Mass spectrometry-based ligand binding assays on adenosine A1 and A2A receptors

Conventional methods to measure ligand-receptor binding parameters typically require radiolabeled ligands as probes. Despite the robustness of radioligand binding assays, they carry inherent disadvantages in terms of safety precautions, expensive synthesis, special lab requirements, and waste disposal. Mass spectrometry (MS) is a method that can selectively detect ligands without the need of a label. The sensitivity of MS equipment increases progressively, and currently, it is possible to detect low ligand quantities that are usually found in ligand binding assays. We developed a label-free MS ligand binding (MS binding) assay on the adenosine A₁ and A_2A receptors (A₁AR and A_2AAR), which are well-characterized members of the class A G protein-coupled receptor (GPCR) family. Radioligand binding assays for both receptors are well established, and ample data is available to compare and evaluate the performance of an MS binding assay. 1,3-Dipropyl-8-cyclopentyl-xanthine (DPCPX) and 4-(2-((7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a]-[1,3,5]triazin-5-yl)amino)ethyl)phenol (ZM-241,385) are high-affinity ligands selective for the A₁AR and A_2AAR, respectively. To proof the feasibility of MS binding on the A₁AR and A_2AAR, we first developed an MS detection method for unlabeled DPCPX and ZM-241,385. To serve as internal standards, both compounds were also deuterium-labeled. Subsequently, we investigated whether the two unlabeled compounds could substitute for their radiolabeled counterparts as marker ligands in binding experiments, including saturation, displacement, dissociation, and competition association assays. Furthermore, we investigated the accuracy of these assays if the use of internal standards was excluded. The results demonstrate the feasibility of the MS binding assay, even in the absence of a deuterium-labeled internal standard, and provide great promise for the further development of label-free assays based on MS for other GPCRs.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-015-9477-0) contains supplementary material, which is available to authorized users.     

Involvement of adenosine A1 and A2A receptors in (-)-linalool-induced antinociception

In recent studies performed in our laboratory we have shown that acute administration of (-)-linalool, the natural occurring enantiomer in essential oils, possesses anti-inflammatory, antihyperalgesic and antinociceptive effects in different animal models. The antihyperalgesic and antinociceptive effects of (-)-linalool have been ascribed to its capacity in stimulating the opioidergic, cholinergic and dopaminergic systems, as well as to its interaction with K+ channels, or to its local anaesthetic activity and/or to the negative modulation of glutamate transmission. Activation of A1 or A2A receptors has been shown to induce antinociceptive effects, and the possible involvement of adenosine in (-)-linalool antinociceptive effect, has not been elucidated yet. Therefore, in the present study, we have investigated the effects of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), a selective adenosine A1 receptor antagonist and the effects of 3,7-dimethyl-1-propargilxanthine (DMPX), a selective adenosine A2A receptor antagonist on the antinociception of (-)-linalool in mice, measured in the hot-plate test. Both DPCPX (0.1 mg/kg; i.p.) and DMPX (0.1 mg/kg; i.p.) pre-treatment significantly depressed the antinociceptive effect of (-)-linalool at the highest doses tested. These findings demonstrated that the effect of (-)-linalool on pain responses is, at least partially, mediated by the activity of adenosine A1 and A2A receptors.     

Discovery of benzothiazole-based adenosine A2B receptor antagonists with improved A2A selectivity

The highly potent but modestly selective N-(2-amino-4-methoxy-benzothiazol-7-yl)-N-ethyl-acetamide derivative 2 was selected as the starting point for the design of novel selective A_2B antagonists, due to its excellent potency, and good drug-like properties. A series of compounds containing nonaromatic amides or ureas of five- or six-membered rings, and also bearing an m-trifluoromethyl-phenyl group (shown to impart superior potency) was prepared and evaluated for their selectivity against the A_2A and A₁ receptors. This work resulted in the identification of compound 30, with excellent potency and high selectivity against both A_2A and A₁ receptors.     

