Adenosine A1 Receptor Ligands Research Articles

Screening of Non‐Purine Ligands Binding to Adenosine Receptors A1, A2A, A2B, and A3

Adenosine receptors (ARs) are G‐protein coupled receptors (GPCRs) which have been shown to have therapeutic potential. ARs are made up of four subtypes, A1, A2A, A2B, and A3. A3AR ligands have previously been indicated to lower inflammation, prevent or treat cancer, and produce antihyperalgesic effects in many preclinical pain models, specifically for neuropathy. We have previously published in animal models that ferulic acid dimer, a non‐purine compound with the 3‐methoxy phenyl moiety, has non‐opioid antinociceptive properties through primarily binding to A3ARs. Current literature suggests that the antinociceptive properties of a natural compound, incarvillateine, which contains the 3‐methoxy phenyl moiety is mediated through ARs, however there is a lack of sufficient evidence of in vitro binding of such compounds at ARs. We thus hypothesized that compounds with the 3‐methoxy phenyl moiety show binding to ARs, with an affinity in the submicromolar range. For this study, we performed fluorescent competitive binding assays using cells transfected with ARs: Chinese Hamster Ovary (CHO) cells for A1 or A3 receptors and Human Embryonic Kidney (HEK) cells for A2A or A2B receptors. The compounds tested included trans cinnamic acid analogs and curcumin analogs. The compounds were synthesized using cavitand‐mediated photoisomerization and cavitand‐mediated photodimerization. Our fluorescent binding protocol employed the separation method, in which the unbound ligand was removed after equilibrium was reached, via PBS wash. The binding assays performed suggested that 3‐methoxy cinnamic acid dimer was the most effective in binding to A3ARs, while the monomer did not bind. There was very little to no binding observed at the other receptors. Other analogs of trans cinnamic acid and curcumin did not show binding at any of the ARs so far. In conclusion, 3‐methoxy cinnamic acid dimer binds to G‐inhibitory coupled adenosine receptor, A3. Future studies will investigate structure‐activity‐relationship of 3‐methoxy phenyl dimers to demonstrate why dimerization leads to superior binding compared to similar monomeric compounds.

Therapeutic Potential of Highly Selective A3 Adenosine Receptor Ligands in the Central and Peripheral Nervous System.

Ligands of the Gi protein-coupled adenosine A3 receptor (A3R) are receiving increasing interest as attractive therapeutic tools for the treatment of a number of pathological conditions of the central and peripheral nervous systems (CNS and PNS, respectively). Their safe pharmacological profiles emerging from clinical trials on different pathologies (e.g., rheumatoid arthritis, psoriasis and fatty liver diseases) confer a realistic translational potential to these compounds, thus encouraging the investigation of highly selective agonists and antagonists of A3R. The present review summarizes information on the effect of latest-generation A3R ligands, not yet available in commerce, obtained by using different in vitro and in vivo models of various PNS- or CNS-related disorders. This review places particular focus on brain ischemia insults and colitis, where the prototypical A3R agonist, Cl-IB-MECA, and antagonist, MRS1523, have been used in research studies as reference compounds to explore the effects of latest-generation ligands on this receptor. The advantages and weaknesses of these compounds in terms of therapeutic potential are discussed.

Adenosine A1 receptor ligands bind to \u03b1-synuclein: implications for \u03b1-synuclein misfolding and \u03b1-synucleinopathy in Parkinson\u2019s disease

