardiac death. Many drugs, like pentamidine, interfere with normal Kv11.1 forward trafficking and thus reduce functional Kv11.1 channel densities. Although class III antiarrhythmics, e.g. dofetilide, rescue congenital and acquired forward trafficking defects, this is of little use due to their simultaneous acute channel blocking effect. We aimed to test the ability of a combination of dofetilide plus LUF7244, a Kv11.1 allosteric modulator/activator, to rescue Kv11.1 trafficking and produce functional Kv11.1 current. LUF7244 treatment by itself did not disturb or rescue WT or G601S Kv11.1 trafficking as shown by western blot and immunofluorescence microcopy analysis. Pentamidine-decreased maturation of WT Kv11.1 levels was rescued by 10 μM dofetilide or 10 μM dofetilide + 5 μM LUF7244. In trafficking defective G601S Kv11.1 cells, dofetilide (10 μM) or dofetilide+LUF7244 (10+5 μM) restored Kv11.1 trafficking also, as demonstrated by western blot and immunofluorescence microscopy. LUF7244 (10 μM) increased IKv11.1 despite the presence of dofetilide (1 μM) in WT Kv11.1 cells. In G601S expressing cells, long-term treatment (24-48 h) with LUF7244 (10 μM) and dofetilide (1 μM) increased IKv11.1 compared to non-treated, or acutely treated cells. We conclude that dofetilide plus LUF7244 rescues Kv11.1 trafficking and produces functional IKv11.1. Thus, combined administration of LUF7244 and an IKV11.1 trafficking corrector could serve as a new pharmacological therapy of both congenital and drug-induced Kv11.1 trafficking defects.

Voltage-gated potassium 11.1 (K

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.

Background and Purpose Kv11.1 (hERG) channel blockade is an adverse effect of many drugs and lead compounds, associated with lethal cardiac arrhythmias. LUF7244 is a negative allosteric modulator/activator of Kv11.1 channels that inhibits early afterdepolarizations in vitro. We tested LUF7244 for antiarrhythmic efficacy and potential proarrhythmia in a dog model. Experimental Approach LUF7244 was tested in vitro for (a) increasing human IKv11.1 and canine IKr and (b) decreasing dofetilide‐induced action potential lengthening and early afterdepolarizations in cardiomyocytes derived from human induced pluripotent stem cells and canine isolated ventricular cardiomyocytes. In vivo, LUF7244 was given intravenously to anaesthetized dogs in sinus rhythm or with chronic atrioventricular block. Key Results LUF7244 (0.5–10 μM) concentration dependently increased IKv11.1 by inhibiting inactivation. In vitro, LUF7244 (10 μM) had no effects on IKIR2.1, INav1.5, ICa‐L, and IKs, doubled IKr, shortened human and canine action potential duration by approximately 50%, and inhibited dofetilide‐induced early afterdepolarizations. LUF7244 (2.5 mg·kg−1·15 min−1) in dogs with sinus rhythm was not proarrhythmic and shortened, non‐significantly, repolarization parameters (QTc: −6.8%). In dogs with chronic atrioventricular block, LUF7244 prevented dofetilide‐induced torsades de pointes arrhythmias in 5/7 animals without normalization of the QTc. Peak LUF7244 plasma levels were 1.75 ± 0.80 during sinus rhythm and 2.34 ± 1.57 μM after chronic atrioventricular block. Conclusions and Implications LUF7244 counteracted dofetilide‐induced early afterdepolarizations in vitro and torsades de pointes in vivo. Allosteric modulators/activators of Kv11.1 channels might neutralize adverse cardiac effects of existing drugs and newly developed compounds that display QTc lengthening.

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 A

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.KEYWORDS: A2A adenosine receptor; Adenosine; Covalent antagonist; G protein-coupled receptors; Radioligand binding

BACKGROUND AND PURPOSE\nDrug-induced arrhythmia due to blockade of the Kv 11.1 channel (also known as the hERG K(+) channel) is a frequent side effect. Previous studies have primarily focused on equilibrium parameters, i.e. affinity or potency, of drug candidates at the channel. The aim of this study was to determine the kinetics of the interaction with the channel for a number of known Kv 11.1 blockers and to explore a possible correlation with the affinity or physicochemical properties of these compounds.\nEXPERIMENTAL APPROACH\nThe affinity and kinetic parameters of 15 prototypical Kv 11.1 inhibitors were evaluated in a number of [(3) H]-dofetilide binding assays. The lipophilicity (logKW - C8 ) and membrane partitioning (logKW - IAM ) of these compounds were determined by means of HPLC analysis.\nKEY RESULTS\nA novel [(3) H]-dofetilide competition association assay was set up and validated, which allowed us to determine the binding kinetics of the Kv 11.1 blockers used in this study. Interestingly, the compounds' affinities (Ki values) were correlated to their association rates rather than dissociation rates. Overall lipophilicity or membrane partitioning of the compounds were not correlated to their affinity or rate constants for the channel.\nCONCLUSIONS AND IMPLICATIONS\nA compound's affinity for the Kv 11.1 channel is determined by its rate of association with the channel, while overall lipophilicity and membrane affinity are not. In more general terms, our findings provide novel insights into the mechanism of action for a compound's activity at the Kv 11.1 channel. This may help to elucidate how Kv 11.1-induced cardiotoxicity is governed and how it can be circumvented in the future.

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.