Potent and selective adenosine A(2A) receptor antagonists: [1,2,4]-triazolo[4,3-c]pyrimidin-3-ones

Antagonism of the adenosine A_2A receptor affords a possible treatment of Parkinson’s disease. In the course of investigating pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine A_2A antagonists, we prepared [1,2,4]-triazolo[4,3-c]pyrimidin-3-ones with potent and selective (vs A₁) A_2A antagonist activity. Structure-activity relationships are described for this series.     

Striatal adenosine A2A receptor expression is controlled by S-adenosyl-L-methionine-mediated methylation

Adenosine A_2A receptor (A_2AR) is a G protein-coupled receptor enriched in the striatum for which an increased expression has been demonstrated in certain neurological diseases. Interestingly, previous in vitro studies demonstrated that A_2AR expression levels are reduced after treatment with S-adenosyl-L-methionine (SAM), a methyl donor molecule involved in the methylation of important biological structures such as DNA, proteins, and lipids. However, the in vivo effects of SAM treatment on A_2AR expression are still obscure. Here, we demonstrated that 2 weeks of SAM treatment produced a significant reduction in the rat striatal A_2AR messenger RNA (mRNA) and protein content as well as A_2AR-mediated signaling. Furthermore, when the content of 5-methylcytosine levels in the 5′UTR region of ADORA2A was analyzed, this was significantly increased in the striatum of SAM-treated animals; thus, an unambiguous correlation between SAM-mediated methylation and striatal A_2AR expression could be established. Overall, we concluded that striatal A_2AR functionality can be controlled by SAM treatment, an issue that might be relevant for the management of these neurological conditions that course with increased A_2AR expression.     

Oligomerization of adenosine A2A and dopamine D2 receptors in living cells

We investigated whether oligomerization of adenosine A(2A) receptor (A(2A)R) and dopamine D(2) receptor (D(2)R) exists in living cells using modified bioluminescence resonance energy transfer (BRET(2)) technology. Fusion of these receptors to a donor, Renilla luciferase (Rluc), and to an acceptor, modified green fluorescent protein (GFP(2)), did not affect the ligand binding affinity, subcellular distribution, and coimmunoprecipitation of the receptors. BRET was detected not only between Myc-D(2)R-Rluc and A(2A)R-GFP(2) but also between HA-tagged A(2A)R-Rluc and A(2A)R-GFP(2). These results indicate A(2A)R, either homomeric or heteromeric with D(2)R, exists as an oligomer in living cells.     

Receptor crosstalk: haloperidol treatment enhances A2A adenosine receptor functioning in a transfected cell model

A(2A) adenosine receptors are considered an excellent target for drug development in several neurological and psychiatric disorders. It is noteworthy that the responses evoked by A(2A) adenosine receptors are regulated by D(2) dopamine receptor ligands. These two receptors are co-expressed at the level of the basal ganglia and interact to form functional heterodimers. In this context, possible changes in A(2A) adenosine receptor functional responses caused by the chronic blockade/activation of D(2) dopamine receptors should be considered to optimise the therapeutic effectiveness of dopaminergic agents and to reduce any possible side effects. In the present paper, we investigated the regulation of A(2A) adenosine receptors induced by antipsychotic drugs, commonly acting as D(2) dopamine receptor antagonists, in a cellular model co-expressing both A(2A) and D(2) receptors. Our data suggest that the treatment of cells with the classical antipsychotic haloperidol increased both the affinity and responsiveness of the A(2A) receptor and also affected the degree of A(2A)-D(2) receptor heterodimerisation. In contrast, an atypical antipsychotic, clozapine, had no effect on A(2A) adenosine receptor parameters, suggesting that the two classes of drugs have different effects on adenosine-dopamine receptor interaction. Modifications to A(2A) adenosine receptors may play a significant role in determining cerebral adenosine effects during the chronic administration of antipsychotics in psychiatric diseases and may account for the efficacy of A(2A) adenosine receptor ligands in pathologies associated with dopaminergic system dysfunction. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11302-010-9201-z) contains supplementary material, which is available to authorized users.     

