Human A3 Adenosine Research Articles

2-Aryladenine Derivatives as a Potent Scaffold for Adenosine Receptor Antagonists: The 6-Morpholino Derivatives.

A set of 2-aryl-9-H or methyl-6-morpholinopurine derivatives were synthesized and assayed through radioligand binding tests at human A1, A2A, A2B, and A3 adenosine receptor subtypes. Eleven purines showed potent antagonism at A1, A3, dual A1/A2A, A1/A2B, or A1/A3 adenosine receptors. Additionally, three compounds showed high affinity without selectivity for any specific adenosine receptor. The structure-activity relationships were made for this group of new compounds. The 9-methylpurine derivatives were generally less potent but more selective, and the 9H-purine derivatives were more potent but less selective. These compounds can be an important source of new biochemical tools and/or pharmacological drugs.

Anti-Inflammatory Activities of 8-Benzylaminoxanthines Showing High Adenosine A2A and Dual A1/A2A Receptor Affinity.

Chronic inflammation plays an important role in the development of neurodegenerative diseases, such as Parkinson's disease (PD). In the present study, we synthesized 25 novel xanthine derivatives with variable substituents at the N1-, N3- and C8-position as adenosine receptor antagonists with potential anti-inflammatory activity. The compounds were investigated in radioligand binding studies at all four human adenosine receptor subtypes, A1, A2A, A2B and A3. Compounds showing nanomolar A2A and dual A1/A2A affinities were obtained. Three compounds, 19, 22 and 24, were selected for further studies. Docking and molecular dynamics simulation studies indicated binding poses and interactions within the orthosteric site of adenosine A1 and A2A receptors. In vitro studies confirmed the high metabolic stability of the compounds, and the absence of toxicity at concentrations of up to 12.5 µM in various cell lines (SH-SY5Y, HepG2 and BV2). Compounds 19 and 22 showed anti-inflammatory activity in vitro. In vivo studies in mice investigating carrageenan- and formalin-induced inflammation identified compound 24 as the most potent anti-inflammatory derivative. Future studies are warranted to further optimize the compounds and to explore their therapeutic potential in neurodegenerative diseases.

Subtle Chemical Changes Cross the Boundary between Agonist and Antagonist: New A3 Adenosine Receptor Homology Models and Structural Network Analysis Can Predict This Boundary.

Distinguishing compounds' agonistic or antagonistic behavior would be of great utility for the rational discovery of selective modulators. We synthesized truncated nucleoside derivatives and discovered 6c (Ki = 2.40 nM) as a potent human A3 adenosine receptor (hA3AR) agonist, and subtle chemical modification induced a shift from antagonist to agonist. We elucidated this shift by developing new hA3AR homology models that consider the pharmacological profiles of the ligands. Taken together with molecular dynamics (MD) simulation and three-dimensional (3D) structural network analysis of the receptor-ligand complex, the results indicated that the hydrogen bonding with Thr943.36 and His2727.43 could make a stable interaction between the 3'-amino group with TM3 and TM7, and the corresponding induced-fit effects may play important roles in rendering the agonistic effect. Our results provide a more precise understanding of the compounds' actions at the atomic level and a rationale for the design of new drugs with specific pharmacological profiles.

Design and Synthesis of 2,6-Disubstituted-4'-Selenoadenosine-5'-N,N-Dimethyluronamide Derivatives as Human A3 Adenosine Receptor Antagonists.

A new series of 4′-selenoadenosine-5′-N,N-dimethyluronamide derivatives as highly potent and selective human A3 adenosine receptor (hA3AR) antagonists, is described. The highly selective A3AR agonists, 4′-selenoadenosine-5′-N-methyluronamides were successfully converted into selective antagonists by adding a second N-methyl group to the 5′-uronamide position. All the synthesized compounds showed medium to high binding affinity at the hA3AR. Among the synthesized compounds, 2-H-N6-3-iodobenzylamine derivative 9f exhibited the highest binding affinity at hA3AR. (Ki = 22.7 nM). The 2-H analogues generally showed better binding affinity than the 2-Cl analogues. The cAMP functional assay with 2-Cl-N6-3-iodobenzylamine derivative 9l demonstrated hA3AR antagonist activity. A molecular modelling study suggests an important role of the hydrogen of 5′-uronamide as an essential hydrogen bonding donor for hA3AR activation.