Background and PurposeKv11.1 (hERG) channel blockade is an adverse effect of many drugs and lead compounds, associated with lethal cardiac arrhythmias. LUF7244 is a negative allosteric modulator/activator of Kv11.1 channels that inhibits early afterdepolarizations in vitro. We tested LUF7244 for antiarrhythmic efficacy and potential proarrhythmia in a dog model.Experimental ApproachLUF7244 was tested in vitro for (a) increasing human IKv11.1 and canine IKr and (b) decreasing dofetilide‐induced action potential lengthening and early afterdepolarizations in cardiomyocytes derived from human induced pluripotent stem cells and canine isolated ventricular cardiomyocytes. In vivo, LUF7244 was given intravenously to anaesthetized dogs in sinus rhythm or with chronic atrioventricular block.Key ResultsLUF7244 (0.5–10 μM) concentration dependently increased IKv11.1 by inhibiting inactivation. In vitro, LUF7244 (10 μM) had no effects on IKIR2.1, INav1.5, ICa‐L, and IKs, doubled IKr, shortened human and canine action potential duration by approximately 50%, and inhibited dofetilide‐induced early afterdepolarizations. LUF7244 (2.5 mg·kg−1·15 min−1) in dogs with sinus rhythm was not proarrhythmic and shortened, non‐significantly, repolarization parameters (QTc: −6.8%). In dogs with chronic atrioventricular block, LUF7244 prevented dofetilide‐induced torsades de pointes arrhythmias in 5/7 animals without normalization of the QTc. Peak LUF7244 plasma levels were 1.75 ± 0.80 during sinus rhythm and 2.34 ± 1.57 μM after chronic atrioventricular block.Conclusions and ImplicationsLUF7244 counteracted dofetilide‐induced early afterdepolarizations in vitro and torsades de pointes in vivo. Allosteric modulators/activators of Kv11.1 channels might neutralize adverse cardiac effects of existing drugs and newly developed compounds that display QTc lengthening.

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. Electronic supplementary material The online version of this article (doi:10.1007/s11302-016-9549-9) contains supplementary material, which is available to authorized users.

Contains fulltext : 103842.pdf (Publisher’s version ) (Closed access) The adenosine A1 receptor is a member of the large membrane protein family that signals through G proteins, the G protein-coupled receptors (GPCRs). GPCRs consist of seven transmembrane domains connected by three intracellular and three extracellular loops. Their N-terminus is extracellular, the C-terminal tail is in the cytoplasm. The transmembrane domains in receptor subfamilies that bind the same endogenous ligand, such as dopamine or adenosine, tend to be highly similar. In contrast, the loop regions can vary greatly, both in sequence and in length, and the role these loops have in the activation mechanism of the receptors remains unclear. Here, we investigated the activating role of the second and third extracellular loop of the human adenosine A1 receptor. By means of an (Ala)3 mutagenic scan in which consecutive sets of three amino acids were mutated into alanine residues in EL2 and a classical alanine scan in EL3, we revealed a strong regulatory role for the second extracellular loop (EL2) of the human adenosine A1 receptor. Besides many residues in the second and the third extracellular loops important for adenosine A1 receptor activation, we also identified two residues in EL2, a tryptophan and a glutamate, that affect the influence of the allosteric modulator PD81,723. These results, combined with a comparison of the different receptor loop regions, provide insight in the activation mechanism of this typical class A GPCR and further emphasize the unique pharmacological profile the loops can provide to individual receptors, even within subfamilies of GPCRs.

Chemogenomic approaches, which link ligand chemistry to bioactivity against targets (and, by extension, to phenotypes) are becoming more and more important due to the increasing number of bioactivity data available both in proprietary databases as well as in the public domain. In this article we review chemogenomics approaches applied in four different domains: Firstly, due to the relationship between protein targets from which an approximate relation between their respective bioactive ligands can be inferred, we investigate the extent to which chemogenomics approaches can be applied to receptor deorphanization. In this case it was found that by using knowledge about active compounds of related proteins, in 93% of all cases enrichment better than random could be obtained. Secondly, we analyze different chemin-formatics analysis methods with respect to their behavior in chemogenomics studies, such as subgraph mining and Baye-sian models. Thirdly, we illustrate how chemogenomics, in its particular flavor of 'proteochemometrics', can be applied to extrapolate bioactivity predictions from given data points to related targets. Finally, we extend the concept of 'chemoge-nomics' approaches, relating ligand chemistry to bioactivity against related targets, into phenotypic space which then falls into the area of 'chemical genomics' and 'chemical genetics'; given that this is very often the desired endpoint of approaches in not only the pharmaceutical industry, but also in academic probe discovery, this is often the endpoint the experimental scientist is most interested in. © 2011 Bentham Science Publishers.

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 chemokine receptor CCR3 is believed to play a role in the development of allergic diseases such as asthma, atopic dermatitis, and allergic rhinitis. Despite the conflicting results that have been reported regarding the importance of eosinophils and CCR3 in allergic inflammation, inhibition of this receptor with small molecule antagonists is thought to provide a valuable approach for the treatment of these diseases. This review describes the structure-activity relationships (SAR) of small molecule CCR3 antagonists as reported in the scientific and patent literature. Various chemical classes of small molecule CCR3 antagonists have been described so far, including (bi)piperidine and piperazine derivatives, N-arylalkylpiperidine urea derivatives and (N-ureidoalkyl)benzylpiperidines, phenylalanine derivatives, morpholinyl derivatives, pyrrolidinohydroquinazolines, arylsulfonamides, amino-alkyl amides, imidazole- and pyrimidine-based antagonists, and bicyclic diamines. The (N-ureidoalkyl)benzylpiperidines are the best studied class in view of their generally high affinity and antagonizing potential. For many of these antagonists subnanomolar IC(50) values were reported for binding to CCR3 along with the ability to effectively inhibit intracellular calcium mobilization and eosinophil chemotaxis induced by CCR3 agonist ligands in vitro.

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.