Design, synthesis, and evaluation of fused heterocyclic analogs of SCH 58261 as adenosine A2A receptor antagonists

SCH 58261 is a reported adenosine A_2A receptor antagonist which is active in rat in vivo models of Parkinson’s Disease upon ip administration. However, it has poor selectivity versus the A₁ receptor and does not demonstrate oral activity. Quinoline analogs have improved upon the selectivity and pharmacokinetics of SCH 58261, but were difficult to handle due to poor aqueous solubility. We report the design and synthesis of fused heterocyclic analogs of SCH 58261 with aqueous solubility as well as improved A_2A receptor binding selectivity and pharmacokinetic properties. In particular, the tetrahydronaphthyridine 4s has excellent A_2A receptor in vitro binding affinity and selectivity, is active orally in a rat in vivo model of Parkinson’s Disease, and has aqueous solubility of 100 μM at physiological pH.     

Modulation of Ca2+-currents by sequential and simultaneous activation of adenosine A1 and A2A receptors in striatal projection neurons

D₁- and D₂-types of dopamine receptors are located separately in direct and indirect pathway striatal projection neurons (dSPNs and iSPNs). In comparison, adenosine A₁-type receptors are located in both neuron classes, and adenosine A_2A-type receptors show a preferential expression in iSPNs. Due to their importance for neuronal excitability, Ca²⁺-currents have been used as final effectors to see the function of signaling cascades associated with different G protein-coupled receptors. For example, among many other actions, D₁-type receptors increase, while D₂-type receptors decrease neuronal excitability by either enhancing or reducing, respectively, Ca_V1 Ca²⁺-currents. These actions occur separately in dSPNs and iSPNs. In the case of purinergic signaling, the actions of A₁- and A_2A-receptors have not been compared observing their actions on Ca²⁺-channels of SPNs as final effectors. Our hypotheses are that modulation of Ca²⁺-currents by A₁-receptors occurs in both dSPNs and iSPNs. In contrast, iSPNs would exhibit modulation by both A₁- and A_2A-receptors. We demonstrate that A₁-type receptors reduced Ca²⁺-currents in all SPNs tested. However, A_2A-type receptors enhanced Ca²⁺-currents only in half tested neurons. Intriguingly, to observe the actions of A_2A-type receptors, occupation of A₁-type receptors had to occur first. However, A₁-receptors decreased Ca_V2 Ca²⁺-currents, while A_2A-type receptors enhanced current through Ca_V1 channels. Because these channels have opposing actions on cell discharge, these differences explain in part why iSPNs may be more excitable than dSPNs. It is demonstrated that intrinsic voltage-gated currents expressed in SPNs are effectors of purinergic signaling that therefore play a role in excitability.     

New A2A adenosine receptor antagonists: a structure-based upside-down interaction in the receptor cavity

Adenosine receptor antagonists are generally based on heterocyclic core structures presenting substituents of various volumes and chemical-physical profiles. Adenine and purine-based adenosine receptor antagonists have been reported in literature. In this work we combined various substituents in the 2, 6, and 8-positions of 9-ethylpurine to depict a structure-affinity relationship analysis at the human adenosine receptors. Compounds were rationally designed trough molecular modeling analysis and then synthesized and evaluated at radioligand binding studies at human adenosine receptors. The new compounds showed affinity for the human adenosine receptors, with some derivatives endowed with low nanomolar K_i data, in particular at the A_2AAR subtype. The purine core proves to be a versatile core structure for the development of novel adenosine receptor antagonists with nanomolar affinity for these membrane proteins.     