Design and pharmacological profile of a novel covalent partial agonist for the adenosine A1 receptor

Partial agonists for G protein-coupled receptors (GPCRs) provide opportunities for novel pharmacotherapies with enhanced on-target safety compared to full agonists. For the human adenosine A1 receptor (hA1AR) this has led to the discovery of capadenoson, which has been in phase IIa clinical trials for heart failure. Accordingly, the design and profiling of novel hA1AR partial agonists has become an important research focus. In this study, we report on LUF7746, a capadenoson derivative bearing an electrophilic fluorosulfonyl moiety, as an irreversibly binding hA1AR modulator. Meanwhile, a nonreactive ligand bearing a methylsulfonyl moiety, LUF7747, was designed as a control probe in our study.In a radioligand binding assay, LUF7746’s apparent affinity increased to nanomolar range with longer pre-incubation time, suggesting an increasing level of covalent binding over time. Moreover, compared to the reference full agonist CPA, LUF7746 was a partial agonist in a hA1AR-mediated G protein activation assay and resistant to blockade with an antagonist/inverse agonist. An in silico structure-based docking study combined with site-directed mutagenesis of the hA1AR demonstrated that amino acid Y2717.36 was the primary anchor point for the covalent interaction. Additionally, a label-free whole-cell assay was set up to identify LUF7746’s irreversible activation of an A1 receptor-mediated cell morphological response.These results led us to conclude that LUF7746 is a novel covalent hA1AR partial agonist and a valuable chemical probe for further mapping the receptor activation process. It may also serve as a prototype for a therapeutic approach in which a covalent partial agonist may cause less on-target side effects, conferring enhanced safety compared to a full agonist.

Truncated (N)-Methanocarba Nucleosides as Partial Agonists at Mouse and Human A3 Adenosine Receptors: Affinity Enhancement by N6-(2-Phenylethyl) Substitution.

Dopamine-derived N6-substituents, compared to N6-(2-phenylethyl), in truncated (N)-methanocarba (bicyclo[3.1.0]hexyl) adenosines favored high A3 adenosine receptor (AR) affinity/selectivity, e.g., C2-phenylethynyl analogue 15 (MRS7591, Ki = 10.9/17.8 nM, at human/mouse A3AR). 15 was a partial agonist in vitro (hA3AR, cAMP inhibition, 31% Emax; mA3AR, [35S]GTP-γ-S binding, 16% Emax) and in vivo and also antagonized hA3AR in vitro. Distal H-bonding substitutions of the N6-(2-phenylethyl) moiety particularly enhanced mA3AR affinity by polar interactions with the extracellular loops, predicted using docking and molecular dynamics simulation with newly constructed mA3AR and hA3AR homology models. These hybrid models were based on an inactive antagonist-bound hA1AR structure for the upper part of TM2 and an agonist-bound hA2AAR structure for the remaining TM portions. These species-independent A3AR-selective nucleosides are low efficacy partial agonists and novel, nuanced modulators of the A3AR, a drug target of growing interest.

Conjugable A3 adenosine receptor antagonists for the development of functionalized ligands and their use in fluorescent probes

Compounds able to simultaneously bind a biological target and be conjugated to a second specific moiety are attractive tools for the development of multi-purpose ligands useful as multi-target ligands, receptor probes or drug delivery systems, with both therapeutic and diagnostic applications. The human A3 adenosine receptor is a G protein-coupled receptor involved in many physio-pathological conditions, e.g. cancer and inflammation, thus representing a promising research target. In this work, two series of conjugable hA3AR antagonists, based on the pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine nucleus, were developed. The introduction of an aromatic ring at the 5 position of the scaffold, before (phenylacetamido moiety) or after (1,2,3-triazole obtained by click chemistry) the conjugation is aimed to increase affinity and selectivity towards the hA3AR receptor. As expected, conjugable compounds showed good affinity towards the hA3AR. In order to prove their potential in the development of hA3AR ligands for different purposes, compounds were also functionalized with fluorescent probes. Unfortunately, conjugation decreased affinity and selectivity for the target as compared to the hA2AAR. Computational studies identified specific non-conserved residues of the extracellular loops which constitute a structural barrier able to discriminate between ligands, giving insights into the rational development of new highly selective ligands.