The superfamily of human G protein-coupled receptors (GPCRs) is large and regulates a plethora of important physiological processes by transducing extracellular signals over cell membranes. A diversity of natural variants occurs in these receptors, including rare mutations and common polymorphisms. These variants differ in their impact on DNA, ranging from single nucleotide polymorphisms (SNPs) to copy number variants, and in their impact on protein function. Natural variants furthermore vary in their effects on human phenotypes from neutral to disease-associated. As mutation data are highly dispersed over numerous sources, a single resource for variants would aid investigators of GPCRs. The GPCR NaVa database therefore integrates data on natural variants in human GPCRs from online databases, the scientific literature, and patents. Where available, variants contain information on their location in the DNA (and protein sequence), the involved nucleotides (and amino acids), the average frequency of each allele, reported disease associations, and references to public databases and the scientific literature. The GPCR NaVa database aims to facilitate studies into pharmacogenetics, genotype–phenotype, and structure–function relationships of GPCRs. The GPCR NaVa database is interlinked with the family-specific GPCRDB resource and is accessible as a stand-alone database through a user-friendly website at http://nava.liacs.nl (last accessed 28 August 2007). Hum Mutat 29(1), 39–44, 2008. © 2007 Wiley-Liss, Inc.

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.

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 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.

Clustering problems arise in various domains of science and engineering. A large number of methods have been developed to date. The Kohonen self-organizing map (SOM) is a popular tool that maps a high-dimensional space onto a small number of dimensions by placing similar elements close together, forming clusters. Cluster analysis is often left to the user. In this paper we present the method TreeSOM and a set of tools to perform unsupervised SOM cluster analysis, determine cluster confidence and visualize the result as a tree facilitating comparison with existing hierarchical classifiers. We also introduce a distance measure for cluster trees that allows one to select a SOM with the most confident clusters.

Allosteric modulation of G protein-coupled receptors has recently been recognized as an alternative approach to gain selectivity in drug action. In this overview, allosteric modulators that enhance or diminish the effects of (endogenous) agonists or antagonists on a variety of G protein-coupled receptors are described. Emphasis is placed on the latest developments in this research area, including data on the first clinical studies. It appears that all three major classes of G protein-coupled receptors (A, B and C) are amenable to allosteric modulation by small molecules. This constitutes an attractive and novel means to identify new leads in the drug discovery process. However, it requires a re-engineering of the majority of current assays. Finally, it is suggested to introduce the term 'non-competitive agonism' or 'allosteric agonism' next to allosteric modulation.

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.

Novel 3,8- and 8,9-disubstituted N6-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 A1 receptors. N6-Cyclopentyl-9- methyladenine (N-0840) was used as a lead compound. Binding affinities of the new analogues were determined for human adenosine A1 and A 3 receptors. Their intrinsic activity was assessed in [ 35S]GTPγS 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 A1 receptor affinity. Next, the 8-position was examined in detail, and affinity was increased with appropriate substitution. Most derivatives were A1-selective and 20 of the new compounds (6-9, 15-21, 23-26, 28, 31, 33, 35, and 36) had higher adenosine A1 receptor affinity than the reference substance, N-0840. Compound 31 (N6-cyclopentyl-8-(N-methylisopropylamino)-9- methyladenine, LUF 5608) had the highest adenosine A1 receptor affinity, 7.7 nM. In the [35S]GTPγS binding experiments, derivatives 5, 14, 22, 23, 25, 26, 33 and 34 did not significantly change basal [35S]GTPγS binding, thus behaving as neutral antagonists. Moreover, four of these compounds (23, 25, 26, and 33) displayed a 4- to 10-fold increased adenosine A1 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 A1 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]GTPγS binding studies. Thus, these compounds have improved characteristics as neutral adenosine A1 receptor antagonists. © 2003 Elsevier Ltd. All rights reserved.

Characterization of the pharmacokinetics, brain distribution, and therapeutic efficacy of the adenosine A1 receptor partial agonist 2′-deoxy-N6-cyclopentyladenosine in sarin-poisoned rats. Bueters, T.J.H., IJzerman, A.P., Van Helden, H.P.M., and Danhof, M. (2003). The objective of the present study was to determine (1) the influence of sarin poisoning (144 μg/kg sc) on the pharmacokinetics and brain distribution of the adenosine A1 receptor partial agonist 2′-deoxy-N6-cyclopentyladenosine (2′dCPA), and (2) the effect of 2′dCPA (20 mg/kg iv) on the central acetylcholine (ACh) release and protection against sarin toxicity. A five-compartment model successfully described the pharmacokinetic profile of 2′dCPA in blood and brain microdialysate. A covariate analysis revealed that the volume of distribution of 2′dCPA in blood was different in sarin-poisoned rats, 177 ± 7 versus 148 ± 8 ml in control rats. However, the transport of 2′dCPA from blood to the brain was unaffected as reflected by the values of the intercompartmental transport clearances, 0.21 ± 0.02 and 0.21 ± 0.04 μl/min in control and sarin-poisoned rats, respectively. Also the area-under-curve (AUC) ratios of brain microdialysate and blood were identical with values of 0.02 ± 0.001 and 0.02 ± 0.002, respectively, demonstrating the restricted transport of 2′dCPA into the brain in both treatment groups. Treatment of sarin-poisoned rats by 2′dCPA did not adequately prevent the accumulation of ACh in the central nervous system. 2′dCPA delayed the emergence of concomitant symptoms compared to untreated rats, but eventually only 29% of the animals survived 24 h. In conclusion, the pharmacokinetic profile of 2′dCPA in blood was slightly changed by sarin, but not the distribution of 2′dCPA into the brain. The therapeutic efficacy of 2′dCPA against sarin was limited, presumably due to insufficient quantities of 2′dCPA reaching the brain.

In this review the latest developments in ligand design for the adenosine A(1) receptor are summarized. Novel series of agonists and antagonists are discussed, leading to the conclusion that ligands truly selective for the human adenosine A(1) receptor are still scarce.

