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 7.5 nM, respectively). Among the boron cluster-containing compounds, the highest A3 affinity (Ki 206 nM) 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.

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.

Structural Characterization of Agonist Binding to an A3 Adenosine Receptor through Biomolecular Simulations and Mutagenesis Experiments.

The adenosine A3 receptor (A3R) binds adenosine and is a drug target against cancer cell proliferation. Currently, there is no experimental structure of A3R. Here, we have generated a molecular model of A3R in complex with two agonists, the nonselective 1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-ß-d-ribofuranuronamide (NECA) and the selective 1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-ß-d-ribofuranuronamide (IB-MECA). Molecular dynamics simulations of the wild-type A3R in complex with both agonists, combined with in vitro mutagenic studies revealed important residues for binding. Further, molecular mechanics-generalized Born surface area calculations were able to distinguish mutations that reduce or negate agonistic activity from those that maintained or increased the activity. Our studies reveal that selectivity of IB-MECA toward A3R requires not only direct interactions with residues within the orthosteric binding area but also with remote residues. Although V1695.30 is considered to be a selectivity filter for A3R binders, when it was mutated to glutamic acid or alanine, the activity of IB-MECA increased by making new van der Waals contacts with TM5. This result may have implications in the design of new A3R agonists.

Probing structure-activity relationship in ß-arrestin2 recruitment of diversely substituted adenosine derivatives.

In the adenosine receptor (AR) subfamily of G protein-coupled receptors (GPCRs), biased agonism has been described for the human A1AR, A2BAR and A3AR. While diverse A3AR agonists have been evaluated for receptor binding and Gi-mediated cAMP signalling, the ß-arrestin2 (ßarr2) pathway has been left largely unexplored. We screened nineteen diverse adenosine derivatives for ßarr2 recruitment using a stable hA3AR-NanoBit®-ßarr2 HEK293T cell line. Their activity profiles were compared with a cAMP accumulation assay in stable hA3AR CHO cells. Structural features linked to ßarr2 activation were further investigated by the evaluation of an additional ten A3AR ligands. The A3AR-selective reference agonist 2-Cl-IB-MECA, which is a full agonist in terms of cAMP inhibition, only showed partial agonist behaviour in ßarr2 recruitment. Highly A3AR-selective (N)-methanocarba 5'-uronamide adenosine derivatives displayed higher potency in both cAMP signalling and ßarr2 recruitment than reference agonists NECA and 2-Cl-IB-MECA. Their A3AR-preferred conformation tolerates C2-position substitutions, for increased ßarr2 efficacy, better than the flexible scaffolds of ribose derivatives. The different amino functionalities in the adenosine scaffold of these derivatives each seem to be important for signalling as well. In conclusion, we have provided insights into ligand features that can help to guide the future therapeutic development of biased A3AR ligands with respect to G-protein and ßarr2 signalling.

A binding kinetics study of human adenosine A3 receptor agonists.

The human adenosine A3 (hA3) receptor has been suggested as a viable drug target in inflammatory diseases and in cancer. So far, a number of selective hA3 receptor agonists (e.g. IB-MECA and 2-Cl-IB-MECA) inducing anti-inflammatory or anticancer effects are under clinical investigation. Drug-target binding kinetics is increasingly recognized as another pharmacological parameter, next to affinity, for compound triage in the early phases of drug discovery. However, such a kinetics-driven analysis has not yet been performed for the hA3 receptor. In this study, we first validated a competition association assay for adenosine A3 receptor agonists to determine the target interaction kinetics. Affinities and Kinetic Rate Index (KRI) values of 11 ribofurano and 10 methanocarba nucleosides were determined in radioligand binding assays. Afterwards, 15 analogues were further selected (KRI <0.70 or KRI >1.35) for full kinetics characterization. The structure-kinetics relationships (SKR) were derived and longer residence times were associated with methanocarba and enlarged adenine N6 and C2 substitutions. In addition, from a kon-koff-KD kinetic map we divided the agonists into three subgroups. A residence time "cliff" was observed, which might be relevant to (N)-methanocarba derivatives' rigid C2-arylalkynyl substitutions. Our findings provide substantial evidence that, next to affinity, additional knowledge of binding kinetics is useful for developing and selecting new hA3R agonists in the early phase of the drug discovery process.

Role and Function of A2A and A3 Adenosine Receptors in Patients with Ankylosing Spondylitis, Psoriatic Arthritis and Rheumatoid Arthritis.

