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Among still comparatively few G protein-coupled receptors, the adenosine A 2A receptor has been co-crystallized with several ligands, agonists as well as antagonists. It can thus serve as a template with a well-described orthosteric ligand binding region for adenosine receptors. As not all subtypes have been crystallized yet, and in order to investigate the usability of homology models in this context, multiple adenosine A 1 receptor (A 1 AR) homology models had been previously obtained and a library of lead-like compounds had been docked. As a result, a number of potent and one selective ligand toward the intended target have been identified. However, in in vitro experimental verification studies, many ligands also bound to the A 2A AR and the A 3 AR subtypes. In this work we asked the question whether a classification of the ligands according to their selectivity was possible based on docking scores. Therefore, we built an A 3 AR homology model and docked all previously found ligands to all three receptor subtypes. As a metric, we employed an in vitro/in silico selectivity ranking system based on taxicab geometry and obtained a classification model with reasonable separation. In the next step, the method was validated with an external library of, selective ligands with similarly good performance. This classification system might also be useful in further screens.

Adenosine receptors (ARs), like many otherGprotein-coupledreceptors (GPCRs), are targets of primary interest indrug design. However, one of the main limits for the development of drugs for this class of GPCRs is the complex selectivity profile usually displayed by ligands. Numerous efforts have been madefor clarifying the selectivity of ARs, leading to the development of many ligand-based models. The structure of the AR subtype A 1 (A 1 AR) has been recently solved,providing important structural insights. In the present work, we rationalized the selectivity profile of two selective A 1 AR and A 2A AR antagonists, investigating their recognition trajectories obtained by Supervised Molecular Dynamics from an unbound state and monitoring the role of the water molecules in the binding site., Competing Interests: The authors declare no conflict of interest.

A series of fourteen methoxy substituted 2-benzoyl-1-benzofuran derivatives were synthesised and their affinities determined for adenosine A 1 and A 2A receptors via radioligand binding assays to establish the structure activity relationships pertinent for A 1 and A 2A affinity. Compound 3j (6,7-dimethoxybenzofuran-2-yl)(3-methoxyphenyl)methanone exhibited A 1 affinity (A 1 K i (rat) = 6.880 µM) as well as A 2A affinity (A 2A K i (rat) = 0.5161 µM). Compounds 3a-b &3i-k exhibited selective affinity towards A 1 with K i values below 10 µM. The results indicate that C6,7-diOCH 3 substitution on ring A in combination with meta (C3')-OCH 3 substitution on ring B is beneficial for A 1 and A 2A affinity and activity. Compounds 3a-b &3j-k showed low cytotoxicity. Upon in vitro and in silico evaluation, compound 3j may be considered lead-like (i.e. a molecular entity suitable for optimization) and, thus, of value in the design of novel, potent and selective adenosine A 1 and A 2A receptor antagonists., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In earlier studies, we generated concentration-response (E/c) curves with CPA ( N 6 -cyclopentyladenosine; a selective A 1 adenosine receptor agonist) or adenosine, in the presence or absence of S -(2-hydroxy-5-nitrobenzyl)-6-thioinosine (NBTI, a selective nucleoside transport inhibitor), and with or without a pretreatment with 8-cyclopentyl- N 3 -[3-(4-(fluorosulfonyl)-benzoyloxy)propyl]- N 1 -propylxanthine (FSCPX, a chemical known as a selective, irreversible A 1 adenosine receptor antagonist), in isolated, paced guinea pig left atria. Meanwhile, we observed a paradoxical phenomenon, i.e. the co-treatment with FSCPX and NBTI appeared to enhance the direct negative inotropic response to adenosine. In the present in silico study, we aimed to reproduce eight of these E/c curves. Four models (and two additional variants of the last model) were constructed, each one representing a set of assumptions, in order to find the model exhibiting the best fit to the ex vivo data, and to gain insight into the paradoxical phenomenon in question. We have obtained in silico evidence for an interference between effects of FSCPX and NBTI upon our ex vivo experimental setting. Regarding the mechanism of this interference, in silico evidence has been gained for the assumption that FSCPX inhibits the effect of NBTI on the level of endogenous (but not exogenous) adenosine. As an explanation, it may be hypothesized that FSCPX inhibits an enzyme participating in the interstitial adenosine formation. In addition, our results suggest that NBTI does not stop the inward adenosine flux in the guinea pig atrium completely., Competing Interests: The authors declare no conflict of interest.

Despite intense interest in designing positive allosteric modulators (PAMs) as selective drugs of the adenosine A 1 receptor (A 1 AR), structural binding modes of the receptor PAMs remain unknown. Using the first X-ray structure of the A 1 AR, we have performed all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) technique to determine binding modes of the A 1 AR allosteric drug leads. Two prototypical PAMs, PD81723 and VCP171, were selected. Each PAM was initially placed at least 20 Å away from the receptor. Extensive GaMD simulations using the AMBER and NAMD simulation packages at different acceleration levels captured spontaneous binding of PAMs to the A 1 AR. The simulations allowed us to identify low-energy binding modes of the PAMs at an allosteric site formed by the receptor extracellular loop 2 (ECL2), which are highly consistent with mutagenesis experimental data. Furthermore, the PAMs stabilized agonist binding in the receptor. In the absence of PAMs at the ECL2 allosteric site, the agonist sampled a significantly larger conformational space and even dissociated from the A 1 AR alone. In summary, the GaMD simulations elucidated structural binding modes of the PAMs and provided important insights into allostery in the A 1 AR, which will greatly facilitate the receptor structure-based drug design.