The objective of the present study was to develop a kinetics of pharmacodynamics model to properly describe and investigate the in vivo interaction between the selective adenosine A(1) agonist N(6)-cyclopentyladenosine (CPA), acetylcholinesterase (AChE) in blood and brain, and the AChE-inhibitor sarin (isopropylmethylphosphonofluoridate). The direct interaction of CPA (2 microM) on the inhibition of AChE by sarin was studied in vitro in heparinized rat blood and in 10% (w/v) brain homogenate. CPA did not directly influence the sarin-mediated inactivation of AChE in either system. In sarin-poisoned (144 microg/kg s.c.) rats not treated with CPA, AChE was completely inactivated in blood and brain within 7 min. CPA (2 mg/kg i.m.) treatment, 1 min after sarin administration, caused a small delay in the inhibition of AChE in blood. Treatment with CPA, 2 min before sarin, protected the neuronal AChE partially from being inhibited, but not the enzyme localized in blood. With a dose-response-time model the proportion of the dose of sarin reaching the site of action was estimated to be 48 +/- 12 or 13 +/- 3% after CPA post- or pretreatment, respectively. A correlation between the residual AChE activity in the brain and the incidence of cholinergic symptoms could be established with logistic regression analysis: lower inhibition of AChE in the brain precluded the onset of critical symptoms. In conclusion, CPA affects the concentration of sarin reaching the site of action, which contributes to the protection previously observed in sarin-poisoned rats.

The objective of the present study was to characterize the adenosine A(1) receptor allosteric enhancing and antagonistic actions of (2-amino-4,5,6,7-tetrahydrobenzo[b]thiophen-3-yl)(3,4-dichlorophenyl)methanone (LUF 5484) and (2-amino-4,5-dimethyl-3-thienyl)-[3-(trifluoromethyl)phenyl]methanone (PD 81,723) on striatal acetylcholine release. Upon local administration in conscious rats, LUF 5484 or PD 81,723 caused a concentration-dependent increase of extracellular acetylcholine levels of approximately 40%, which was similar to that obtained by the selective adenosine A(1) receptor antagonists 8-cyclopentyl-1,3-dimethylxanthine (8CPT) and N(6)-cyclopentyl-9-methyladenine (N0840). In interaction experiments, LUF 5484 or PD 81,723 did not change the inhibition of acetylcholine release by the adenosine A(1) receptor agonist N(6)-cyclopentyladenosine (CPA), whereas 8CPT caused an eightfold rightward shift. Acetylcholine concentrations were diminished with 62+/-3%, 48+/-11% and 56+/-9% by CPA, CPA+LUF 5484 and CPA+PD 81,723, respectively. In conclusion, the antagonistic action of LUF 5484 and PD 81,723 seems to counteract the putative allosteric actions with respect to the reduction of striatal acetylcholine release.

1H,3H-Pyrido[2,1-f]purine-2,4-diones, which can be described as fused xanthine structures, have been synthesized by a novel synthetic procedure, and their affinities for the human adenosine A(1), A(2A), and A(3) receptors have been evaluated in radioligand binding studies. The synthetic procedure employed was developed in our laboratory and involved a two-step one-pot reaction that consists of the treatment of 6-aminouracil derivatives with N-bromosuccinimide to generate a 5,5-dibromo-6-imino intermediate that reacts "in situ" with pyridine, 4-methoxypyridine, 4-tert-butylpyridine, or 4-phenylpyridine to afford the corresponding 1H,3H-pyrido[2,1-f]purine-2,4-diones (2-5). Functionalization at the N(3) position in compounds 2-5 was performed by reaction with DBU and different alkyl, alkenyl, alkynyl, or benzyl halides. Binding studies at human adenosine A(1), A(2A), and A(3) receptors revealed significant antagonist effects in the low nanomolar range, in particular against the A(3) receptor. Thus, the 1-benzyl-3-propyl-1H,3H-pyrido[2,1-f]purine-2,4-dione derivative 6, which can be considered a lead compound in this series, exhibited a K(i) value of 4.0 +/- 0.3 nM against the hA(3) receptor. Because xanthine derivatives have traditionally been considered poor A(3) antagonists, the described pyrido[2,1-f]purine-2,4-dione derivatives represent a new family of adenosine receptor antagonists which deserves further exploration.

Adenosine triphosphate (ATP) can be released in large amounts from (damaged) cells, leading to locally high concentrations. In this study, we investigated the effect of such high concentrations of ATP on neuroblastoma cells. ATP (≥30 μM) induced apoptosis in the mouse neuroblastoma cell line N1E-115. Activation of the ATP receptor P2X 7 is one of the routes via which ATP has been shown to induce apoptosis. Although the P2X 7 receptor was present in N1E-115 cells, both at the protein and mRNA level, studies with the P2X 7 receptor agonist benzoyl–benzoyl ATP showed that this receptor was not involved in ATP-induced apoptosis. It has been shown previously that adenosine induces apoptosis in N1E-115 cells after transport inside the cell. In this study, both dipyridamole, a nucleoside transport protein blocker, and uridine, a substrate for this transporter, were able to block ATP-induced apoptosis. This indicated that ATP had to be broken down to adenosine to induce apoptosis. The ecto-nucleotidase inhibitors 6- N , N -diethyl-β-dibromomethylene- d -adenosine-5′-triphosphate (ARL67156) and α,β-methylene adenosine 5′-diphosphate (AOPCP) commonly used to slow breakdown of ATP did not inhibit ATP breakdown appreciably, while the ATP antagonist PPADS inhibited the breakdown of AMP to adenosine; PPADS was also the only compound capable of inhibiting ATP-induced apoptosis. We conclude that the main route of ATP-induced apoptosis in N1E-115 cells was via breakdown to adenosine.

