(N)-Methanocarba adenosine 5′-methyluronamides containing known A3 AR (adenosine receptor)-enhancing modifications, i.e. 2-(arylethynyl)adenine and N6-methyl or N6-(3-substituted-benzyl), were nanomolar full agonists of human (h) A3AR and highly selective (Ki ~0.6 nM, N6-methyl 2-(halophenylethynyl) analogues 13, 14). Combined 2-arylethynyl-N6-3-chlorobenzyl substitutions preserved A3AR affinity/selectivity in the (N)-methanocarba series (e.g. 3,4-difluoro full agonist MRS5698 31, Ki 3 nM, human and mouse A3) better than for ribosides. Polyaromatic 2-ethynyl N6-3-chlorobenzyl analogues, such as potent linearly extended 2-p-biphenylethynyl MRS5679 34 (Ki hA3 3.1 nM; A1, A2A: inactive) and fluorescent 1-pyrene adduct MRS5704 35 (Ki hA3 68.3 nM) were conformationally rigid; receptor docking identified a large, mainly hydrophobic binding region. The vicinity of receptor-bound C2 groups was probed by homology modeling based on recent X-ray structure of an agonist-bound A2AAR, with a predicted helical rearrangement requiring an agonist-specific outward displacement of TM2 resembling opsin. Thus, X-ray structure of related A2AAR is useful in guiding design of new A3AR agonists.

G protein-coupled receptors (GPCRs) are attractive targets for pharmaceutical research. With the recent determination of several GPCR X-ray structures, the applicability of structure-based computational methods for ligand identification, such as docking, has increased. Yet, as only about 1% of GPCRs have a known structure, receptor homology modeling remains necessary. In order to investigate the usability of homology models and the inherent selectivity of a particular model in relation to close homologs, we constructed multiple homology models for the A1 adenosine receptor (A1AR) and docked ∼2.2 M lead-like compounds. High-ranking molecules were tested on the A1AR as well as the close homologs A2AAR and A3AR. While the screen yielded numerous potent and novel ligands (hit rate 21% and highest affinity of 400 nM), it delivered few selective compounds. Moreover, most compounds appeared in the top ranks of only one model. These findings have implications for future screens.

A1 adenosine receptor (AR) agonists display antiischemic and antiepileptic neuroprotective activity, but peripheral cardiovascular side effects impeded their development. SAR study of N6-cycloalkylmethyl 4′-truncated (N)-methanocarba-adenosines identified 10 (MRS5474, N6-dicyclopropylmethyl, Ki 47.9 nM) as a moderately A1AR-selective full agonist. Two stereochemically defined N6-methynyl group substituents displayed narrow SAR; larger than cyclobutyl greatly reduced AR affinity, and larger or smaller than cyclopropyl reduced A1AR selectivity. Nucleoside docking to A1AR homology model characterized distinct hydrophobic cyclopropyl subpockets, the larger “A” forming contacts with Thr270 (7.35), Tyr271 (7.36), Ile274 (7.39) and carbon chains of glutamates (EL2), and smaller subpocket “B” between TM6 and TM7. 10 suppressed minimal clonic seizures (6 Hz mouse model) without typical rotarod impairment of A1AR agonists. Truncated nucleosides, an appealing preclinical approach, have more drug-like physicochemical properties than other A1AR agonists. Thus, we identified highly restricted regions for substitution around N6 suitable for an A1AR agonist with anticonvulsant activity.

C2-Arylethynyladenosine-5′-N-methyluronamides containing a bicyclo[3.1.0]hexane ((N)-methanocarba) ring are selective A3 adenosine receptor (AR) agonists. Similar 4′-truncated C2-arylethynyl-(N)-methanocarba nucleosides containing alkyl or alkylaryl groups at the N6 position were low-efficacy agonists or antagonists of the human A3AR with high selectivity. Higher hA3AR affinity was associated with N6-methyl and ethyl (Ki 3–6 nM), than with N6-arylalkyl groups. However, combined C2-phenylethynyl and N6-2-phenylethyl substitutions in selective antagonist 15 provided a Ki of 20 nM. Differences between 4′-truncated and nontruncated analogues of extended C2-p-biphenylethynyl substitution suggested a ligand reorientation in AR binding, dominated by bulky N6 groups in analogues lacking a stabilizing 5′-uronamide moiety. Thus, 4′-truncation of C2-arylethynyl-(N)-methanocarba adenosine derivatives is compatible with general preservation of A3AR selectivity, especially with small N6 groups, but reduced efficacy in A3AR-induced inhibition of adenylate cyclase.