BackgroundAccumulating α-synuclein (α-syn) aggregates in neurons and glial cells are the staples of many synucleinopathy disorders, such as Parkinson’s disease (PD). Since brain adenosine becomes greatly elevated in ageing brains and chronic adenosine A1 receptor (A1R) stimulation leads to neurodegeneration, we determined whether adenosine or A1R receptor ligands mimic the action of known compounds that promote α-syn aggregation (e.g., the amphetamine analogue 2-aminoindan) or inhibit α-syn aggregation (e.g., Rasagiline metabolite 1-aminoindan). In the present study, we determined whether adenosine, A1R receptor agonist N6-Cyclopentyladenosine (CPA) and antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) could directly interact with α-syn to modulate α-syn aggregation and neurodegeneration of dopaminergic neurons in the substantia nigra (SN).MethodsNanopore analysis and molecular docking were used to test the binding properties of CPA and DPCPX with α-syn in vitro. Sprague–Dawley rats were administered with 7-day intraperitoneal injections of the A1R ligands and 1- and 2-aminoindan, and levels of α-syn aggregation and neurodegeneration were examined in the SN pars compacta and hippocampal regions using confocal imaging and Western blotting.ResultsUsing nanopore analysis, we showed that the A1R agonists (CPA and adenosine) interacted with the N-terminus of α-syn, similar to 2-aminoindan, which is expected to promote a “knot” conformation and α-syn misfolding. In contrast, the A1R antagonist DPCPX interacted with the N- and C-termini of α-syn, similar to 1-aminoindan, which is expected to promote a “loop” conformation that prevents α-syn misfolding. Molecular docking studies revealed that adenosine, CPA and 2-aminoindan interacted with the hydrophobic core of α-syn N-terminus, whereas DPCPX and 1-aminoindan showed direct binding to the N- and C-terminal hydrophobic pockets. Confocal imaging and Western blot analyses revealed that chronic treatments with CPA alone or in combination with 2-aminoindan increased α-syn expression/aggregation and neurodegeneration in both SN pars compacta and hippocampus. In contrast, DPCPX and 1-aminoindan attenuated the CPA-induced α-syn expression/aggregation and neurodegeneration in SN and hippocampus.ConclusionsThe results indicate that A1R agonists and drugs promoting a “knot” conformation of α-syn can cause α-synucleinopathy and increase neuronal degeneration, whereas A1R antagonists and drugs promoting a “loop” conformation of α-syn can be harnessed for possible neuroprotective therapies to decrease α-synucleinopathy in PD.

Multipotent Stromal Cells from Subcutaneous Adipose Tissue of Normal Weight and Obese Subjects: Modulation of Their Adipogenic Differentiation by Adenosine A1 Receptor Ligands.

Adenosine A1 receptor (A1R) activation, stimulating lipogenesis and decreasing insulin resistance, could be useful for metabolic syndrome management in obese subjects. Since full A1R agonists induce harmful side-effects, while partial agonists show a better pharmacological profile, we investigated the influence of two derivatives of the full A1R agonist 2-chloro-N6-cyclopentyladenosine (CCPA), C1 and C2 behaving as A1R partial agonists in animal models, on the adipogenic differentiation of stromal/stem cells (ASCs) from human subcutaneous adipose tissue, which mainly contribute to increase fat mass in obesity. The ASCs from normal-weight subjects showed increased proliferation and A1R expression but reduced adipogenic differentiation compared to obese individual-derived ASCs. Cell exposure to CCPA, C1, C2 or DPCPX, an A1R antagonist, did not affect ASC proliferation, while mainly C2 and DPCPX significantly decreased adipogenic differentiation of both ASC types, reducing the activity of glycerol-3-phosphate dehydrogenase and the expression of PPARγ and FABP-4, all adipogenic markers, and phosphorylation of Akt in the phosphatidylinositol-3-kinase pathway, which plays a key-role in adipogenesis. While requiring confirmation in in vivo models, our results suggest that A1R partial agonists or antagonists, by limiting ASC differentiation into adipocytes and, thereby, fat mass expansion, could favor development/worsening of metabolic syndrome in obese subjects without a dietary control.

Synthesis and evaluation of adenosine derivatives as A1, A2A, A2B and A3 adenosine receptor ligands containing boron clusters as phenyl isosteres and selective A3 agonists.

A series of adenosine and 2'-deoxyadenosine pairs modified with a 1,12-dicarba-closo-dodecaborane cluster or alternatively with a phenyl group at the same position was synthesized, and their affinity was determined at A1, A2A, A2B and A3 adenosine receptors (ARs). While AR affinity differences were noted, a general tendency to preferentially bind A3 AR over other ARs was observed for most tested ligands. In particular, 5'-ethylcarbamoyl-N6-(3-phenylpropyl)adenosine (18), N6-(3-phenylpropyl)-2-chloroadenosine (24) and N6-(3-phenylpropyl)adenosine (40) showed nanomolar A3 affinity (Ki 4.5, 6.4 and 7.5nM, respectively). Among the boron cluster-containing compounds, the highest A3 affinity (Ki 206nM) was for adenosine derivative 41 modified at C2. In the matched molecular pairs, analogs bearing boron clusters were found to show lower binding affinity for adenosine receptors than the corresponding phenyl analogs. Nevertheless, interestingly, several boron cluster modified adenosine ligands showed significantly higher A3 receptor selectivity than the corresponding phenyl analogs: 7vs. 8, 15vs. 16, 17vs. 18.