Design, Synthesis and Evaluation of New Indolylpyrimidylpiperazines for Gastrointestinal Cancer Therapy.

Human A3 adenosine receptor hA3AR has been implicated in gastrointestinal cancer, where its cellular expression has been found increased, thus suggesting its potential as a molecular target for novel anticancer compounds. Observation made in our previous work indicated the importance of the carbonyl group of amide in the indolylpyrimidylpiperazine (IPP) for its human A2A adenosine receptor (hA2AAR) subtype binding selectivity over the other AR subtypes. Taking this observation into account, we structurally modified an indolylpyrimidylpiperazine (IPP) scaffold, 1 (a non-selective adenosine receptors’ ligand) into a modified IPP (mIPP) scaffold by switching the position of the carbonyl group, resulting in the formation of both ketone and tertiary amine groups in the new scaffold. Results showed that such modification diminished the A2A activity and instead conferred hA3AR agonistic activity. Among the new mIPP derivatives (3–6), compound 4 showed potential as a hA3AR partial agonist, with an Emax of 30% and EC50 of 2.89 ± 0.55 μM. In the cytotoxicity assays, compound 4 also exhibited higher cytotoxicity against both colorectal and liver cancer cells as compared to normal cells. Overall, this new series of compounds provide a promising starting point for further development of potent and selective hA3AR partial agonists for the treatment of gastrointestinal cancers.

2-Aryladenine derivatives as a potent scaffold for A1, A3 and dual A1/A3 adenosine receptor antagonists: Synthesis and structure-activity relationships

From a collection containing more than 1500 academic compounds, in silico screening identified a hit for the human A1 adenosine receptor containing a new purine scaffold. To study the structure activity relationships of this new chemical series for adenosine receptors, a library of 24 purines was synthesized and tested in radioligand binding assays at human A1, A2A, A2B and A3 adenosine receptor subtypes. Fourteen molecules showed potent antagonism at A1, A3 or dual A1/A3 adenosine receptors. This purine scaffold is an important source for novel biochemical tools and/or therapeutic drugs.

Novel 8-amino-1,2,4-triazolo[4,3-a]pyrazin-3-one derivatives as potent human adenosine A1 and A2A receptor antagonists. Evaluation of their protective effect against β-amyloid-induced neurotoxicity in SH-SY5Y cells

In this work, an enlarged series of 1,2,4-triazolo[4,3-a]pyrazin-3-ones was designed to target the human (h) A2A adenosine receptor (AR) or both hA1 and hA2A ARs. The novel 8-amino-1,2,4-triazolopyrazin-3-one derivatives 1–25 featured a phenyl or a benzyl pendant at position 2 while different aryl/heteroaryl substituents were placed at position 6. Two compounds (8 and 10) endowed with high affinity (Ki = 7.2 and 10.6 nM) and a complete selectivity for the hA2A AR were identified. Moreover, several derivatives possessed nanomolar affinity for both hA1 and hA2A ARs (both Ki < 20 nM) and different degrees of selectivity versus the hA3 AR. Two selected compounds (10 and 25) demonstrated ability in preventing β-amyloid peptide (25–35)-induced neurotoxicity in SH-SY5Y cells. Results of docking studies at the hA2A and hA1 AR crystal structures helped us to rationalize the observed affinity data and to highlight that the steric hindrance of the substituents at the 2- and 6-position of the bicyclic core affects the binding mode in the receptor cavity.