The physiological role of the A3 adenosine receptor (AR) was explored in cardiac ischaemia, inflammatory diseases and cancer. We report a new fluorophore-conjugated human (h) A3AR antagonist for application to cell-based assays in ligand discovery and for receptor imaging. Fluorescent pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-ylamine (pyrazolo-triazolo-pyrimidine, PTP) and triazolo[1,5-c]quinazolin-5-yl)amine (triazolo-quinazoline, TQ) AR antagonists were compared. A chain-extended and click-conjugated Alexa Fluor-488 TQ derivative (MRS5449) displayed a radioligand binding Ki value of 6.4 ± 2.5 nM in hA3AR-expressing CHO cell membranes. MRS5449 antagonized hA3AR agonist-induced inhibition of cyclic AMP accumulation in a concentration-dependent manner (KB 4.8 nM). Using flow cytometry (FCM), MRS5449 saturated hA3ARs with very high specific-to-nonspecific binding ratio with an equilibrium binding constant 5.15 nM, comparable to the Kd value of 6.65 nM calculated from kinetic experiments. Ki values of known AR antagonists in inhibition of MRS5449 binding in whole cell FCM were consistent with radioligand binding in membranes, but agonist binding was 5–20 fold weaker than obtained with agonist radioligand [125I]I-AB-MECA. Further binding analysis of MRS5549 suggested multiple agonist binding states of the A3AR. Molecular docking predicted binding modes of these fluorescent antagonists. Thus, MRS5449 is a useful tool for hA3AR characterization.

A1 adenosine receptor (AR) agonists are neuroprotective, cardioprotective, and anxiolytic. (N)-Methanocarba adenine nucleosides designed to bind to human A1AR were truncated to eliminate 5′-CH2OH. This modification previously converted A3AR agonists into antagonists, but the comparable effect at A1AR is unknown. In comparison to ribosides, affinity at the A1AR was less well preserved than at the A3AR, although a few derivatives were moderately A1AR selective, notably full agonist 21 (N6-dicyclopropylmethyl, Ki 47.9 nM). Thus, at the A1AR recognition elements for nucleoside binding depend more on 5′region interactions, and in their absence A3AR selectivity predominates. Based on the recently reported agonist-bound AR structure, this difference between subtypes likely correlates with an essential His residue in transmembrane domain 6 of A1 but not A3AR. The derivatives ranged from partial to full agonists in A1AR-mediated adenylate cyclase inhibition. Truncated derivatives have more drug-like physical properties than other A1AR agonists; this approach is appealing for preclinical development.

Molecular modeling of agonist binding to the human A2A adenosine receptor (AR) was assessed and extended in light of crystallographic structures. Heterocyclic adenine nitrogens of co-crystallized agonist overlayed corresponding positions of the heterocyclic base of a bound triazolotriazine antagonist, and ribose moiety was coordinated in a hydrophilic region, as previously predicted based on modeling using the inactive receptor. Automatic agonist docking of 20 known potent nucleoside agonists to agonist-bound A2AAR crystallographic structures predicted new stabilizing protein interactions, to provide a structural basis for previous empirical structure activity relationships consistent with previous mutagenesis results. We predicted binding of novel C2 terminal amino acid conjugates of A2AAR agonist CGS21680 and used these models to interpret effects on binding affinity of newly-synthesized agonists. D-Amino acid conjugates were generally more potent than L- stereoisomers, and free terminal carboxylates more potent than corresponding methyl esters. Amino acid moieties were coordinated close to extracellular loops 2 and 3. Thus, molecular modeling is useful in probing ligand recognition and rational design of GPCR–targeting compounds with specific pharmacological profiles.

Structures of G protein-coupled receptors (GPCRs) have a proven utility in the discovery of new antagonists and inverse agonists modulating signaling of this important family of clinical targets. Applicability of active-state GPCR structures to virtual screening and rational optimization of agonists, however, remains to be assessed. In this study of adenosine 5′ derivatives, we evaluated the performance of an agonist-bound A2A adenosine receptor (AR) structure in retrieval of known agonists and then employed the structure to screen for new fragments optimally fitting the corresponding subpocket. Biochemical and functional assays demonstrate high affinity of new derivatives that include polar heterocycles. The binding models also explain modest selectivity gain for some substituents toward the closely related A1AR subtype and the modified agonist efficacy of some of these ligands. The study suggests further applicability of in silico fragment screening to rational lead optimization in GPCRs.