Adenosine and Inflammation: Here, There and Everywhere.

Adenosine is a ubiquitous endogenous modulator with the main function of maintaining cellular and tissue homeostasis in pathological and stress conditions. It exerts its effect through the interaction with four G protein-coupled receptor (GPCR) subtypes referred as A1, A2A, A2B, and A3 adenosine receptors (ARs), each of which has a unique pharmacological profile and tissue distribution. Adenosine is a potent modulator of inflammation, and for this reason the adenosinergic system represents an excellent pharmacological target for the myriad of diseases in which inflammation represents a cause, a pathogenetic mechanism, a consequence, a manifestation, or a protective factor. The omnipresence of ARs in every cell of the immune system as well as in almost all cells in the body represents both an opportunity and an obstacle to the clinical use of AR ligands. This review offers an overview of the cardinal role of adenosine in the modulation of inflammation, showing how the stimulation or blocking of its receptors or agents capable of regulating its extracellular concentration can represent promising therapeutic strategies for the treatment of chronic inflammatory pathologies, neurodegenerative diseases, and cancer.

Allosterism vs. Orthosterism: Recent Findings and Future Perspectives on A2B AR Physio-Pathological Implications.

The development of GPCR (G-coupled protein receptor) allosteric modulators has attracted increasing interest in the last decades. The use of allosteric modulators in therapy offers several advantages with respect to orthosteric ones, as they can fine-tune the tissue responses to the endogenous agonist. Since the discovery of the first A1 adenosine receptor (AR) allosteric modulator in 1990, several efforts have been made to develop more potent molecules as well as allosteric modulators for all adenosine receptor subtypes. There are four subtypes of AR: A1, A2A, A2B, and A3. Positive allosteric modulators of the A1 AR have been proposed for the cure of pain. A3 positive allosteric modulators are thought to be beneficial during inflammatory processes. More recently, A2A and A2B AR allosteric modulators have also been disclosed. The A2B AR displays the lowest affinity for its endogenous ligand adenosine and is mainly activated as a consequence of tissue damage. The A2B AR activation has been found to play a crucial role in chronic obstructive pulmonary disease, in the protection of the heart from ischemic injury, and in the process of bone formation. In this context, allosteric modulators of the A2B AR may represent pharmacological tools useful to develop new therapeutic agents. Herein, we provide an up-to-date highlight of the recent findings and future perspectives in the field of orthosteric and allosteric A2B AR ligands. Furthermore, we compare the use of orthosteric ligands with positive and negative allosteric modulators for the management of different pathological conditions.

Species Differences in Microsomal Metabolism of Xanthine-Derived A1 Adenosine Receptor Ligands.

Tracer development for positron emission tomography (PET) requires thorough evaluation of pharmacokinetics, metabolism, and dosimetry of candidate radioligands in preclinical animal studies. Since variations in pharmacokinetics and metabolism of a compound occur in different species, careful selection of a suitable model species is mandatory to obtain valid data. This study focuses on species differences in the in vitro metabolism of three xanthine-derived ligands for the A1 adenosine receptor (A1AR), which, in their 18F-labeled form, can be used to image A1AR via PET. In vitro intrinsic clearance and metabolite profiles of 8-cyclopentyl-3-(3-fluoropropyl)-1-propylxanthine (CPFPX), an established A1AR-ligand, and two novel analogs, 8-cyclobutyl-3-(3-fluoropropyl)-1-propylxanthine (CBX) and 3-(3-fluoropropyl)-8-(1-methylcyclobutyl)-1-propylxanthine (MCBX), were determined in liver microsomes from humans and preclinical animal species. Molecular mechanisms leading to significant differences between human and animal metabolite profiles were also examined. The results revealed significant species differences regarding qualitative and quantitative aspects of microsomal metabolism. None of the tested animal species fully matched human microsomal metabolism of the three A1AR ligands. In conclusion, preclinical evaluation of xanthine-derived A1AR ligands should employ at least two animal species, preferably rodent and dog, to predict in vivo behavior in humans. Surprisingly, rhesus macaques appear unsuitable due to large differences in metabolic activity towards the test compounds.

Potent and selective A3 adenosine receptor antagonists bearing aminoesters as heterobifunctional moieties.