Based on in silico results, recently we have assumed that FSCPX, an irreversible A₁ adenosine receptor antagonist, inhibits the action of NBTI that is apparent on E / c curves of adenosine receptor agonists. As a mechanism for this unexpected effect, we hypothesized that FSCPX might modify the equilibrative and NBTI-sensitive nucleoside transporter (ENT1) in a way that allows ENT1 to transport adenosine but impedes NBTI to inhibit this transport. This assumption implies that our method developed to estimate receptor reserve for agonists with short half-life such as adenosine, in its original form, overestimates the receptor reserve. In this study, therefore, our goals were to experimentally test our assumption on this effect of FSCPX, to improve our receptor reserve-estimating method and then to compare the original and improved forms of this method. Thus, we improved our method and assessed the receptor reserve for the direct negative inotropic effect of adenosine with both forms of this method in guinea pig atria. We have found that FSCPX inhibits the effects of NBTI that are mediated by increasing the interstitial concentration of adenosine of endogenous (but not exogenous) origin. As a mechanism for this action of FSCPX, inhibition of enzymes participating in the interstitial adenosine production can be hypothesized, while modification of ENT1 can be excluded. Furthermore, we have shown that, in comparison with the improved form, the original version of our method overestimates receptor reserve but only to a small extent. Nevertheless, use of the improved form is recommended in the future.

New 7-amino-2-phenylpyrazolo[4,3-d]pyrimidine derivatives, substituted at the 5-position with aryl(alkyl)amino- and 4-substituted-piperazin-1-yl- moieties, were synthesized with the aim of targeting human (h) adenosine A 1 and/or A 2A receptor subtypes. On the whole, the novel derivatives 1-24 shared scarce or no affinities for the off-target hA 2B and hA 3 ARs. The 5-(4-hydroxyphenethylamino)- derivative 12 showed both good affinity (K i = 150 nM) and the best selectivity for the hA 2A AR while the 5-benzylamino-substituted 5 displayed the best combined hA 2A (K i = 123 nM) and A 1 AR affinity (K i = 25 nM). The 5-phenethylamino moiety (compound 6) achieved nanomolar affinity (K i = 11 nM) and good selectivity for the hA 1 AR. The 5-(N 4 -substituted-piperazin-1-yl) derivatives 15-24 bind the hA 1 AR subtype with affinities falling in the high nanomolar range. A structure-based molecular modeling study was conducted to rationalize the experimental binding data from a molecular point of view using both molecular docking studies and Interaction Energy Fingerprints (IEFs) analysis.[Formula: see text].

Adenosine A 1 and A 2A receptors are attracting great interest as drug targets for their role in cognitive and motor deficits, respectively. Antagonism of both these adenosine receptors may offer therapeutic benefits in complex neurological diseases, such as Alzheimer's and Parkinson's disease. The aim of this study was to explore the affinity and selectivity of 2-benzylidene-1-tetralone derivatives as adenosine A 1 and A 2A receptor antagonists. Several 5-hydroxy substituted 2-benzylidene-1-tetralone analogues with substituents on ring B were synthesized and assessed as antagonists of the adenosine A 1 and A 2A receptors via radioligand binding assays. The results indicated that hydroxy substitution in the meta and para position of phenyl ring B, displayed the highest selectivity and affinity for the adenosine A 1 receptor with K i values in the low micromolar range. Replacement of ring B with a 2-amino-pyrimidine moiety led to compound 12 with an increase of affinity and selectivity for the adenosine A 2A receptor. These substitution patterns led to enhanced adenosine A 1 and A 2A receptor binding affinity. The para-substituted 5-hydroxy analogue 3 behaved as an adenosine A 1 receptor antagonists in a GTP shift assay performed with rat whole brain membranes expressing adenosine A 1 receptors. In conclusion, compounds 3 and 12, showed the best adenosine A 1 and A 2A receptor affinity respectively, and therefore represent novel adenosine receptor antagonists that may have potential with further structural modifications as drug candidates for neurological disorders., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The adenosine A 1 receptor (A 1 -AR) is a G-protein-coupled receptor that plays a vital role in cardiac, renal, and neuronal processes but remains poorly targeted by current drugs. We determined a 3.2 Å crystal structure of the A 1 -AR bound to the selective covalent antagonist, DU172, and identified striking differences to the previously solved adenosine A 2A receptor (A 2A -AR) structure. Mutational and computational analysis of A 1 -AR revealed a distinct conformation of the second extracellular loop and a wider extracellular cavity with a secondary binding pocket that can accommodate orthosteric and allosteric ligands. We propose that conformational differences in these regions, rather than amino-acid divergence, underlie drug selectivity between these adenosine receptor subtypes. Our findings provide a molecular basis for AR subtype selectivity with implications for understanding the mechanisms governing allosteric modulation of these receptors, allowing the design of more selective agents for the treatment of ischemia-reperfusion injury, renal pathologies, and neuropathic pain., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Respiratory complications in patients with spinal cord injury (SCI) are common and have a negative impact on the quality of patients' lives. Systemic administration of drugs that improve respiratory function often cause deleterious side effects. The present study examines the applicability of a novel nanotechnology-based drug delivery system, which induces recovery of diaphragm function after SCI in the adult rat model. We developed a protein-coupled nanoconjugate to selectively deliver by transsynaptic transport small therapeutic amounts of an A1 adenosine receptor antagonist to the respiratory centers. A single administration of the nanoconjugate restored 75% of the respiratory drive at 0.1% of the systemic therapeutic drug dose. The reduction of the systemic dose may obviate the side effects. The recovery lasted for 4 weeks (the longest period studied). These findings have translational implications for patients with respiratory dysfunction after SCI., Significance Statement: The leading causes of death in humans following SCI are respiratory complications secondary to paralysis of respiratory muscles. Systemic administration of methylxantines improves respiratory function but also leads to the development of deleterious side effects due to actions of the drug on nonrespiratory sites. The importance of the present study lies in the novel drug delivery approach that uses nanotechnology to selectively deliver recovery-inducing drugs to the respiratory centers exclusively. This strategy allows for a reduction in the therapeutic drug dose, which may reduce harmful side effects and markedly improve the quality of life for SCI patients., (Copyright © 2016 the authors 0270-6474/16/363441-12$15.00/0.)