Rheumatoid arthritis (RA), ankylosing spondylitis (AS) and psoriatic arthritis (PsA) are chronic inflammatory rheumatic diseases that affect joints, causing debilitating pain and disability. Adenosine receptors (ARs) play a key role in the mechanism of inflammation, and the activation of A2A and A3AR subtypes is often associated with a reduction of the inflammatory status. The aim of this study was to investigate the involvement of ARs in patients suffering from early-RA (ERA), RA, AS and PsA. Messenger RNA (mRNA) analysis and saturation binding experiments indicated an upregulation of A2A and A3ARs in lymphocytes obtained from patients when compared with healthy subjects. A2A and A3AR agonists inhibited nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) activation and reduced inflammatory cytokines release, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß and IL-6. Moreover, A2A and A3AR activation mediated a reduction of metalloproteinases (MMP)-1 and MMP-3. The effect of the agonists was abrogated by selective antagonists demonstrating the direct involvement of these receptor subtypes. Taken together, these data confirmed the involvement of ARs in chronic autoimmune rheumatic diseases highlighting the possibility to exploit A2A and A3ARs as therapeutic targets, with the aim to limit the inflammatory responses usually associated with RA, AS and PsA.

Selectivity is species-dependent: Characterization of standard agonists and antagonists at human, rat, and mouse adenosine receptors.

Adenosine receptors (ARs) have emerged as new drug targets. The majority of data on affinity/potency and selectivity of AR ligands described in the literature has been obtained for the human species. However, preclinical studies are mostly performed in mouse or rat, and standard AR agonists and antagonists are frequently used for studies in rodents without knowing their selectivity in the investigated species. In the present study, we selected a set of frequently used standard AR ligands, 8 agonists and 16 antagonists, and investigated them in radioligand binding studies at all four AR subtypes, A1, A2A, A2B, and A3, of three species, human, rat, and mouse. Recommended, selective agonists include CCPA (for A1AR of rat and mouse), CGS-21680 (for A2A AR of rat), and Cl-IB-MECA (for A3AR of all three species). The functionally selective partial A2B agonist BAY60-6583 was found to additionally bind to A1 and A3AR and act as an antagonist at both receptor subtypes. The antagonists PSB-36 (A1), preladenant (A2A), and PSB-603 (A2B) displayed high selectivity in all three investigated species. MRS-1523 acts as a selective A3AR antagonist in human and rat, but is only moderately selective in mouse. The comprehensive data presented herein provide a solid basis for selecting suitable AR ligands for biological studies.

Metabolic mapping of A3 adenosine receptor agonist MRS5980.

(1S,2R,3S,4R,5S)-4-(2-((5-Chlorothiophen-2-yl)ethynyl)-6-(methylamino)-9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo[3.1.0]hexane-1-carboxamide (MRS5980) is an A3AR selective agonist containing multiple receptor affinity- and selectivity-enhancing modifications and a therapeutic candidate drug for many inflammatory diseases. Metabolism-related poor pharmacokinetic behavior and toxicities are a major reason for drug R&D failure. Metabolomics with UPLC-MS was employed to profile the metabolism of MRS5980 and MRS5980-induced disruption of endogenous compounds. Recombinant drug-metabolizing enzymes screening experiment were used to determine the enzymes involved in MRS5980 metabolism. Analysis of lipid metabolism-related genes was performed to investigate the reason for MRS5980-induced lipid metabolic disorders. Unsupervised principal components analysis separated the control and MRS5980 treatment groups in feces, urine, and liver samples, but not in bile and serum. The major ions mainly contributing to the separation of feces and urine were oxidized MRS5980, glutathione (GSH) conjugates and cysteine conjugate (degradation product of the GSH conjugates) of MRS5980. The major ions contributing to the group separation of liver samples were phosphatidylcholines. In vitro incubation experiments showed the involvement of CYP3A enzymes in the oxidative metabolism of MRS5980 and direct GSH reactivity of MRS5980. The electrophilic attack by MRS5980 is a minor pathway and did not alter GSH levels in liver or liver histology, and thus may be of minor clinical consequence. Gene expression analysis further showed decreased expression of PC biosynthetic genes choline kinase a and b, which further accelerated conversion of lysophosphatidylcholine to phosphatidylcholines through increasing the expression of lysophosphatidylcholine acyltransferase 3. These data will be useful to guide rational design of drugs targeting A3AR, considering efficacy, metabolic elimination, and electrophilic reactivity.

Synthesis and evaluation of N6-substituted apioadenosines as potential adenosine A3 receptor modulators.

Adenosine receptors (ARs) trigger signal transduction pathways inside the cell when activated by extracellular adenosine. Selective modulation of the A3AR subtype may be beneficial in controlling diseases such as colorectal cancer and rheumatoid arthritis. Here, we report the synthesis and evaluation of ß-D-apio-D-furano- and α-D-apio-L-furanoadenosines and derivatives thereof. Introduction of a 2-methoxy-5-chlorobenzyl group at N(6) of ß-D-apio-D-furanoadenosine afforded an A3AR antagonist (10c, Ki=0.98 µM), while a similar modification of an α-D-apio-L-furanoadenosine gave rise to a partial agonist (11c, Ki=3.07 µM). The structural basis for this difference was examined by docking to an A3AR model; the antagonist lacked a crucial interaction with Thr94.