A3 adenosine receptors were found to have a role in different pathological states, such as glaucoma, renal fibrosis, neuropathic pain and cancer. Consequently, it is important to utilize any molecular tool which could help to study these conditions. In the present study we continue our search for potent A3 adenosine receptor ligands which could be successively conjugated to other molecules with the aim of obtaining more potent (e.g. allosteric ligand conjugation) or detectable ligands (e.g. fluorescent molecule or biotin conjugation). Specifically, different aminoester moieties were introduced at the 5 position of the pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine core. The ester functionalization represents the candidate for the subsequent conjugation. All the reported compounds are potent hA3 adenosine receptor antagonists and some of them exhibited high selectivity against the other adenosine receptors. The main structural terms of ligand recognition and selectivity were disclosed by molecular modelling studies. Molecular docking results led to the characterization of an alternative binding mode for antagonists at the orthosteric binding site of the hA3 adenosine receptor, evaluated and assessed by classical molecular dynamics simulations.

Targeting Adenosine Receptors: A Potential Pharmacological Avenue for Acute and Chronic Pain.

Adenosine is a purine nucleoside, responsible for the regulation of multiple physiological and pathological cellular and tissue functions by activation of four G protein-coupled receptors (GPCR), namely A1, A2A, A2B, and A3 adenosine receptors (ARs). In recent years, extensive progress has been made to elucidate the role of adenosine in pain regulation. Most of the antinociceptive effects of adenosine are dependent upon A1AR activation located at peripheral, spinal, and supraspinal sites. The role of A2AAR and A2BAR is more controversial since their activation has both pro- and anti-nociceptive effects. A3AR agonists are emerging as promising candidates for neuropathic pain. Although their therapeutic potential has been demonstrated in diverse preclinical studies, no AR ligands have so far reached the market. To date, novel pharmacological approaches such as adenosine regulating agents and allosteric modulators have been proposed to improve efficacy and limit side effects enhancing the effect of endogenous adenosine. This review aims to provide an overview of the therapeutic potential of ligands interacting with ARs and the adenosinergic system for the treatment of acute and chronic pain.

Influence of incubation conditions on microsomal metabolism of xanthine-derived A1 adenosine receptor ligands

IntroductionIn vitro metabolism models such as liver microsomes represent an important tool for the development of novel radioligands. Comparability and physiological relevance of in vitro metabolism data critically depend on the careful evaluation and optimization of assay protocols. We therefore investigated the influence of incubation conditions on the microsomal stability of xanthine-derived A1 adenosine receptor (A1AR) ligands which have been developed for positron emission tomography (PET). MethodsSubstrate depletion assays using rat liver microsomes (RLM) were performed for three analogous compounds which differ with regard to the metabolically vulnerable substituent at the xanthine C8 position. Incubation conditions were varied systematically. Additionally, the stability of the cofactor NADPH during incubation was investigated. ResultsMicrosomal metabolism was strongly influenced by buffer pH, organic solvents and preincubation time. Substrate depletion values varied up to 5-fold depending on incubation matrix composition, however, the rank order of metabolic stability remained unchanged. Prolonged incubation periods led to drastic loss in enzyme activity which could not be prevented by addition of metal chelators or antioxidants. Cofactor NADPH was rapidly oxidized in microsomal matrix, even in the absence of cytochrome P450 substrates. DiscussionIn summary, short incubation times, precise pH control and minimal concentrations of organic solvents are mandatory to obtain reliable microsomal stability data. Furthermore, in vitro metabolic stability of the tested A1AR ligands varied largely depending on the particular C8 substituent. Consequently, structural modifications at the xanthine C8 position appear to be a promising strategy for the improvement of A1AR PET radioligands.

Polypharmacology of N6-(3-Iodobenzyl)adenosine-5′-N-methyluronamide (IB-MECA) and Related A3 Adenosine Receptor Ligands: Peroxisome Proliferator Activated Receptor (PPAR) γ Partial Agonist and PPARδ Antagonist Activity Suggests Their Antidiabetic Potential

A3 adenosine receptor (AR) ligands including A3 AR agonist, N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (1a, IB-MECA) were examined for adiponectin production in human bone marrow mesenchymal stem cells (hBM-MSCs). In this model, 1a significantly increased adiponectin production, which is associated with improved insulin sensitivity. However, A3 AR antagonists also promoted adiponectin production in hBM-MSCs, indicating that the A3 AR pathway may not be directly involved in the adiponectin promoting activity. In a target deconvolution study, their adiponectin-promoting activity was significantly correlated to their binding activity to both peroxisome proliferator activated receptor (PPAR) γ and PPARδ. They functioned as both PPARγ partial agonists and PPARδ antagonists. In the diabetic mouse model, 1a and its structural analogues A3 AR antagonists significantly decreased the serum levels of glucose and triglyceride, supporting their antidiabetic potential. These findings indicate that the polypharmacophore of these compounds may provide therapeutic insight into their multipotent efficacy against various human diseases.