Application of the functionalized congener approach to dendrimer-based signaling agents acting through A2A adenosine receptors

As a continued effort to develop multivalent ligands to enhance the pharmacological effects of monomeric drugs, DITC-APEC, a chemically reactive nucleoside A(2A) adenosine receptor (AR) agonist, was employed to derivatize the surface of third-generation (G3) polyamidoamine (PAMAM) dendrimers. The resulting conjugates carried multiple copies of the agonist attached through a thiourea linkage and differed in the number of attachments and in the presence of a fluorophore or additional surface modification. Computer modeling studies suggested that these DITC-APEC-loaded dendrimers extended the overall diameter of the previously reported PAMAM-CGS21680 dendrimer derivatives (Kim et al., Bioconjugate Chem 2008 19:406-411) by ca. 20 A, potentially increasing the conformational flexibility of the appended ligands to achieve optimal geometry for efficient binding at A(2A) ARs. Increased affinity and selectivity in binding in comparison to the CGS21680 conjugate were envisioned, due to the presence of an extended linker, i.e., a dithioureylenephenyl functionality. In vitro radioligand competition experiments showed effective binding of these PAMAM-DITC-APEC dendrimer conjugates at the human A(2A) and A(3) ARs with submicromolar K (i) values and selectivity in comparison to the human A(1) AR. Furthermore, these nucleoside-loaded dendrimers exhibited an A(2A) AR-mediated inhibitory effect on ADP-induced aggregation of human platelets. The present study demonstrates the potential of applying the functionalized congener concept to engineer dendrimer-based multivalent ligands for G protein-coupled receptors.     

Novel selective antagonist radioligands for the pharmacological study of A2B adenosine receptors

The adenosine A_2B receptor is the least well characterized of the four adenosine subtypes due to the lack of potent and selective agonists and antagonists. Despite the widespread distribution of A_2B receptor mRNA, little information is available with regard to their function. The characterization of A_2B receptors, through radioligand binding studies, has been performed, until now, by using low-affinity and non-selective antagonists like 1,3-dipropyl-8-cyclopentylxanthine ([³H]DPCPX),(4-(2-[7-amino-2-(2-furyl)-[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl)-phenol ([³H]ZM 241385) and 3-(3,4-aminobenzyl)-8-(4-oxyacetate)phenyl-1-propyl-xanthine ([¹²⁵I]ABOPX). Recently, high-affinity radioligands for A_2B receptors, [N-(4-cyanophenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)-phenoxy]acetamide ([³H]MRS 1754), N-(2-(2-Phenyl-6-[4-(2,2,3,3-tetratritrio-3-phenylpropyl)-piperazine-1-carbonyl]-7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-ethyl)-acetamide ([³H]OSIP339391) and N-benzo[1,3]dioxol-5-yl-2-[5-(1,3-dipropyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yloxy]-acetamide] ([³H]MRE 2029F20), have been introduced. This minireview offers an overview of these recently developed radioligands and the most important applications of drugs towards A_2B receptors.     

Biaryl and heteroaryl derivatives of SCH 58261 as potent and selective adenosine A2A receptor antagonists

SCH 58261 is a reported adenosine A_2A receptor antagonist, which is active in rat in vivo models of Parkinson’s Disease upon ip administration. However, it has poor selectivity versus the A₁ receptor and does not demonstrate oral activity. We report the design and synthesis of biaryl and heteroaryl analogs of SCH 58261 which improve the A_2A receptor binding selectivity as well as the pharmacokinetic properties of SCH 58261. In particular, the quinoline 25 has excellent A_2A receptor in vitro binding affinity and selectivity, sustained rat plasma levels upon oral dosing, and is active orally in a rat behavioral assay.     

Adenosine A1 and A2A receptors are involved on guanosine protective effects against oxidative burst and mitochondrial dysfunction induced by 6-OHDA in striatal slices

