of rigidified A3 adenosine receptor (AR)
agonists with a 2-((5-chlorothiophen-2-yl)ethynyl) or a 2-(4-(5-chlorothiophen-2-yl)-1H-1,2,3-triazol-1-yl) group provides prolonged protection
in a model of chronic neuropathic pain. These agonists contain a bicyclo[3.1.0]hexane
((N)-methanocarba) ring system in place of ribose,
which adopts a receptor-preferred conformation. N6-Small alkyl derivatives were newly optimized for A3AR affinity and the effects of a 1-deaza-adenine modification
probed. 1-Deaza-N6-ethyl alkyne 20 (MRS7144, Ki 1.7 nM) and 1-aza N6-propyl alkyne 12 (MRS7154, Ki 1.1 nM) were highly efficacious in vivo. Thus,
the presence of N1 is not required for nanomolar binding affinity
or potent, long-lasting functional activity. Docking of 1-deaza compounds
to a receptor homology model confirmed a similar binding mode as previously
reported 1-aza derivatives. This is the first demonstration in nonribose
adenosine analogues that the 1-deaza modification can maintain high
A3AR affinity, selectivity, and efficacy.
G protein-coupled receptor; purines; chronic
neuropathic pain; molecular modeling; adenosine
receptor; crystallographic structure
2-Arylethynyl derivatives of (N)-methanocarba adenosine 5′-uronamides are selective A3AR (adenosine receptor) agonists. Here we substitute a 1,2,3-triazol-1-yl linker in place of the rigid, linear ethynyl group to eliminate its potential metabolic liability. Docking of nucleosides containing possible short linker moieties at the adenine C2 position using a hybrid molecular model of the A3AR (based on the A2AAR agonist-bound structure) correctly predicted that a triazole would maintain the A3AR selectivity, due to its ability to fit a narrow cleft in the receptor. The analogues with various N6 and C2-aryltriazolyl substitution were synthesized and characterized in binding (Ki at hA3AR 0.3 – 12 nM) and in vivo to demonstrate efficacy in controlling chronic neuropathic pain (chronic constriction injury). Among N6-methyl derivatives, a terminal pyrimidin-2-yl group in 9 (MRS7116) increased duration of action (36% pain protection at 3 h) in vivo. N6-Ethyl 5-chlorothien-2-yl analogue 15 (MRS7126) preserved in vivo efficacy (85% protection at 1 h) with short duration. Larger N6 groups, e.g. 17 (MRS7138, >90% protection at 1 and 3 h), greatly enhanced in vivo activity. Thus, we have combined structure-based methods and phenotypic screening to identify nucleoside derivatives having translational potential.
G protein-coupled receptor; nucleoside; click chemistry; neuropathic pain; molecular modeling
adenosine 5′-methyluronamides containing 2-arylethynyl groups
were synthesized as A3 adenosine receptor (AR) agonists
and screened in vivo (po) for reduction of neuropathic pain. A small N6-methyl group maintained binding affinity,
with human > mouse A3AR and MW < 500 and other favorable
physicochemical properties. Emax (maximal
efficacy in a mouse chronic constriction injury pain model) of previously
characterized A3AR agonist, 2-(3,4-difluorophenylethynyl)-N6-(3-chlorobenzyl) derivative 6a, MRS5698, was surpassed. More efficacious analogues (in vivo) contained
the following C2-arylethynyl groups: pyrazin-2-yl 23 (binding Ki, hA3AR, nM 1.8), fur-2-yl 27 (0.6), thien-2-yl 32 (0.6) and its 5-chloro 33, MRS5980 (0.7) and 5-bromo 34 (0.4) equivalents, and physiologically unstable ferrocene 36, MRS5979 (2.7). 33 and 36 displayed
particularly long in vivo duration (>3 h). Selected analogues were
docked to an A3AR homology model to explore the environment
of receptor-bound C2 and N6 groups. Various analogues bound with μM affinity at off-target
biogenic amine (M2, 5HT2A, β3, 5HT2B, 5HT2C, and α2C) or other receptors.
Thus, we have expanded the structural range of orally active A3AR agonists for chronic pain treatment.
(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 N6-p-sulfo-phenylethyl 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. N6-3-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 i.p. reduced mouse chronic neuropathic pain that was ascribed to either A3 or A1/A3ARs 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.
Molecular modeling; G protein-coupled receptor; neuropathic pain; purines; radioligand binding; 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 N6 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 38 min in mA3AR-HEK293 cells; 26.4 min in sucrose-treated hA3AR-CHO cells (Kd 31 nM). Membrane binding indicated moderate mA3AR affinity, but not selectivity. Specific accumulation of fluorescence (50 nM 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.
purines; fluorescence; G protein-coupled receptor; A3 adenosine receptor; flow cytometry
G protein coupled receptors play crucial roles in mediating cellular responses to external stimuli, and increasing evidence suggests that they function as multiple units comprising homo/heterodimers and hetero-oligomers. Adenosine and β-adrenergic receptors are co-expressed in numerous tissues and mediate important cellular responses to the autocoid adenosine and sympathetic stimulation, respectively. The present study was undertaken to examine whether adenosine A1ARs heterodimerize with β1-and/or β2-adrenergic receptors (β1R and β2R), and whether such interactions lead to functional consequences. Co-immunoprecipitation and co-localization studies with differentially epitope-tagged A1, β1, and β2 receptors transiently co-expressed in HEK-293 cells indicate that A1AR forms constitutive heterodimers with both β1R and β2R. This heterodimerization significantly influenced orthosteric ligand binding affinity of both β1R and β2R without altering ligand binding properties of A1AR. Receptor-mediated ERK1/2 phosphorylation significantly increased in cells expressing A1AR/β1R and A1AR/β2R heteromers. β-receptor-mediated cAMP production was not altered in A1AR/β1R expressing cells, but was significantly reduced in the A1AR/β2R cells. The inhibitory effect of the A1AR on cAMP production was abrogated in both A1AR/β1R and A1AR/β2R expressing cells in response to the A1AR agonist CCPA. Co-immunoprecipitation studies conducted with human heart tissue lysates indicate that endogenous A1AR, β1R, and β2R also form heterodimers. Taken together, our data suggest that heterodimerization between A1 and β receptors leads to altered receptor pharmacology, functional coupling, and intracellular signaling pathways. Unique and differential receptor cross-talk between these two important receptor families may offer the opportunity to fine-tune crucial signaling responses and development of more specific therapeutic interventions.