Rational design of sulfonated A3 adenosine receptor-selective nucleosides as pharmacological tools to study chronic neuropathic pain.

(N)-Methanocarba(bicyclo[3.1.0]hexane)adenosine derivatives were probed for sites of charged sulfonate substitution, which precludes diffusion across biological membranes, e.g., blood-brain barrier. Molecular modeling predicted that sulfonate groups on C2-phenylethynyl substituents would provide high affinity at both mouse (m) and human (h) A3 adenosine receptors (ARs), while a N(6)-p-sulfophenylethyl substituent would determine higher hA3AR vs mA3AR affinity. These modeling predictions, based on steric fitting of the binding cavity and crucial interactions with key residues, were confirmed by binding/efficacy studies of synthesized sulfonates. N(6)-3-Chlorobenzyl-2-(3-sulfophenylethynyl) derivative 7 (MRS5841) bound selectively to h/m A3ARs (Ki(hA3AR) = 1.9 nM) as agonist, while corresponding p-sulfo isomer 6 (MRS5701) displayed mixed A1/A3AR agonism. Both nucleosides administered ip reduced mouse chronic neuropathic pain that was ascribed to either A3AR or A1/A3AR using A3AR genetic deletion. Thus, rational design methods based on A3AR homology models successfully predicted sites for sulfonate incorporation, for delineating adenosine's CNS vs peripheral actions.

Characterization by flow cytometry of fluorescent, selective agonist probes of the A(3) adenosine receptor.

Various fluorescent nucleoside agonists of the A3 adenosine receptor (AR) were compared as high affinity probes using radioligands and flow cytometry (FCM). They contained a fluorophore linked through the C2 or N(6) position and rigid A3AR-enhancing (N)-methanocarba modification. A hydrophobic C2-(1-pyrenyl) derivative MRS5704 bound nonselectively. C2-Tethered cyanine5-dye labeled MRS5218 bound selectively to hA3AR expressed in whole CHO cells and membranes. By FCM, binding was A3AR-mediated (blocked by A3AR antagonist, at least half through internalization), with t1/2 for association 38min in mA3AR-HEK293 cells; 26.4min in sucrose-treated hA3AR-CHO cells (Kd 31nM). Membrane binding indicated moderate mA3AR affinity, but not selectivity. Specific accumulation of fluorescence (50nM MRS5218) occurred in cells expressing mA3AR, but not other mouse ARs. Evidence was provided suggesting that MRS5218 detects endogenous expression of the A3AR in the human promyelocytic leukemic HL-60 cell line. Therefore, MRS5218 promises to be a useful tool for characterizing the A3AR.

Polyamidoamine (PAMAM) dendrimer conjugate specifically activates the A3 adenosine receptor to improve post-ischemic/reperfusion function in isolated mouse hearts.

BACKGROUND: When stimulated by small molecular agonists, the A3 adenosine receptor (AR) mediates cardioprotective effects without inducing detrimental hemodynamic side effects. We have examined pharmacologically the protective properties of a multivalent dendrimeric conjugate of a nucleoside as a selective multivalent agonist for the mouse A3AR. RESULTS: A PAMAM dendrimer fully substituted by click chemistry on its peripheral groups with 64 moieties of a nucleoside agonist was shown to be potent and selective in binding to the mouse A3AR and effective in cardioprotection in an isolated mouse heart model of ischemia/reperfusion (I/R) injury. This conjugate MRS5246 and a structurally related model compound MRS5233 displayed binding Ki values of 0.04 and 3.94 nM, respectively, and were potent in in vitro functional assays to inhibit cAMP production. A methanocarba (bicyclo[3.1.0]hexane) ring system in place of ribose maintained a North conformation that is preferred at the A3AR. These analogues also contained a triazole linker along with 5'-N-methyl-carboxamido and 2-alkynyl substitution, previously shown to be associated with species-independent A3AR selectivity. Both MRS5233 and MRS5246 (1 and 10 nM) were effective at increasing functional recovery of isolated mouse hearts after 20 min ischemia followed by 45 min reperfusion. A statistically significant greater improvement in the left ventricular developed pressure (LVDP) by MRS5246 compared to MRS5233 occurred when the hearts were observed throughout reperfusion. Unliganded PAMAM dendrimer alone did not have any effect on functional recovery of isolated perfused mouse hearts. 10 nM MRS5246 did not improve functional recovery after I/R in hearts from A3AR gene "knock-out" (A3KO) mice compared to control, indicating the effects of MRS5246 were A3AR-specific. CONCLUSIONS: Covalent conjugation to a versatile drug carrier enhanced the functional potency and selectivity at the mouse A3AR and maintained the cardioprotective properties. Thus, this large molecular weight conjugate is not prevented from extravasation through the coronary microvasculature.