6-Hydroxydopamine (6-OHDA) is the most used toxin in experimental Parkinson’s disease (PD) models. 6-OHDA shows high affinity for the dopamine transporter and once inside the neuron, it accumulates and undergoes non-enzymatic auto-oxidation, promoting reactive oxygen species (ROS) formation and selective damage of catecholaminergic neurons. In this way, our group has established a 6-OHDA in vitro protocol with rat striatal slices as a rapid and effective model for screening of new drugs with protective effects against PD. We have shown that co-incubation with guanosine (GUO, 100 μM) prevented the 6-OHDA-induced damage in striatal slices. As the exact GUO mechanism of action remains unknown, the aim of this study was to investigate if adenosine A₁ (A₁R) and/or A_2A receptors (A_2AR) are involved on GUO protective effects on striatal slices. Pre-incubation with DPCPX, an A₁R antagonist prevented guanosine effects on 6-OHDA-induced ROS formation and mitochondrial membrane potential depolarization, while CCPA, an A₁R agonist, did not alter GUO effects. Regarding A_2AR, the antagonist SCH58261 had similar protective effect as GUO in ROS formation and mitochondrial membrane potential. Additionally, SCH58261 did not affect GUO protective effects. The A_2AR agonist CGS21680, although, completely blocked GUO effects. Finally, the A₁R antagonist DPCPX, and the A_2AR agonist CGS21680 also abolished the preventive guanosine effect on 6-OHDA-induced ATP levels decrease. These results reinforce previous evidence for a putative interaction of GUO with A₁R-A_2AR heteromer as its molecular target and clearly indicate a dependence on adenosine receptors modulation to GUO protective effect.

     

Lack of adenosine A3 receptors causes defects in mouse peripheral blood parameters

The role of the adenosine A₃ receptor in hematopoiesis was studied using adenosine A₃ receptor knockout (A₃AR KO) mice. Hematological parameters of peripheral blood and femoral bone marrow of irradiated and untreated A₃AR KO mice and their wild-type (WT) counterparts were investigated. Irradiation of the mice served as a defined hematopoiesis-damaging means enabling us to evaluate contingent differences in the pattern of experimentally induced hematopoietic suppression between the A₃AR KO mice and WT mice. Defects were observed in the counts and/or functional parameters of blood cells in the A₃AR KO mice. These defects include statistically significantly lower values of blood neutrophil and monocyte counts, as well as those of mean erythrocyte volume, mean erythrocyte hemoglobin, blood platelet counts, mean platelet volume, and plateletcrit, and can be considered to bear evidence of the lack of a positive role played by the adenosine A₃ receptor in the hematopoietic system. Statistically significantly increased values of the bone marrow parameters studied in A₃AR KO mice (femoral bone marrow cellularity, granulocyte/macrophage progenitor cells, and erythrocyte progenitor cells) can probably be explained by compensatory mechanisms attempting to offset the disorders in the function of blood elements in these mice. The pattern of the radiation-induced hematopoietic suppression was very similar in A₃AR KO mice and their WT counterparts.     

Effect of 2-(6-cyano-1-hexyn-1-yl)adenosine on ocular blood flow in rabbits

Previously, we reported that a relatively selective adenosine A(2A) receptor agonist 2-(6-cyano-1-hexyn-1-yl)adenosine (2-CN-Ado) elicited ocular hypotension in rabbits (Journal of Pharmacological Sciences 2005;97:501-509). In the present study, we investigated the effect of 2-CN-Ado on ocular blood flow in rabbit eyes. An intravitreal injection of 2-CN-Ado increased ocular blood flow, measured by a non-contact laser flowmeter. 2-CN-Ado-induced increase in ocular blood flow was accompanied with the retinal vasodilation. The increase in ocular blood flow was inhibited by an adenosine A(2A) receptor antagonist 1,3,7-trimethyl-8-(3-chlorostyryl)xanthine, but not by an adenosine A(2B) receptor antagonist alloxazine or an adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. The repetitive applications of topical 2-CN-Ado twice a day for 7 days produced a persistent increase in ocular blood flow with ocular hypotension. These results suggest that 2-CN-Ado increases the ocular blood flow mainly via adenosine A(2A) receptor, and that the topical application of 2-CN-Ado for several days not only increases the ocular blood flow but also prolong ocular hypotension, indicating that 2-CN-Ado may be a useful lead compound for the treatment of ischemic retinal diseases such as glaucoma.     

