Prediction of human CNS pharmacokinetics using a physiologically-based pharmacokinetic modeling approach.

Knowledge of drug concentration-time profiles at the central nervous system (CNS) target-site is critically important for rational development of CNS targeted drugs. Our aim was to translate a recently published comprehensive CNS physiologically-based pharmacokinetic (PBPK) model from rat to human, and to predict drug concentration-time profiles in multiple CNS compartments on available human data of four drugs (acetaminophen, oxycodone, morphine and phenytoin). Values of the system-specific parameters in the rat CNS PBPK model were replaced by corresponding human values. The contribution of active transporters for the four selected drugs was scaled based on differences in expression of the pertinent transporters in both species. Model predictions were evaluated with available pharmacokinetic (PK) data in human brain extracellular fluid and/or cerebrospinal fluid, obtained under physiologically healthy CNS conditions (acetaminophen, oxycodone, and morphine) and under pathophysiological CNS conditions where CNS physiology could be affected (acetaminophen, morphine and phenytoin). The human CNS PBPK model could successfully predict their concentration-time profiles in multiple human CNS compartments in physiological CNS conditions within a 1.6-fold error. Furthermore, the model allowed investigation of the potential underlying mechanisms that can explain differences in CNS PK associated with pathophysiological changes. This analysis supports the relevance of the developed model to allow more effective selection of CNS drug candidates since it enables the prediction of CNS target-site concentrations in humans, which are essential for drug development and patient treatment.

Predicting Drug Concentration-Time Profiles in Multiple CNS Compartments Using a Comprehensive Physiologically-Based Pharmacokinetic Model.

Drug development targeting the central nervous system (CNS) is challenging due to poor predictability of drug concentrations in various CNS compartments. We developed a generic physiologically based pharmacokinetic (PBPK) model for prediction of drug concentrations in physiologically relevant CNS compartments. System-specific and drug-specific model parameters were derived from literature and in silico predictions. The model was validated using detailed concentration-time profiles from 10 drugs in rat plasma, brain extracellular fluid, 2 cerebrospinal fluid sites, and total brain tissue. These drugs, all small molecules, were selected to cover a wide range of physicochemical properties. The concentration-time profiles for these drugs were adequately predicted across the CNS compartments (symmetric mean absolute percentage error for the model prediction was <91%). In conclusion, the developed PBPK model can be used to predict temporal concentration profiles of drugs in multiple relevant CNS compartments, which we consider valuable information for efficient CNS drug development.

A prospective cross-screening study on G-protein-coupled receptors: lessons learned in virtual compound library design.

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 ß-2 adrenoreceptor (ADRB2), the adenosine A(2A) 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.

GPCR structure and activation: an essential role for the first extracellular loop in activating the adenosine A2B receptor.

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 ß 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.

Project management of life-science research projects: project characteristics, challenges and training needs.

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.

Characterization of [3H]LUF5834: A novel non-ribose high-affinity agonist radioligand for the adenosine A1 receptor.

The adenosine A(1) 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 A(1) receptor (hA(1)R) has challenged this dogma (Beukers et al., 2004). In this study we characterize the tritiated form of one of these compounds, [(3)H]LUF5834, as the first non-ribose partial agonist radioligand with nanomolar affinity for the hA(1)R. Due to its partial agonist efficacy, [(3)H]LUF5834 labeled both G protein-coupled and uncoupled receptors with a similar high affinity. Using [(3)H]LUF5834 we performed competition binding experiments to characterize a range of A(1)R 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 1mM 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 A(1)R. Consequently, [(3)H]LUF5834 represents a novel high affinity radioligand for the A(1)R and may prove a useful tool to provide estimates of inverse agonist efficacy at this receptor.

A novel chemogenomics analysis of G protein-coupled receptors (GPCRs) and their ligands: a potential strategy for receptor de-orphanization.

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

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

2-Amino-6-furan-2-yl-4-substituted nicotinonitriles as A2A adenosine receptor antagonists.

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.

Internalization and desensitization of adenosine receptors.

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.

A new generation of adenosine receptor antagonists: from di- to trisubstituted aminopyrimidines.

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.

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

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

Structure-affinity relationships of adenosine A2B receptor ligands.

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.

A two-entropies analysis to identify functional positions in the transmembrane region of class A G protein-coupled receptors.

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.

Allosteric modulators affect the internalization of human adenosine A1 receptors.

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

Techniques: how to boost GPCR mutagenesis studies using yeast.