Adenosine A2A antagonists are believed to have therapeutic potential in the treatment of Parkinson's disease (PD). We have characterized the dual adenosine A2A/A1 receptor antagonist JNJ-40255293 (2-amino-8-[2-(4-morpholinyl)ethoxy]-4-phenyl-5H-indeno[1,2-d]pyrimidin-5-one). JNJ-40255293 was a high-affinity (7.5 nM) antagonist at the human A2A receptor with 7-fold in vitro selectivity versus the human A1 receptor. A similar A2A:A1 selectivity was seen in vivo (ED50's of 0.21 and 2.1 mg/kg p.o. for occupancy of rat brain A2A and A1 receptors, respectively). The plasma EC50 for occupancy of rat brain A2A receptors was 13 ng/mL. In sleep-wake encephalographic (EEG) studies, JNJ-40255293 dose-dependently enhanced a consolidated waking associated with a subsequent delayed compensatory sleep (minimum effective dose: 0.63 mg/kg p.o.). As measured by microdialysis, JNJ-40255293 did not affect dopamine and noradrenaline release in the prefrontal cortex and the striatum. However, it was able to reverse effects (catalepsy, hypolocomotion, and conditioned avoidance impairment in rats; hypolocomotion in mice) produced by the dopamine D2 antagonist haloperidol. The compound also potentiated the agitation induced by the dopamine agonist apomorphine. JNJ-40255293 also reversed hypolocomotion produced by the dopamine-depleting agent reserpine and potentiated the effects of l-dihydroxyphenylalanine (L-DOPA) in rats with unilateral 6-hydroxydopamine-induced lesions of the nigro-striatal pathway, an animal model of Parkinson's disease. Extrapolating from the rat receptor occupancy dose-response curve, the occupancy required to produce these various effects in rats was generally in the range of 60-90%. The findings support the continued research and development of A2A antagonists as potential treatments for PD.

FLAP fingerprints are applied in the ligand-, structure- and pharmacophore-based mode in a case study on antagonists of all four adenosine receptor (AR) subtypes. Structurally diverse antagonist collections with respect to the different ARs were constructed by including binding data to human species only. FLAP models well discriminate "active" (=highly potent) from "inactive" (=weakly potent) AR antagonists, as indicated by enrichment curves, numbers of false positives, and AUC values. For all FLAP modes, model predictivity slightly decreases as follows: A(2B)R > A(2A)R > A(3)R > A(1)R antagonists. General performance of FLAP modes in this study is: ligand- > structure- > pharmacophore- based mode. We also compared the FLAP performance with other common ligand- and structure-based fingerprints. Concerning the ligand-based mode, FLAP model performance is superior to ECFP4 and ROCS for all AR subtypes. Although focusing on the early first part of the A(2A), A(2B) and A(3) enrichment curves, ECFP4 and ROCS still retain a satisfactory retrieval of actives. FLAP is also superior when comparing the structure-based mode with PLANTS and GOLD. In this study we applied for the first time the novel FLAPPharm tool for pharmacophore generation. Pharmacophore hypotheses, generated with this tool, convincingly match with formerly published data. Finally, we could demonstrate the capability of FLAP models to uncover selectivity aspects although single AR subtype models were not trained for this purpose.