Novel 2,5'-disubstituted adenosine derivatives were synthesized in good overall yields starting from commercially available guanosine. Binding affinities were determined for rat adenosine A(1) and A(2A) receptors and human A(3) receptors. E(max) values were determined for the stimulation or inhibition of cAMP production in CHO cells expressing human adenosine A(2A) (EC(50) values as well) or A(3) receptors, respectively. The compounds displayed affinities in the nanomolar range for both the adenosine A(2A) and A(3) receptor, without substantial preference for either receptor. The derivatives with a 2-(1-hexynyl) group had the highest affinities for both receptors; compound 4 (2-(1-hexynyl)adenosine) had the highest affinity for the adenosine A(2A) receptor with a K(i) value of 6 nM (A(3)/A(2A) selectivity ratio of approximately 3), whereas compound 37 (2-(1-hexynyl)-5'-S-methyl-5'-thioadenosine) had the highest affinity for the adenosine A(3) receptor with a K(i) value of 15 nM (A(2A)/A(3) selectivity ratio of 4). In general, compounds with a relatively small 5'-S-alkyl-5'-thio substituent (methyl-5'-thio) displayed the highest affinities for both the adenosine A(2A) and A(3) receptor; the larger ones (n- or i-propyl-5'-thio) increased the selectivity for the adenosine A(3) receptor. The novel compounds were also evaluated in cAMP assays for their (partial) agonistic behavior. Overall, the disubstituted derivatives behaved as partial agonists for both the adenosine A(2A) and A(3) receptor. The compounds showed somewhat higher intrinsic activities on the adenosine A(2A) receptor than on the A(3) receptor. Compounds 37, 40 and 45, 48, with either a 5'-S-methyl-5'-thio or a 5'-S-i-propyl-5'-thio substituent had the lowest intrinsic activities on the adenosine A(2A) receptor. For the A(3) receptor, compounds 34, 35, 38, 39, and 46, 47, with a 5'-S-ethyl-5'-thio or a 5'-S-n-propyl-5'-thio substituent had the lowest intrinsic activities.

New N,5'-di- and N,2,5'-trisubstituted adenosine derivatives were synthesized in good overall yields. Appropriate 5-O-alkyl-substituted ribose moieties were coupled to 6-chloropurine or 2,6-dichloropurine via Vorbrüggen's glycosylation method. Subsequent amination and deprotection of the intermediates yielded compounds 18-35. Binding affinities were determined for rat adenosine A1 and A2A receptors and the human A3 receptor. The ability of compounds 18-35 to inhibit forskolin-induced (10 microM) cyclic AMP (cAMP) production and their ability to stimulate guanosine 5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding, via either the adenosine A1 receptor or the adenosine A3 receptor, were assessed. N-Cyclopentyl-substituted adenosine derivatives displayed affinities in the low nanomolar range for the adenosine A1 receptor, whereas N-(3-iodobenzyl)-substituted derivatives had high affinity for the adenosine A3 receptor. Compound 22 had the highest affinity for the adenosine A1 receptor (K(i) value of 16 nM), and compounds 20 and 26 had the highest affinities for the adenosine A3 receptor (K(i) values of 4 and 3 nM, respectively). A chlorine substituent at the 2-position either did not affect or slightly increased the adenosine A1 receptor affinity, whereas the A3 receptor affinity was affected differently, depending on the N-substituent. Furthermore, the introduction of chlorine slightly increased the A3/A1 selectivity ratio. At the 5'-position, an O-methyl substituent induced the highest adenosine A1 receptor affinity, whereas an O-ethyl substituent did so for the A3 receptor. All compounds showed partial agonistic effects in both the cAMP and [35S]GTPgammaS assays, although more marked in the latter assay. In general, the 2-chloro derivatives seemed to have lower intrinsic activities compared to the 2-H-substituted compounds on both the adenosine A1 and the adenosine A3 receptors. The compounds with an N-(3-iodobenzyl) substituent displayed the lowest intrinsic activities. Finally, all compounds also showed partially antagonistic behavior in the [35S]GTPgammaS assay.

Allosteric modulation of G protein-coupled receptors is a relatively novel and unexplored pharmacological concept that may lead to more selective and more ‘natural’ drugs for these receptors. In particular, allosteric enhancers may serve as tools to intensify selectively a weakened hormone or neurotransmitter signal caused by a localized deficit, such as in Alzheimer's or Parkinson's disease. In this paper, attention is paid to the adenosine A 1 receptor, for which novel allosteric enhancers were synthesized and characterized that proved superior to the prototypic allosteric enhancer PD 81,723.

The induction of apoptosis by adenosine was studied in the mouse neuroblastoma cell line N1E-115. Apoptosis was characterized by fluorescence and electron microscopy, fluorescence-activated cell sorter (FACS) analysis, and caspase activity assays. A sixteen-hour exposure to 100 μM of adenosine led to chromatin condensation and caspase activation. However, selective agonists for all four adenosine receptors were ineffective. Caspase activation could be blocked partially by an inhibitor of the nucleoside transporter, dipyridamole, and completely by uridine, a competing substrate for adenosine transport. 2′-Deoxycoformycin, an inhibitor of adenosine deaminase, enhanced caspase activation by adenosine but had no effect by itself. Caspase activation could be blocked by 5′-amino-5′-deoxyadenosine, which inhibits the phosphorylation of adenosine by adenosine kinase. These results indicate that adenosine receptors are not involved in adenosine-induced apoptosis in N1E-115 cells, but that uptake of adenosine and its subsequent phosphorylation is required.