G-protein-coupled receptors (GPCRs) are the major targets of today's medicines. To elucidate the mechanism of activation of GPCRs and the interaction of these receptors with their G proteins, mutagenesis studies have proven to be a powerful tool and have provided insight into the structure and function of GPCRs. Random mutagenesis is useful in this respect particularly when combined with a robust screening assay that is based on the functional properties of the mutants. In this article, the use of random mutagenesis combined with a functional screening assay in yeast is described and compared with alternative approaches such as site-directed mutagenesis per se, alanine/cysteine scanning and another screening assay, receptor selection and amplification technology (R-SAT). Screening in yeast of randomly mutated GPCRs has proven successful in the identification of ligands for orphan receptors and in high-throughput approaches. Moreover, it has provided substantial insight into G-protein coupling and receptor activation.

A series of ligands displaying a remarkable agonistic-antagonistic profile at the adenosine A1 receptor.

Adenosine receptor agonists are usually variations of the natural ligand, adenosine. The ribose moiety in the ligand has previously been shown to be of great importance for the agonistic effects of the compound. In this paper, we present a series of nonadenosine ligands selective for the adenosine A(1) receptor with an extraordinary pharmacological profile. 2-Amino-4-benzo[1,3]dioxol-5-yl-6-(2-hydroxyethylsulfanyl)pyridine-3,5-dicarbonitrile (70, LUF 5853) shows full agonistic behavior comparable with the reference compound CPA, while also displaying comparable receptor binding affinity (K(i) = 11 nM). In contrast, compound 58 (2-amino-4-(3-trifluoromethylphenyl)-6-(2-hydroxyethylsulfanyl)pyridine-3,5-dicarbonitrile, LUF 5948) has a binding affinity of 14 nM and acts as an inverse agonist. Also present within this same series are compounds that show neutral antagonism of the adenosine A(1) receptor, for example compound 65 (2-amino-4-(4-difluoromethoxyphenyl)-6-(2-hydroxyethylsulfanyl)pyridine-3,5-dicarbonitrile, LUF 5826).

2,4,6-trisubstituted pyrimidines as a new class of selective adenosine A1 receptor antagonists.

Adenosine receptor antagonists usually possess a bi- or tricyclic heteroaromatic structure at their core with varying substitution patterns to achieve selectivity and/or greater affinity. Taking into account molecular modeling results from a series of potent adenosine A1 receptor antagonists, a pharmacophore was derived from which we show that a monocyclic core can be equally effective. To achieve a compound that may act at the CNS we propose imposing a restriction related to its polar surface area (PSA). In consequence, we have synthesized two novel series of pyrimidines, possessing good potency at the adenosine A1 receptor and desirable PSA values. In particular, compound 30 (LUF 5735) displays excellent A1 affinity (Ki = 4 nM) and selectivity (< or =50% displacement of 1 muM concentrations of the radioligand at the other three adenosine receptors) and has a PSA value of 53 A2.

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

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

Random mutagenesis of the human adenosine A2B receptor followed by growth selection in yeast. Identification of constitutively active and gain of function mutations.

To gain insight in spontaneous as well as agonist-induced activation of the human adenosine A2B receptor, we applied a random mutagenesis approach in yeast to create a large number of receptor mutants and selected mutants of interest with a robust screening assay based on growth. The amino acid sequence of 14 mutated receptors was determined. All these mutated receptors displayed constitutive activity. In particular, single-point mutations at T42A, V54L, and F84S and a triple-point mutation at N36S, T42A, and T66A resulted in high constitutive activity. In addition, a C-terminally truncated (after Lys269) mutant, Q214L I230N V240M V250M N254Y T257S K269stop, was highly constitutively active. The T42A, V54L, and F84S mutants showed a considerable decrease, 4.9- to 6.9-fold, in the EC50 value of 5'-N-ethylcarboxamidoadenosine (NECA), an adenosine analog. Combined mutation of I242T, K269R, V284A, and H302Q, as well as F84L together with S95G, resulted in an even greater potency of NECA of 10- and 18-fold, respectively. In fact, all constitutively active mutants had an increased potency for NECA. This suggests that the wild-type (wt) human A2B receptor itself is rather silent, which may explain the low affinity of agonists for this receptor. To verify the ability of the mutant receptors to activate mammalian second messenger systems, cAMP experiments were performed in CHO cells stably expressing the wt and T42A receptors. These experiments confirmed the increased sensitivity of T42A for NECA, because the EC50 values of T42A and the wt receptor were 0.15 +/- 0.04 and 1.3 +/- 0.4 microM, respectively.