A Comparison in the Use of the Crystallographic Structure of the Human A1 or the A2A Adenosine Receptors as a Template for the Construction of a Homology Model of the A3 Subtype

In the last decades, the field of therapeutic application in targeting the human A3 adenosine receptor has represented a rapidly growing area of research in adenosine field. Both agonists and antagonists have been described to have a potential application in the treatment of several diseases, including, for example, glaucoma, cancer, and autoimmune inflammations. To date, the most severe factor limiting the accuracy of the structure-based molecular modeling approaches is the fact that the three-dimensional human A3 structure has not yet been solved. However, the crystallographic structures of either human A1 or A2A subtypes are available as potential templates for the construction of its homology model. In this study, we have compared the propensity of both models to accommodate a series of known potent and selective human A3 agonists and antagonists. As described, on the basis of the results obtained from this preliminary study, it is possible to affirm that the human A3 receptor model based on the crystallographic structure of the A1 subtype can represent a valid alternative to the one conventionally used today, based on the available A2A structures.

1,2,4]Triazolo[1,5-c]pyrimidines as adenosine receptor antagonists: Modifications at the 8 position to reach selectivity towards A3 adenosine receptor subtype

[1,2,4]Triazolo[1,5-c]pyrimidine is a promising platform to develop adenosine receptor antagonists. Here, we tried to investigate the effect of the substituent at the 8 position of [1,2,4]triazolo[1,5-c]pyrimidine derivatives on affinity and selectivity at the human A3 adenosine receptor subtype. In particular, we have introduced both esters and amides, principally with a benzylic nature. In addition, a small series of 5-substituted [1,2,4]triazolo[1,5-c]pyrimidines was designed in order to complete the structure-activity relationship analysis. Several of these new compounds showed affinity towards human A3 adenosine receptor in the low nanomolar range, with the most potent derivative of the series bringing a 4-ethylbenzylester at the 8 position (compound 18, hA3AR Ki = 1.21 nM). Docking studies performed on the synthesized compounds inside models of human A1, A2A and A3 adenosine receptors showed similar binding modes, comparable with the typical crystallographic binding mode of the inverse agonist ZM-241,385.

Identification of novel thiazolo[5,4-d]pyrimidine derivatives as human A1 and A2A adenosine receptor antagonists/inverse agonists

In this study a new set of thiazolo[5,4-d]pyrimidine derivatives was synthesized. These derivatives bear different substituents at positions 2 and 5 of the thiazolopyrimidine core while maintaining a free amino group at position-7. The new compounds were tested for their affinity and potency at human (h) A1, A2A, A2B and A3 adenosine receptors expressed in CHO cells. The results reveal that the higher affinity of these new set of thiazolopyrimidines is toward the hA1 and hA2A adenosine receptors subtypes and is tuned by the substitution pattern at both the 2 and 5 positions of the thiazolopyrimidine nucleus. Functional studies evidenced that the compounds behaved as dual A1/A2A antagonists/inverse agonists. Compound 3, bearing a 5-((2-methoxyphenyl) methylamino) group and a phenyl moiety at position 2, displayed the highest affinity (hA1 Ki = 10.2 nM; hA2A Ki = 4.72 nM) and behaved as a potent A1/A2A antagonist/inverse agonist (hA1 IC50 = 13.4 nM; hA2A IC50 = 5.34 nM).

Molecular dissection of the human A3 adenosine receptor coupling with β-arrestin2

Besides classical G protein coupling, G protein-coupled receptors (GPCRs) are nowadays well known to show significant signalling via other adaptor proteins, such as β-arrestin2 (βarr2). The elucidation of the molecular mechanism of the GPCR-βarr2 interaction is a prerequisite for the structure-activity based design of biased ligands, which introduces a new chapter in drug discovery. The general mechanism of the interaction is believed to rely on phosphorylation sites, exposed upon agonist binding. However, it is not known whether this mechanism is universal throughout the GPCR family or if GPCR-specific patterns are involved. In recent years, promising orally active agonists for the human A3 adenosine receptor (A3AR), a GPCR highly expressed in inflammatory and cancer cells, have been evaluated in clinical trials for the treatment of rheumatoid arthritis, psoriasis, and hepatocellular carcinoma. In this study, the effect of cytoplasmic modifications of the A3AR on βarr2 recruitment was evaluated in transiently transfected HEK293T cells, using a live-cell split-reporter system (NanoBit®, Promega), based on the structural complementation of NanoLuc luciferase, allowing real-time βarr2 monitoring. The A3AR-selective reference agonist 2-Cl-IB-MECA yielded a robust, concentration dependent (5 nM–1 µM) recruitment of βarr2 (logEC50: −7.798 ± 0.076). The role of putative phosphorylation sites, located in the C-terminal part and cytoplasmic loops, and the role of the ‘DRY’ motif was evaluated. It was shown that the A3AR C-terminus was dispensable for βarr2 recruitment. This contrasts with studies in the past for the rat A3AR, which pointed at crucial C-terminal phosphorylation sites. When combining truncation of the A3AR with modification of the ‘DRY’ motif to ‘AAY’, the βarr2 recruitment was drastically reduced. Recruitment could be partly rescued by back-mutation to ‘NQY’, or by extending the C-terminus again. In conclusion, other parts of the human A3AR, either cytosolic or exposed upon receptor activation, rather than the C-terminus alone, are responsible for βarr2 recruitment in a complementary or synergistic way.