Adenosine A(2A) receptor activation limits chronic granulomatous disease-induced hyperinflammation

Chronic granulomatous disease (CGD) is caused by defects in the NADPH oxidase complex and is characterized by an increased susceptibility to infection. Other significant complications of CGD include autoimmunity and non-infectious hyperinflammatory disorders. We show that a gp91^phox deficiency leads to the development of phenotypically altered T lymphocytes in mice and that this abnormal, hyperactive phenotype can be modulated by activation of the adenosine A_2A receptor. T cells isolated from CGD mice produce significantly higher levels of the pro-inflammatory cytokines IFN-γ, IL-2, TNF-α, IL-4 and IL-13 than do WT cells after TCR-mediated activation; treatment with the selective adenosine A_2A receptor agonist, CGS21680, potently inhibits this response. Additionally, the over exuberant inflammatory response elicited by thioglycollate challenge in gp91^phox deficient mice is attenuated by CGS21680. These data suggest that treatment with A_2AR agonists may be an effective therapy by which to regulate the immune system hyperactivity that results from a gp91^phox deficiency.     

G protein-coupled adenosine (P1) and P2Y receptors: ligand design and receptor interactions

The medicinal chemistry and pharmacology of the four subtypes of adenosine receptors (ARs) and the eight subtypes of P2Y receptors (P2YRs, activated by a range of purine and pyrimidine mono- and dinucleotides) has recently advanced significantly leading to selective ligands. X-ray crystallographic structures of both agonist- and antagonist-bound forms of the A(2A)AR have provided unprecedented three-dimensional detail concerning molecular recognition in the binding site and the conformational changes in receptor activation. It is apparent that this ubiquitous cell signaling system has implications for understanding and treating many diseases. ATP and other nucleotides are readily released from intracellular sources under conditions of injury and organ stress, such as hypoxia, ischemia, or mechanical stress, and through channels and vesicular release. Adenosine may be generated extracellularly or by cellular release. Therefore, depending on pathophysiological factors, in a given tissue, there is often a tonic activation of one or more of the ARs or P2YRs that can be modulated by exogenous agents for a beneficial effect. Thus, this field has provided fertile ground for pharmaceutical development, leading to clinical trials of selective receptor ligands as imaging agents or for conditions including cardiac arrhythmias, ischemia/reperfusion injury, diabetes, pain, thrombosis, Parkinson's disease, rheumatoid arthritis, psoriasis, dry eye disease, pulmonary diseases such as cystic fibrosis, glaucoma, cancer, chronic hepatitis C, and other diseases.     

A selective adenosine A2A receptor agonist, ATL-146e, prevents concanavalin A-induced acute liver injury in mice

BACKGROUND AND AIMS: Concanavalin A (Con A) activates T lymphocytes and induces CD4+ T cell-mediated hepatic injury in mice. Pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), and interleukin-6 (IL-6), are critical mediators in this experimental model. Activation of adenosine A2A receptors reduces the production of various pro-inflammatory cytokines and suppresses T cell activation. A selective adenosine A2A receptor agonist (ATL-146e) has been shown to be a potent inhibitor of inflammation by increasing intracellular cyclic AMP (cAMP) in leukocytes. The aim of the present study was to determine whether ATL-146e could ameliorate Con A-induced hepatic injury, reduction of pro-inflammatory cytokine production. METHODS: Balb/c mice were injected with 25mg/kg Con A with or without a single injection of ATL-146e (0.5-50 microg/kg), 5 min prior to Con A administration. Liver enzymes, histology, and serum levels of tumor necrosis factor-alpha, interferon-gamma, and interleukin-6 were examined. We also assessed the effects of ATL-146e on pro-inflammatory cytokine production with CD4+ T cell. RESULTS: Pretreatment with ATL-146e significantly reduced serum levels of liver enzymes (P<0.001). The serum pro-inflammatory cytokines were all increased after Con A administration and reduced to near normal levels by ATL-146e. ATL-146e also inhibited CD4+ T cell pro-inflammatory cytokine production. CONCLUSION: A selective adenosine A2A receptor agonist, ATL-146e, can prevent concanavalin A-induced hepatic injury that is presumably mediated by its anti-inflammatory properties.