Novel Approach to Repeated Arterial Blood Sampling in Small Animal PET: Application in a Test-Retest Study with the Adenosine A1 Receptor Ligand [(11)C]MPDX.

PurposeSmall animal positron emission tomography (PET) can be used to detect small changes in neuroreceptor availability. This often requires rapid arterial blood sampling. However, current catheterization procedures do not allow repeated blood sampling. We have developed a procedure which allows arterial sampling on repeated occasions in the same animal.ProceduresEleven male Wistar rats were two times catheterized via a superficial branch of a femoral artery and scanned with [11C]MPDX and blood sampling. PET images were co-registered to a magnetic resonance imaging (MRI) template. Regional tracer distribution volumes (VT) in the brain were calculated by the Logan analysis. The procedure was repeated after 1 week.ResultsSurgery was successful in 90 % of the cases, and discomfort was minor. The VT data showed small differences between test and retest, low between subject variability, and a strong agreement between and within subjects.ConclusionRepeated quantitative imaging with a high reproducibility is possible with this approach.

Topological sub-structural molecular design (TOPS-MODE): a useful tool to explore key fragments of human A3 adenosine receptor ligands.

Adenosine regulates tissue function by activating four G-protein-coupled adenosine receptors (ARs). Selective agonists and antagonists for A3 ARs have been investigated for the treatment of a variety of immune disorders, cancer, brain, and heart ischemic conditions. We herein present a QSAR study based on a Topological sub-structural molecular design (TOPS-MODE) approach, intended to predict the A3 ARs of a diverse dataset of 124 (94 training set/ 30 prediction set) adenosine derivatives. The final model showed good fit and predictive capability, displaying 85.1 % of the experimental variance. The TOPS-MODE approach afforded a better understanding and interpretation of the developed model based on the useful information extracted from the analysis of the contribution of different molecular fragments to the affinity.

Development of novel adenosine receptor ligands based on the 3-amidocoumarin scaffold

With the aim of finding new adenosine receptor (AR) ligands presenting the 3-amidocoumarin scaffold, a study focusing on the discovery of new chemical entities was carried out. The synthesized compounds 1–8 were evaluated in radioligand binding (A1, A2A and A3) and adenylyl cyclase activity (A2B) assays in order to determine their affinity for human AR subtypes. The 3-benzamide derivative 4 showed the highest affinity of the whole series and was more than 30-fold selective for the A3 AR (Ki=3.24μM). The current study supported that small structural changes in this scaffold allowed modulating the affinity resulting in novel promising classes of A1, A2A, and/or A3 AR ligands. We also performed docking calculations in hA2A and hA3 to identify the hypothetical binding mode for the most active compounds. In addition, some ADME properties were calculated in order to better understand the potential of these compounds as drug candidates.

Insight into the interactions between novel coumarin derivatives and human A3 adenosine receptors.

A study focused on the discovery of new chemical entities based on the 3-arylcoumarin scaffold was performed with the aim of finding new adenosine receptor (AR) ligands. Thirteen synthesized compounds were evaluated by radioligand binding (A1, A2A, and A3) and adenylyl cyclase activity (A2B) assays in order to study their affinity for the four human AR (hAR) subtypes. Seven of the studied compounds proved to be selective A3 AR ligands, with 3-(4'-methylphenyl)-8-(2-oxopropoxy)coumarin (12) being the most potent (Ki =634 nM). None of the compounds showed affinity for the A2B receptor, while four compounds were found to be nonselective AR ligands for the other three subtypes. Docking simulations were carried out to identify the hypothetical binding mode and to rationalize the interaction of these types of coumarin derivatives with the binding site of the three ARs to which binding was observed. The results allowed us to conclude that the 3-arylcoumarin scaffold composes a novel and promising class of A3 AR ligands. ADME properties were also calculated, with the results suggesting that these compounds are promising leads for the identification of new drug candidates.

