We present the systematic prospective evaluation of a protein-based and a ligand-based virtual screening platform against a set of three G-protein-coupled receptors (GPCRs): the β-2 adrenoreceptor (ADRB2), the adenosine A2A receptor (AA2AR), and the sphingosine 1-phosphate receptor (S1PR1). Novel bioactive compounds were identified using a consensus scoring procedure combining ligand-based (frequent substructure ranking) and structure-based (Snooker) tools, and all 900 selected compounds were screened against all three receptors. A striking number of ligands showed affinity/activity for GPCRs other than the intended target, which could be partly attributed to the fuzziness and overlap of protein-based pharmacophore models. Surprisingly, the phosphodiesterase 5 (PDE5) inhibitor sildenafil was found to possess submicromolar affinity for AA2AR. Overall, this is one of the first published prospective chemogenomics studies that demonstrate the identification of novel cross-pharmacology between unrelated protein targets. The lessons learned from this study can be used to guide future virtual ligand design efforts.

Thirty-four project managers of life-science research projects were interviewed to investigate the characteristics of their projects, the challenges they faced and their training requirements. A set of ten discriminating parameters were identified based on four project categories: contract research, development, discovery and call-based projects--projects set up to address research questions defined in a call for proposals. The major challenges these project managers are faced with relate to project members, leadership without authority and a lack of commitment from the respective organization. Two-thirds of the project managers indicated that they would be interested in receiving additional training, mostly on people-oriented, soft skills. The training programs that are currently on offer, however, do not meet their needs.

BACKGROUND:G protein-coupled receptors (GPCRs) represent a family of well-characterized drug targets with significant therapeutic value. Phylogenetic classifications may help to understand the characteristics of individual GPCRs and their subtypes. Previous phylogenetic classifications were all based on the sequences of receptors, adding only minor information about the ligand binding properties of the receptors. In this work, we compare a sequence-based classification of receptors to a ligand-based classification of the same group of receptors, and evaluate the potential to use sequence relatedness as a predictor for ligand interactions thus aiding the quest for ligands of orphan receptors.RESULTS:We present a classification of GPCRs that is purely based on their ligands, complementing sequence-based phylogenetic classifications of these receptors. Targets were hierarchically classified into phylogenetic trees, for both sequence space and ligand (substructure) space. The overall organization of the sequence-based tree and substructure-based tree was similar; in particular, the adenosine receptors cluster together as well as most peptide receptor subtypes (e.g. opioid, somatostatin) and adrenoceptor subtypes. In ligand space, the prostanoid and cannabinoid receptors are more distant from the other targets, whereas the tachykinin receptors, the oxytocin receptor, and serotonin receptors are closer to the other targets, which is indicative for ligand promiscuity. In 93% of the receptors studied, de-orphanization of a simulated orphan receptor using the ligands of related receptors performed better than random (AUC > 0.5) and for 35% of receptors de-orphanization performance was good (AUC > 0.7).CONCLUSIONS:We constructed a phylogenetic classification of GPCRs that is solely based on the ligands of these receptors. The similarities and differences with traditional sequence-based classifications were investigated: our ligand-based classification uncovers relationships among GPCRs that are not apparent from the sequence-based classification. This will shed light on potential cross-reactivity of GPCR ligands and will aid the design of new ligands with the desired activity profiles. In addition, we linked the ligand-based classification with a ligand-focused sequence-based classification described in literature and proved the potential of this method for de-orphanization of GPCRs.

The highly variable extracellular loops in G protein-coupled receptors (GPCRs) have been implicated in receptor activation, the mechanism of which is poorly understood. In a random mutagenesis screen on the human adenosine A(2B) receptor (A(2B)R) using the MMY24 Saccharomyces cerevisiae strain as a read-out system, we found that two residues in the first extracellular loop, a phenylalanine and an aspartic acid at positions 71 and 74, respectively, are involved in receptor activation. We subsequently performed further site-directed and site-saturation mutagenesis. These experiments revealed that the introduction of mutations at either of the identified positions results in a wide variety of receptor activation profiles, with changes in agonist potency, constitutive activity, and intrinsic activity. Radioligand binding studies showed that the changes in activation were not due to changes in receptor expression. We interpret these data in the light of the recently revealed structure of the adenosine A(2A)R, the closest homologue of the A(2B)R. The two residues are suggested to be vital in maintaining the tertiary structure of a β sheet in the extracellular domain of the A(2B)R. We hypothesize that deterioration of structure in the extracellular domains of GPCRs compromises overall receptor structure with profound consequences for receptor activation and constitutive activity.