G protein coupled receptors; heterodimerization; adenosine receptor; β-adrenergic receptors
(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 receptor; purines; molecular modeling; structure activity relationship; radioligand binding; adenylate cyclase
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.
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.
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.
Adenosine is released from injured or hypoxic tissues where it exerts numerous anti-inflammatory effects including suppression of neutrophil functions. Although most previous work has implicated the A2AAR, we have recently shown that selective activation of the abundantly expressed A3AR inhibits neutrophil superoxide production and chemotaxis providing a potential mechanistic explanation for the efficacy of A3AR agonists in experimental animal models of inflammation. In this study, we hypothesized that the A3AR suppresses neutrophil functions by inhibiting the monomeric GTPase Rac, a central regulator of chemokine-directed neutrophil migration and superoxide production. We found that pre-treating neutrophils with the highly selective A3AR agonist CP-532,903 reduced fMLP-induced Rac activation using an ELISA-based assay that detects all three Rac isoforms. CP-532,903 also inhibited fMLP-induced F-actin formation, a downstream effector function of Rac relevant to neutrophil migration, but not activation of ERK1/2 or p38. Pre-treating neutrophils with CP-532,903 did not stimulate cAMP production or alter fMLP-induced calcium transients, implicating that A3AR stimulation does not inhibit Rac activation or neutrophil activities by suppressing Ca2+ signaling, elevating the intracellular concentration of cAMP, or by cross-desensitizing fMLP receptors. Our results suggest that activation of the A3AR signals to suppress neutrophil functions by interfering with the monomeric GTPase Rac, thus contributing to the ant-inflammatory actions of adenosine.
adenosine; adenosine receptors; neutrophils; inflammation; Rac; cell signaling
A recently reported selective agonist of the human A3 adenosine receptor (hA3AR), MRS5127, (1′R,2′R,3′S,4′R,5′S)-4′-[2-chloro-6-(3-iodobenzylamino)-purine]-2′,3′-O-dihydroxy-bicyclo-[3.1.0]hexane, was radioiodinated and characterized pharmacologically. It contains a rigid bicyclic ring system in place of a 5′-truncated ribose moiety, and was selected for radiolabeling due to its nanomolar binding affinity at both human and rat A3ARs. The radioiodination of the N6-3-iodobenzyl substituent by iododestannylation of a 3- (trimethylstannyl)benzyl precursor was achieved in 73% yield, measured after purification by HPLC. [125I]MRS5127 bound to the human A3AR expressed in membranes of stably transfected HEK 293 cells. Specific binding was saturable, competitive, and followed a one-site binding model, with a Kd value of 5.74 ± 0.97 nM. At a concentration equivalent to its Kd, non-specific binding comprised 27±2% of total binding. In kinetic studies, [125I]MRS5127 rapidly associated with the hA3AR (t1/2 = 0.514 ± 0.014 min), and the affinity calculated from association and dissociation rate constants was 3.50 ± 1.46 nM. The pharmacological profile of ligands in competition experiments with [125I]MRS5127 was consistent with the known structure-activity-relationship profile of the hA3AR. [125I]MRS5127 bound with similar high affinity (Kd, nM) to recombinant A3ARs from mouse (4.90 ± 0.77), rabbit (2.53 ± 0.11), and dog (3.35 ± 0.54). For all of the species tested, MRS5127 exhibited A3AR agonist activity based on negative coupling to cAMP production. Thus, [125I]MRS5127 represents a new species-independent agonist radioligand for the A3AR. The major advantage of [125I]MRS5127 compared with previously used A3AR radioligands is its high affinity, low degree of non-specific binding, and improved A3AR selectivity.
nucleoside; G protein-coupled receptor; adenosine receptor; radioligand binding
2-Chloro-5′ -N-methylcarboxamidoadenosine analogues containing the (N)-methanocarba (bicyclo[3.1.0]hexane) ring system as a ribose substitute display increased selectivity as agonists of the human A3 adenosine receptor (AR). However, the selectivity in mouse was greatly reduced due to an increased tolerance of this ring system at the mouse A1AR. Therefore, we varied substituents at the N6 and C2 positions in search of compounds that have improved A3AR selectivity and are species independent. An N6-methyl analogue was balanced in affinity at mouse A1/A3ARs, with high selectivity in comparison to the A2AAR. Substitution of the 2-chloro atom with larger and more hydrophobic substituents, such as iodo and alkynyl groups, tended to increase the A3AR selectivity (up to 430-fold) in mouse and preserve it in human. Extended and chemically functionalized alkynyl chains attached at the C2 position of the purine moiety preserved A3AR selectivity more effectively than similar chains attached at the 3 position of the N6–benzyl group.
nucleoside; G protein-coupled receptor; mouse; adenosine receptor; radioligand binding