The 2-amino-benzoylthiophene derivative PD81,723 [(2-amino-4,5-dimethyl-trienyl)[3-(trifluoromethyl) phenyl]methanone] has been shown to allosterically enhance agonist binding and function at the adenosine A 1 receptor. The aim of the present study was to elucidate the effects of PD81,723 both as an allosteric enhancer and as an antagonist on the adenosine A 1 receptor. We investigated its effect on the human wild-type in relation to a mutant (T277A) adenosine A 1 receptor for which agonists have a greatly diminished affinity. Binding (saturation and displacement experiments) and functional adenosine 3′,5′-cyclic monophosphate studies were performed, and differential effects of allosteric enhancer PD81,723 on agonists and antagonists were observed on the wild-type (wt) and mutant adenosine A 1 receptor. Our results showed opposite effects of PD81,723 on the binding of agonists and antagonists. Within the concept of a simplified two-state receptor model, it is possible that the effects of PD81,723 are mainly “allosteric”, enhancing the binding of adenosine A 1 agonists and inhibiting the binding of antagonists/inverse agonists. However, the suggestion that PD81,723 acts as an allosteric inhibitor of DPCPX (1,3-dipropyl-8-cyclopentylxanthine) binding cannot be confirmed by kinetic studies, since PD81,723 does not seem to affect the dissociation kinetics of [ 3 H]DPCPX. Nevertheless, our results show that the action of PD81,723 on DPCPX binding is due to more than mere competitive antagonistic activity, i.e. binding to the ligand-binding site and competing with the binding of DPCPX, as suggested previously. The effect of PD81,723 on the mutant receptor was much less pronounced. Mutation of Thr277 to Ala not only decreased agonist affinity but also inhibited the effects of PD81,723. Insensitivity of the mutT277A to PD81,723 may be linked to the fact that this mutant appears to be uncoupled from G proteins. It further supported a differential binding mode of PD81,723 compared to competitive antagonists for the adenosine A 1 receptor.

The interaction of 2-amino-3-benzoyl-thiophene derivative, PD81,723, as well as other G protein-coupled receptor (GPCR)-modulating agents such as suramin (SUR), N-ethylmaleimide (NEM), GTP, and NaCl with the rat adenosine A1 receptor was investigated using kinetic, saturation, as well as displacement experiments of various agonists, partial agonists or antagonists. PD81,723 enhanced agonist (CPA, R-PIA, NECA) binding ∼2-fold, while its effect on CPA binding was increased (4–11-fold) with other modulators present. In contrast, binding of antagonists (DPCPX, CPT, N-0840) was inhibited, while binding of partial agonists (8BCPA, MeSCPA) remained uninfluenced. The effect of PD81,723 is consistent with shifting A1 receptors to an “active” (R*) state with high affinity for agonists and low for antagonists. Further, all described allosteric modulators influenced both agonist and antagonist binding. The IC50 values observed for the agonist CPA, ranging from 4.7 nM in the presence of PD81,723 to a high value of 2.9 μM in the combined presence of NEM, NaCl, and GTP, represented a greater than 600-fold affinity shift. We suggest that the latter micromolar IC50 value may approximate CPA’s “true” affinity (KA) for the rat adenosine A1 receptor. Drug Dev. Res. 51:207–215, 2000. © 2001 Wiley-Liss, Inc.

The influence of nitro substituents on the properties of adenosine and 1-deazaadenosine was studied. Combination of a nitro group at the 2-position with several N6 substituents such as cyclopentyl and m-iodobenzyl gave a series of analogues with good adenosine receptor affinity, showing directable selectivity for the A1, A2A and A3 adenosine receptor subtypes.

The largest family of cell surface receptors involved in signal transduction, G protein coupled receptors (GPCRs), are one of the major targets for current drugs as well as new drug development. Ligands interacting with for e.g. adrenergic, histamine, adenosine, opioid, dopamine or serotonin receptors, constitute a large portion of currently used therapeutics. A common property of GPCRs is that upon activation (agonist binding) they transmit signals across the plasma membrane via an interaction with heterotrimeric G proteins (Stadel et al., 1997). The corresponding activated G protein subsequently interacts with an intracellular effector system, such as adenylate cyclase or phospholipase C, leading to a wide variety of distinct physiological responses. Recent evidence suggests that GPCRs have the potential to be 'active' even in the absence of an agonist. This exhibition of spontaneous receptor activity has led to the observation that various ligands, previously considered as antagonists with no intrinsic activity, actually can inhibit this spontaneous activity, appearing to possess 'negative intrinsic activity'. This phenomenon has been termed inverse agonism and the corresponding ligands are referred to as inverse agonists. Although intrinsic constitutive receptor activity and inverse agonism have unequivocally been demonstrated in vitro, (patho)physiological consequences are far from self-evident. Thus, in this review we should like to focus on the expression of inverse agonism under more 'physiological conditions', since it appears timely to address the physiological relevance and consequences of this new concept, both in GPCR research and drug discovery. Historical overview Traditional receptor theory has postulated on a single, 'quiescent' receptor state to which agonists bind inducing a conformational change of the receptor to an activated and 'functional' state. This view, initially formed in the early 1950s, was more clearly expressed by Del Castillo & Katz (1957), and stood as the foundation of receptor pharmacology for decades. It was believed that antagonists interact with the receptor, thereby preventing agonist binding, without having an effect on conformational changes of the receptor that remained in its 'quiescent' state. The first evidence of a ligand ('antagonist') producing opposite effects to those of an agonist, and thus not merely inhibiting agonist binding, stems from the GABA-benzodiazepine field. In Braestrup et al. 1982, reported on the discovery of an agent, DMCM (methyl 6,7-dimethoxy-4-ethyl-β-carboline-e-carboxylate), which in contrast to benzodiazepines, not only was a potent convulsant in vivo, but seemed to favour binding to benzodiazepine receptors that were in the non-GABA(agonist)-stimulated conformation. For the first time, it was elaborated that GABA-benzodiazepine receptors may perhaps exist in two conformations which are in equilibrium, an open chloride channel form (activated conformation) and a closed one (inactivated conformation) for which DMCM may have a high affinity and a tendency to stabilize, thereby decreasing binding of GABA to the activated conformation. Interestingly, the above concept of an 'agonist-independent' two-state receptor conformation, introduced initially for ion-channel-coupled receptors, soon found support from those studying the large family of GPCRs. Costa & Herz (1989) were pioneers in setting the grounds for what later would be referred to as 'inverse agonism'. They demonstrated for the first time that some antagonists of the δ opioid receptor had 'negative intrinsic activity' in vitro, in contrast to others that lacked any intrinsic activity. Moreover, such ligands diminished even further the 'basal' or constitutive activity of the receptor defined as the activity of the receptor in the absence of any ligand. It was apparent that the concept of 'negative intrinsic activity' implied a pre-existing equilibrium between (at least) two states of a receptor. These two states could easily be defined as either a G protein-bound or a free form of the receptor, the first one active and the latter inactive. In their studies, basal GTPase activity in NG108-15 cell membranes was suggested to be due to stimulated activity resulting from a spontaneous interaction between empty or free receptors and G proteins. Their data lent support to the receptor model of Wregget & De Lean (1984) which predicted that 'antagonists may be active by hindering the ability of receptors to associate spontaneously with G proteins in membranes'. Since then, and especially over the past few years, disclosure of negative intrinsic activity has corroborated even further this two-state model of GPCR activation. Hence, a large number of publications within this decade are concerned with, and demonstrate with a variety of systems or means, the phenomenon of constitutive receptor activity and its implication for inverse agonism. In this review we will first address some of the more seminal papers, showing that in most cases genetically engineered cell systems were pivotal for the development of this new concept. These and many more studies have been adequately and thoroughly reviewed recently by Milligan et al. (1997) and Leurs et al. (1998). We will then gradually move to more 'physiological' systems, in order to address the central issue of this review whether inverse agonism and spontaneous receptor activity are relevant phenomena in health and disease, and hence for drug discovery. It should be pointed out, however, that a classification of ligands based on their (negative) intrinsic activity is not an easy task. Due to the large influence of receptor systems and experimental conditions (whole cells versus membranes, stoichiometry of receptors/G protein, signalling proteins etc.) the same ligand may behave as an inverse agonist, a neutral antagonist or even a (partial) agonist.