The adenosine A1 receptor is a promising therapeutic target for neurological disorders such as cognition deficits and is involved in cardiovascular preconditioning. Classically adenosine receptor agonists were all derivatives of adenosine, and thought to require a D-ribose moiety. More recently, however, the discovery of non-adenosine agonists for the human adenosine A1 receptor (hA1R) has challenged this dogma (Beukers et al., 2004). In this study we characterize the tritiated form of one of these compounds, [3H]LUF5834, as the first non-ribose partial agonist radioligand with nanomolar affinity for the hA1R. Due to its partial agonist efficacy, [3H]LUF5834 labeled both G protein-coupled and uncoupled receptors with a similar high affinity. Using [3H]LUF5834 we performed competition binding experiments to characterize a range of A1R ligands varying in efficacy from the full agonist CPA to the inverse agonist DPCPX. Surprisingly, in the control condition both agonists and inverse agonists displayed biphasic isotherms. With the addition of 1 mM GTP the high affinity isotherm of agonists or the low affinity isotherm of inverse agonists was lost revealing the mechanism of action of such inverse agonists at the A1R. Consequently, [3H]LUF5834 represents a novel high affinity radioligand for the A1R and may prove a useful tool to provide estimates of inverse agonist efficacy at this 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 A3 (hA3R) receptor. The endocannabinoid 2-AG was able to inhibit agonist ([125I]N6-(4-amino-3-iodobenzyl) adenosine-5′-(N-methyluronamide) – [125I] AB MECA) binding at the hA3R. 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 hA3R. Moreover, by measuring intracellular cAMP levels, we demonstrate that 2-AG decreases both the potency of an agonist at the hA3R and the basal signalling of this receptor. Since the hA3R 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.

The adenosine A1 receptor is a typical example of a G protein coupled receptor (GPCR). Despite a wealth of biochemical data the general mechanism of GPCR signaling has not been fully clarified. Whether GPCR signaling takes place in membrane microdomains, and whether the respective G proteins are precoupled, is still heavily debated. Both mechanisms would explain the fast receptor G protein interaction that is observed in experiments. Using single-molecule microscopy in live CHO cells and our recently developed analysis technique (PICS, Semrau, Schmidt., Biophys. J., 2007) we unraveled the first steps of the A1 receptor signaling. We found that at least 7% of the receptors are precoupled to the G protein already before stimulation with an agonist. Furthermore, 9% of the receptors translocate to membrane microdomains upon agonist stimulation. These domains, which are about 150 nm in size, are related to the cytoskeleton. We believe that this knowledge about the molecular mechanisms of GPCR signaling will open up new ways to manipulate GPCRs and develop new, potent drugs.

A 2A adenosine receptor antagonists usually have bi- or tricyclic N aromatic systems with varying substitution patterns to achieve desired receptor affinity and selectivity. Using a pharmacophore model designed by overlap of nonxanthine type of previously known A 2A antagonists, we synthesized a new class of compounds having a 2-amino nicotinonitrile core moiety. From our data, we conclude that the presence of at least one furan group rather than phenyl is beneficial for high affinity on the A 2A adenosine receptor. Compounds 39 (LUF6050) and 44 (LUF6080) of the series had K i values of 1.4 and 1.0 nM, respectively, with reasonable selectivity toward the other adenosine receptor subtypes, A 1, A 2B, and A 3. The high affinity of 44 was corroborated in a cAMP second messenger assay, yielding subnanomolar potency for this compound.

Until now, more than 800 distinct G protein-coupled receptors (GPCRs) have been identified in the human genome. The four subtypes of the adenosine receptor (A(1), A(2A), A(2B) and A(3) receptor) belong to this large family of GPCRs that represent the most widely targeted pharmacological protein class. Since adenosine receptors are widespread throughout the body and involved in a variety of physiological processes and diseases, there is great interest in understanding how the different subtypes are regulated, as a basis for designing therapeutic drugs that either avoid or make use of this regulation. The major GPCR regulatory pathway involves phosphorylation of activated receptors by G protein-coupled receptor kinases (GRKs), a process that is followed by binding of arrestin proteins. This prevents receptors from activating downstream heterotrimeric G protein pathways, but at the same time allows activation of arrestin-dependent signalling pathways. Upon agonist treatment, adenosine receptor subtypes are differently regulated. For instance, the A(1)Rs are not (readily) phosphorylated and internalize slowly, showing a typical half-life of several hours, whereas the A(2A)R and A(2B)R undergo much faster downregulation, usually shorter than 1 h. The A(3)R is subject to even faster downregulation, often a matter of minutes. The fast desensitization of the A(3)R after agonist exposure may be therapeutically equivalent to antagonist occupancy of the receptor. This review describes the process of desensitization and internalization of the different adenosine subtypes in cell systems, tissues and in vivo studies. In addition, molecular mechanisms involved in adenosine receptor desensitization are discussed.

New adenosine receptor ligands were designed as hybrid structures between previously synthesized substituted dicyanopyridines and aminopyrimidines, yielding two series of cyano-substituted diphenylaminopyrimidines. We were interested in assessing the effect of this substitution pattern on both affinity and intrinsic activity, as the dicyanopyridines comprised both agonists and inverse agonists, whereas the original aminopyrimidines were exclusively inverse agonists. It was found that the new compounds were generally selective for adenosine A(1) receptors, although affinity for the adenosine A(2A) receptor was also noticed for some of the compounds. In a cAMP second messenger assay the compounds behaved as inverse agonists rather than agonists. Among the more A(1) receptor-selective compounds were 5 (LUF6048), 27 (LUF6040) and 53 (LUF6056) with K(i) values of 8.1, 1.2 and 5.7nM, respectively.