Synthesis, spectroscopic characterization and X-ray structure of novel 7-methoxy-4-oxo-N-phenyl-4H-chromene-2-carboxamides

The chromone scaffold has been found to be an important tool in the drug discovery process through its relevant pharmacological activities. Chromone carboxamide derivatives synthesized within our group have shown noteworthy results as inhibitors of monoamino oxidase-B and as ligands for adesonine receptors. Specifically, chromone-2-carboxamide has been shown to be a privileged structure for the development of selective A3 adenosine receptor ligands. In this work two novel substituted 4-oxo-N-phenyl-4H-chromene-2-carboxamides have been synthesized and a complete structural characterization was performed using Fourier Transform Infrared Spectroscopy, one-dimensional and two-dimensional Nuclear Magnetic Resonance techniques and Mass Spectroscopy. Finally, the molecular and supramolecular structures were determined by X-ray analysis. The X-ray crystallographic analysis describes in detail the molecular conformation and supramolecular structure of a hemihydrate of 7-methoxy-4-oxo-N-phenyl-4H-chromene-2-carboxamide.The data acquired from this study add valuable information to our chromone database. The unambiguous identification of the compounds will support the understanding of the structure–activity relationships of these compounds.

The Selective A3AR Antagonist LJ-1888 Ameliorates UUO-Induced Tubulointerstitial Fibrosis

Adenosine in the normal kidney significantly elevates in response to cellular damage. The renal A3 adenosine receptor (A3AR) is up-regulated under stress, but the therapeutic effects of A3AR antagonists on chronic kidney disease are not fully understood. The present study examined the effect of LJ-1888 [(2R,3R,4S)-2-[2-chloro-6-(3-iodobenzylamino)-9H-purine-9-yl]-tetrahydrothiophene-3,4-diol], a newly developed potent, selective, species-independent, and orally active A3AR antagonist, on unilateral ureteral obstruction (UUO)-induced renal fibrosis. Pretreatment with LJ-1888 inhibited UUO-induced fibronectin and collagen I up-regulation in a dose-dependent manner. Masson's trichrome staining confirmed that LJ-1888 treatment effectively reduced UUO-induced interstitial collagen accumulation. Furthermore, delayed administration of LJ-1888 showed an equivalent therapeutic effect on tubulointerstitial fibrosis to that of losartan. Small-interfering A3AR transfection effectively inhibited transforming growth factor-β1 (TGF-β1)-induced fibronectin and collagen I up-regulation in proximal tubular cells similar to LJ-1888, confirming that the renoprotective effect of LJ-1888 resulted from A3AR blockade. UUO- or TGF-β1-induced c-Jun N-terminal kinase and extracellular signal-regulated kinase phosphorylation decreased significantly after LJ-1888 administration. A3AR blockade reduced UUO- or TGF-β1-induced up-regulation of lysyl oxidase, which induces cross-linking of extracellular matrix, suggesting that LJ-1888 may also regulate extracellular matrix accumulation via post-translational regulation. In conclusion, the present data demonstrate that the A3AR antagonist, LJ-1888, blocked the development and attenuated the progression of renal fibrosis, and they suggest that LJ-1888 may become a new therapeutic modality for renal interstitial fibrosis.

Recent Advances on QSAR-Based Profiling of Agonist and Antagonist A3 Adenosine Receptor Ligands

Adenosine receptors (ARs) are signaling molecules ubiquitously expressed in a wide variety of tissues in the human body. ARs mediate physiological functions by interacting with four subtypes of G-protein-coupled receptors, namely A1, A2A, A2B and A3. The A3 AR, probably the most studied subtype, is also ubiquitously expressed, with high levels in peripheral organs and low levels in the brain. This type of AR is involved in a variety of important pathophysiological processes, ranging from modulation of cerebral and cardiac ischemic damage to regulation of immunosuppression and inflammation. Consequently, the development of potent and selective A3 AR ligands as promising therapeutic options for a variety of diseases has been a prime subject of medicinal chemistry research for more than two decades. Among the plethora of approaches applied quantitative structure activity relationships (QSAR) stands out for being largely employed due to their potential to increase the efficiency at initial stages of the drug discovery process. So, we provide a review of the main QSAR studies devoted to the design, discovery and development of agonist and antagonist A3 adenosine receptor ligands. Common pitfalls of these QSAR applications and the current trends in this area are also analyzed.

Functional selectivity of adenosine A1 receptor ligands?

The concept of functional selectivity offers great potential for the development of drugs that selectively activate a specific intracellular signaling pathway. During the last few years, it has become possible to systematically analyse compound libraries on G protein-coupled receptors (GPCRs) for this 'biased' form of signaling. We screened over 800 compounds targeting the class of adenosine A(1) receptors using a 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.