A series of phenyl-substituted N 6 -phenyladenosines and N 6 -phenyl-5'-N-ethylcarboxamidoadenosines were synthesized and tested at adenosine receptor subtypes. EC 50 values were determined for cyclic AMP production in CHO cells expressing human A 2B receptors. Binding affinities were determined for rat A 1 and A 2A receptors and human A 3 receptors. N 6 -phenyladenosine displayed an EC 50 value at A 2B receptors of 6.3 μM. Several N 6 -phenyladenosine derivatives were more active than N 6 -phenyladenosine, while two analogs were also more potent than 5'-N-ethylcarboxamidoadenosine (NECA, 0.76 μM), i.e., the 4-iodophenyl (10, 0.37 μM) and the 4-aminosulfonylphenyl (20, 0.44 μM) derivatives. N 6 -phenyl-NECA derivatives were as active as their analogous adenosine derivatives.

Novel 2-amino-3-benzoylthiophene derivatives, with variable substitution on the thiophene as well as benzoyl ring, were synthesized and evaluated both as allosteric enhancers of agonist binding to the rat adenosine A1 receptor, and as antagonists on this receptor. Structural features were identified on the novel derivatives that favored allosteric enhancing activity, such as benzoyl lipophilic substitution and thiophene 4-alkyl substitution. In contrast, antagonistic properties were favored by thiophene 5-bulky substitution. Upon further analysis, a significant correlation was found between antagonistic activity and hydrophobic fragment constants (π values) of substituent R5, in contrast to a negative correlation with those of R4. Comparison of low energy conformations of some of the 2-amino-3-benzoylthiophene derivatives (PD81,723 and 4f) with known adenosine A1 antagonists (theophylline and 8-cyclohexyltheophylline) indicated that thiophene 5-substituents may interact with the same lipophilic domain of the adenosine A1 receptor accommodating 8-substituents of xanthine antagonists. Drug Dev. Res. 49:227–237, 2000. © 2000 Wiley-Liss, Inc.

A series of novel and known 5-substituted 7-amino-2-(2-furyl)[1,2,4]triazolo[1,5-a][1,3,5]triazine derivatives were synthesized and tested for adenosine receptor antagonism in radioligand binding assays at all four adenosine receptor subtypes and for inhibition of the agonist-induced cyclic AMP response at human A2B receptors. The known potent adenosine A2A receptor antagonist, 7-amino-2-(2-furyl)-5-[2-(4-hydroxyphenyl)ethyl]amino[1,2,4]triazolo[1,5-a][1,3,5]triazine (ZM241385, KiA2A = 1.78 nM) had a Ki value of 16.5 nM at A2B receptors in radioligand binding studies on Chinese hamster ovary cells expressing A2B receptors. A pA2 value of 7.9 was measured for the inhibition of the cyclic AMP response by A2B receptors induced by 5′-N-ethylcarboxamidoadenosine (NECA). In a series of 5-phenyl(alkyl)amino analogs the 5-(2-phenylethyl)amino analog LUF5452 and the 5-benzylamino analog LUF5451 were both more potent than ZM241385 in the cyclic AMP assay at A2B receptors. Moreover, Ki values of 9.9 and 7.6 nM were found in binding studies at this receptor subtype, indicating that LUF5451 and LUF5452 are more potent A2B receptor antagonists than ZM241385. The affinity of LUF5451 for the A2A receptor (Ki value = 13 nM) showed that the selectivity for this receptor subtype was lost and that a modest A2B receptor selectivity was achieved. The 5-(2-phenylhydrazino) derivative LUF5475 showed a high A2B receptor affinity (Ki = 7.6 nM), while it was equally active at A2A receptors, being A2B receptor-selective with respect to A1 and A3 receptors. Drug Dev. Res. 48:95–103, 1999. © 1999 Wiley-Liss, Inc.