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.

Many selective and high affinity agonists and antagonists have been developed for the adenosine A(1), A(2A), and A(3) receptors. Very recently such compounds have been identified for the adenosine A(2B) receptors. This review presents an overview of the structure-affinity relationships of antagonists and agonists for this receptor subtype as published in the scientific and patent literature. To date the most selective >370-fold, high affinity adenosine A(2B) receptor antagonist is the xanthine analog, compound 16 (8-(1-(3-phenyl-1,2,4-oxadiazol-5-yl)methyl)-1H-pyrazol-4-yl)-1,3-dipropyl-1H-purine-2,6(3H,7H)-dione). The pyrrolopyrimidine analog OSIP339391 (73) is slightly less selective, 70-fold, but has a higher affinity 0.41 nM compared to 1 nM for compound 16. Other promising classes of compounds with selectivities ranging from 10- to 160-fold and affinities ranging from 3 to 112 nM include triazolo, aminothiazole, quinazoline, and pyrimidin-2-amine analogs. Progress has also been achieved concerning the development of selective high affinity agonists for the adenosine A(2B) receptor. For years the most potent, albeit non-selective adenosine A(2B) receptor agonist was (S)PHPNECA (88). Last year, a new class of non-ribose ligands was reported. Several compounds displayed selectivity with respect to adenosine A(2A) and A(3) receptors. In addition, full and partial agonists for the adenosine A(2B) receptor were identified with EC(50) values of 10 nM (LUF5835, 103) and 9 nM (LUF5845, 105), respectively.

Residues in the transmembrane region of G protein-coupled receptors (GPCRs) are important for ligand binding and activation, but the function of individual positions is poorly understood. Using a sequence alignment of class A GPCRs (grouped in subfamilies), we propose a so-called "two-entropies analysis" to determine the potential role of individual positions in the transmembrane region of class A GPCRs. In our approach, such positions appear scattered, while largely clustered according to their biological function. Our method appears superior when compared to other bioinformatics approaches, such as the evolutionary trace method, entropy-variability plot, and correlated mutation analysis, both qualitatively and quantitatively.

Biological data such as DNA and protein sequences can be analyzed using a variety of methods. This paper combines phylogenetic trees, experience-based classification and self-organizing maps for cluster analysis of G protein-coupled receptors (GPCRs), a class of pharmacologically relevant transmembrane proteins with specific characteristics. This combination allows to gain additional insights.

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Ecto-nucleotidases are plasma membrane-bound enzymes that sequentially dephosphorylate extracellular nucleotides such as ATP. This breakdown of ATP and other nucleotides obscures the characterization and classification of P2 (nucleotide) receptors. We therefore studied suramin and several of its analogs, divalent cations and ATP gamma S for their ability to inhibit ecto-ATPase in human blood cells. Suramin itself and Ni2+ were the more potent, non-competitive inhibitors with micromolar affinity. ATP gamma S also displayed micromolar affinity and inhibited ecto-ATPase competitively. The data obtained with the divalent cations demonstrate that coordination of the phosphate chain but not the N7 of the adenine ring is required for the breakdown of ATP by ecto-ATPase. Divalent cations that coordinate both the phosphate chain and N7 inhibit ecto-ATPase in a non-competitive manner.

Amiloride, a potassium sparing diuretic, inhibits the specific binding of [3H]8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and [3H]N6-R-1-phenyl-2-propyladenosine (PIA) to adenosine A1 receptors in calf brain. This interaction is different from the agonist-receptor or the antagonist-receptor interaction as Na+ and H+ counteract the inhibitory effect of amiloride whereas these ions hardly affect the binding of the classic A1 receptor ligands. In the present study, the effects of protein modifiers on the equilibrium inhibition constant of amiloride are compared with effects of these reagents on the affinities of DPCPX and PIA. It is demonstrated that the affinities of amiloride and [3H]DPCPX are changed after treatment with a carboxyl-modifying reagent but unaffected by modification of histidyl, arginyl and cystein residues. The maximal binding capacity of [3H]DPCPX is enhanced by sulfhydryl modification, whereas the number of [3H]DPCPX binding sites is reduced by treatment with a histidine-modifying reagent. The histidyl residues of the [3H]DPCPX binding site can be partially protected against modification by 300 microM amiloride, present during treatment of the membranes. An equivalent concentration of 8-phenyltheophylline results in complete protection. The apparent affinity of PIA is altered by modification of histidyl, carboxyl, arginyl and cystein residues. In the latter two cases, uncoupling of the G protein seems to be the major reason for the decrease in affinity of PIA. The results suggest that amiloride is an A1 antagonist with binding characteristics that differ from the classic A1 antagonists such as DPCPX.