Studies on enantioselectivity of chiral 4-acetylamino-6-alkyloxy-2-alkylthiopyrimidines acting as antagonists of the human A3 adenosine receptor.

Three A3 adenosine receptor (AR) antagonists (1-3) selected from 4-acylamino-6-alkyloxy-2-alkylthiopyrimidines previously investigated by us were modified by inserting a methyl group on their ether or thioether side chains. These compounds gave us the chance to evaluate whether their higher lipophilicity, reduced conformational freedom and chirality might improve the potency towards the A3 AR. Racemic mixtures of 1-3 were resolved using chiral HPLC methods and the absolute configurations of the enantiomers were assigned by chiroptical spectroscopy and density functional theory calculations. We measured the affinity for human A1, A2A, A2B and A3 ARs of the racemic mixtures and the pure enantiomers of 1-3 by radioligand competition binding experiments. Cell-based assays of the most potent enantiomers confirmed their A3 AR antagonist profiles. Our research led to the identification of (S)-1 with high potency (0.5 nM) and selectivity as an A3 AR antagonist. Moreover we built a docking-model useful to design new pyrimidine derivatives.

Determination and validation of LJ-2698, a potent human A3 adenosine receptor antagonist, in rat plasma by liquid chromatography-tandem massspectrometry and its application in pharmacokinetic study.

LJ-2698, a highly potent human A3 adenosine receptor antagonist with nucleoside structure, was designed to have a minimal species dependence. For further pre-clinical studies, analytical method for the detection of LJ-2698 in rat plasma was developed by liquid chromatography-tandem mass. Plasma samples were processed by protein precipitation method with acetonitrile, using losartan as the internal standard (IS). Chromatographic separation was carried out using a Kinetex C18 column (100×4.6mm; 100 Å; 2.6 μ) with acetonitrile/water with 0.2% (v/v) formic acid (65:35, v/v) in the isocratic mode at a flow rate of 0.4mL/min. Mass spectrometric detection in multiple reaction monitoring mode was performed with positive electrospray ionization. The mass transitions of LJ-2698 and IS were m/z 412.3→294.1 and m/z 423.1→207.2, respectively. The calibration curves were linear in the range 5.00-5000ng/mL (r 2≥0.998). The lower limit of quantification was established as 5.00ng/mL. Within- and between-run precisions were<7.01%, as relative standard deviation; and accuracies were in the range 3.37-3.64%, as relative error. The validated method was successfully applied to its pharmacokinetic evaluation after intravenous and oral administration in rats, and the dose-dependent pharmacokinetic behavior of LJ-2698 was elucidated for the first time.

The role of 5-arylalkylamino- and 5-piperazino- moieties on the 7-aminopyrazolo[4,3-d]pyrimidine core in affecting adenosine A1 and A2A receptor affinity and selectivity profiles

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 A1 and/or A2A receptor subtypes. On the whole, the novel derivatives 1–24 shared scarce or no affinities for the off-target hA2B and hA3 ARs. The 5-(4-hydroxyphenethylamino)- derivative 12 showed both good affinity (Ki = 150 nM) and the best selectivity for the hA2A AR while the 5-benzylamino-substituted 5 displayed the best combined hA2A (Ki = 123 nM) and A1 AR affinity (Ki = 25 nM). The 5-phenethylamino moiety (compound 6) achieved nanomolar affinity (Ki = 11 nM) and good selectivity for the hA1 AR. The 5-(N4-substituted-piperazin-1-yl) derivatives 15–24 bind the hA1 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.