The structure–activity relationships (SAR) of alkylxanthine derivatives as antagonists at the recombinant human adenosine receptors were explored in order to identify selective antagonists of A2B receptors. The effects of lengthening alkyl substituents from methyl to butyl at 1- and 3-positions and additional substitution at the 7- and 8-positions were probed. Ki values, determined in competition binding in membranes of HEK-293 cells expressing A2B receptors using 125I-ABOPX (125I-3-(4-amino-3-iodobenzyl)-8-(phenyl-4-oxyacetate)-1-propylxanthine), were approximately 10 to 100 nM for 8-phenylxanthine functionalized congeners. Xanthines containing 8-aryl, 8-alkyl, and 8-cycloalkyl substituents, derivatives of XCC (8-[4-[[[carboxy]methyl]oxy]phenyl]-1,3-dipropylxanthine) and XAC (8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl]-oxy]phenyl]-1,3-dipropylxanthine), containing various ester and amide groups, including L- and D-amino acid conjugates, were included. Enprofylline was 2-fold more potent than theophylline in A2B receptor binding, and the 2-thio modification was not tolerated. Among the most potent derivatives examined were XCC, its hydrazide and aminoethyl and fluoroethyl amide derivatives, XAC, N-hydroxyethyl-XAC, and the L-citrulline and D-p-aminophenylalanine conjugates of XAC. An N-hydroxysuccinimide ester of XCC (XCC-NHS, MRS 1204) bound to A2B receptors with a Ki of 9.75 nM and was the most selective (at least 20-fold) in this series. In a functional assay of recombinant human A2B receptors, four of these potent xanthines were shown to fully antagonize the effects of NECA-induced stimulation of cyclic AMP accumulation. Drug Dev. Res. 47:45–53, 1999. Published 1999 Wiley-Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.

As the temperature and humidity rose in the auditorium during the heat-wave of the Italian alps, it became evident that, in fact, more was revealed of the bodies of certain distinguished pharmacologists than of the true nature of Stephenson's efficacy.Nevertheless, real 3D structures of receptors in their empty, apo-, form as well as in their holo-forms, i.e. in complex with different ligands: agonists, partial agonists, antagonists, etc. are currently becoming available, at least in the nuclear receptor field (Fig. 2Fig. 2). Thus, we are rapidly approaching a long-awaited point in time where pharmacologists and medicinal chemists can relate theoretical pharmacological concepts directly to 3D structures. This could soon be the case even for 7TM receptors. On the day of the meeting the structural basis for activation of the G protein, Ras, by the nucleotide exchange factor, Sos, was published[10xBorlack-Sjodin, P.A., Margerit, S.M., Bar-Sagi, D., and Kuriyan, J. Nature. 1998; 394: 337–343Crossref | PubMed | Scopus (430)See all References[10]. The `loosening' of the GDP (corresponding to efficacy—see above) was clearly shown to be achieved through two antiparallel helices extending from the Sos molecule and interacting with a switch domain on the G protein. It could be relevant that the main G protein-interacting domain of 7TM receptors also is represented by two antiparallel helices, i.e. the intracellular extensions of TM5 and TM6 (Ref. [11xAltenbach, C. et al. Biochemistry. 1995; 35: 12470–12478Crossref | Scopus (263)See all References[11]; Fig. 1Fig. 1).

Natural hibernation consists of torpid phases with metabolic suppression alternating with euthermic periods. Induction of torpor holds substantial promise in various medical conditions, including trauma, major surgery, and transplantation. Torpor in mice can be induced pharmacologically by 5′-AMP. Previously, we showed that during natural torpor, the reduction in body temperature results in lymphopenia via a reduction in plasma S1P. Here, we show that during torpor induced by 5′-AMP, there is a similar reduction in the number of circulating lymphocytes that is a result of their retention in secondary lymphoid organs. This lymphopenia could be mimicked by engagement of A2BRs by a selective A2BR agonist (LUF6210) in the absence of changes in temperature and prevented by A2BR antagonists during 5′-AMP-induced torpor. In addition, forced cooling of mice led to peripheral blood lymphopenia, independent of A2BR signaling. The induction of torpor using 5′-AMP impacted the migration of lymphocytes within and between secondary lymphoid organs. During torpor, the homing into LNs was impaired, and two-photon intravital microscopy revealed that cell motility was decreased significantly and rapidly upon 5′-AMP administration. Furthermore, the S1P plasma concentration was reduced by 5′-AMP but not by LUF6210. S1P plasma levels restored upon arousal. Likely, the reduced migration in LNs combined with the reduced S1P plasma level substantially reduces lymphocyte egress after injection of 5′-AMP. In conclusion, 5′-AMP induces a state of pharmacological torpor in mice, during which, lymphopenia is governed primarily by body temperature-independent suppression of lymphocyte egress from LNs.

The radical scavenging properties of adenosine and related nucleosides were determined. Various protocols were used to assess the reactivity of the compounds toward hydroxyl radicals, superoxide ions, and hypochlorous acid. In the 2-deoxy-D-ribose assay (hydroxyl radicals) all nucleosides tested were equally effective in the presence of EDTA, with rate constants between 10 9 and 10 10 M -1 .sec -1 . However, in the absence of EDTA, resembling in vivo conditions, large differences were observed. An intact cis diol function in the sugar moiety of the compounds appeared important to retain full scavenging activity, probably due to firm complexation of iron ions. The reactivity toward hydroxyl radicals was confirmed in electron spin resonance (ESR) experiments with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin-trapping agent. Superoxide ions and hypochlorous acid did not react with the nucleosides. From the results it is hypothesized that the radical scavenging properties of adenosine play a role in its established cardioprotective actions in vivo.

The N 6-cyclopentyladenosine (CPA) analogue (4) was synthesized in 10 steps starting from glucose. The results of the radioligand binding assays are consistent with the thus far published findings that compounds containing a six-membered moiety at N 9 exhibit extremely weak affinity for adenosine receptors. Replacement of the ribofuranosyl moiety of CPA (2) by a 2-deoxy-D-altrohexitol moiety is sufficient to completely abolish its agonist activity.

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 β-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 β-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.

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.