Synthesis and evaluation of N-substituted 2-amino-4,5-diarylpyrimidines as selective adenosine A1 receptor antagonists

We report the synthesis and biological evaluation of new 2-amino-4,5-diarylpyrimidines as selective antagonists at the adenosine A1 receptor. The scaffold they are based upon is a deaza variation of a previously reported collection of 3-amino-5,6-diaryl-1,2,4-triazines, members of which had a subnanomolar affinity but limited selectivity over the A2A subtype. Initially, similar structure-affinity relationships at the 5-aryl ring were established, and then emphasis was put on increasing selectivity at the hA1AR by introducing substituents on the N2-position, all the while maintaining a nanomolar affinity. Compound 3z, bearing a trans 4-hydroxycyclohexyl substituent, was identified as a potent (Ki(hA1AR) = 7.7 nM) and selective (Ki(hA2AAR) = 1389 nM) antagonist at the human adenosine A1 receptor. Computational docking was effected at the A1 and A2A subtypes, rationalizing the effect of the 4-hydroxycyclohexyl substituent on selectivity, in relation with the nature of the substituent on the 5-position of the pyrimidine.

Scintillation proximity assay (SPA) as a new approach to determine a ligand's kinetic profile. A case in point for the adenosine A1 receptor.

Scintillation proximity assay (SPA) is a radio-isotopic technology format used to measure a wide range of biological interactions, including drug-target binding affinity studies. The assay is homogeneous in nature, as it relies on a “mix and measure” format. It does not involve a filtration step to separate bound from free ligand as is the case in a traditional receptor-binding assay. For G protein-coupled receptors (GPCRs), it has been shown that optimal binding kinetics, next to a high affinity of a ligand, can result in more desirable pharmacological profiles. However, traditional techniques to assess kinetic parameters tend to be cumbersome and laborious. We thus aimed to evaluate whether SPA can be an alternative platform for real-time receptor-binding kinetic measurements on GPCRs. To do so, we first validated the SPA technology for equilibrium binding studies on a prototypic class A GPCR, the human adenosine A1 receptor (hA1R). Differently to classic kinetic studies, the SPA technology allowed us to study binding kinetic processes almost real time, which is impossible in the filtration assay. To demonstrate the reliability of this technology for kinetic purposes, we performed the so-called competition association experiments. The association and dissociation rate constants (kon and koff) of unlabeled hA1R ligands were reliably and quickly determined and agreed very well with the same parameters from a traditional filtration assay performed simultaneously. In conclusion, SPA is a very promising technique to determine the kinetic profile of the drug-target interaction. Its robustness and potential for high-throughput may render this technology a preferred choice for further kinetic studies.

Structural refinement of pyrazolo[4,3-d]pyrimidine derivatives to obtain highly potent and selective antagonists for the human A3 adenosine receptor

In previous research, we identified some 7-oxo- and 7-acylamino-substituted pyrazolo[4,3-d]pyrimidine derivatives as potent and selective human (h) A3 adenosine receptor (AR) antagonists.Herein we report on the structural refinement of this class of antagonists aimed at achieving improved receptor-ligand recognition. Hence, substituents with different steric bulk, flexibility and lipophilicity (Me, Ar, heteroaryl, CH2Ph) were introduced at the 5- and 2-positions of the bicyclic scaffold of both the 7-oxo and 7-amino derivatives, and acyl residues were appended on the 7-amino group of the latter. All the 2-phenylpyrazolo[4,3-d]pyrimidin-7-amines and 7-acylamines bearing a 4-methoxyphenyl- or a 2-thienyl group at the 5-position showed high hA3 affinity and selectivity. In particular, the 2-phenyl-5-(2-thienyl)-pyrazolo[4,3-d]pyrimidin-7-(4-methoxybenzoyl)amine 25 (Ki = 0.027 nM) is one of the most potent and selective hA3 antagonists reported so far. By using an in silico receptor-driven approach the obtained binding data were rationalized and the molecular bases of the observed hA3 AR affinities were critically described.