Extracellular ADP is known to play many important physiological roles. In this study, we identified the P2Y13 receptor in a rat mast cell line (RBL-2H3) and explored the functional role of ADP, its endogenous agonist. ADP induced both intracellular calcium mobilization and release of hexosaminidase (Hex). In an assay of intracellular calcium, ADP was 100-fold less potent than and equally efficacious as the P2Y1 receptor-selective agonist MRS2365. However, ADP was more potent and efficacious than MRS2365 in inducing Hex release and in enhancing antigen-induced Hex release. ADP-induced intracellular calcium mobilization was blocked by phospholipase C inhibitor U73122 and by P2Y1 receptor selective antagonist MRS2500, but not by pertussis toxin (PTX), suggesting a mechanism mediated by the Gq-coupled P2Y1 receptor, but not P2Y13 (Gi-coupled) or P2X receptors. ADP-induced Hex release was blocked by PTX and a selective P2Y13 receptor antagonist MRS2211, but not by MRS2500 or P2Y1 receptor-specific siRNA, suggesting a Gi-coupled P2Y13 receptor-related mechanism. Measurement of gene expression confirmed high expression of both P2Y1 and P2Y13 receptors (in comparison to a previously reported P2Y14 receptor) in RBL-2H3 cells. Thus, we demonstrated that ADP-mediated intracellular calcium mobilization and Hex release in RBL-2H3 cells are via P2Y1 and P2Y13 receptors, respectively. Selective antagonists of the P2Y13 receptor might be novel therapeutic agents for various allergic conditions.
P2Y13 receptor; nucleotide; mast cell; degranulation; allergy; G protein-coupled receptor
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.
G protein-coupled receptor; purines; molecular modeling; seizures; in vivo
The structure-activity relationship (SAR) for a novel class of 1,2,4-triazole antagonists of the human A2A adenosine receptor (hA2AAR) was explored. Thirty-three analogs of a ligand that was discovered in a structure-based virtual screen against the hA2AAR were tested in hA1, A2A, and A3 radioligand binding assays and in functional assays for the A2BAR subtype. As a series of closely related analogs of the initial lead, 1, did not display improved binding affinity or selectivity, molecular docking was used to guide the selection of more distantly related molecules. This resulted in the discovery of 32, a hA2AAR antagonist (Ki 200 nM) with high ligand efficiency. In the light of the SAR for the 1,2,4-triazole scaffold, we also investigated the binding mode of these compounds based on docking to several A2AAR crystal structures.
1,2,4-triazole; A2A adenosine receptor; antagonist; molecular docking; structure-activity relationship
The role of the A2B adenosine receptor (AR) in prostate cell death and growth was studied. The A2B AR gene expression quantified by real-time quantitative RT-PCR and Western blot analysis was the highest among four AR subtypes (A1, A2A, A2B, and A3) in all three commonly used prostate cancer cell lines, PC-3, DU145, and LNCaP. We explored the function of the A2B AR using PC-3 cells as a model. The A2B AR was visualized in PC-3 cells by laser confocal microscopy. The nonselective A2B AR agonist NECA and the selective A2B AR agonist BAY60-6583, but not the A2A AR agonist CGS21680, concentration-dependently induced adenosine 3′,5′-cyclic monophosphate (cyclic AMP) accumulation. NECA diminished lactate dehydrogenase (LDH) release, TNF-α-induced increase of caspase-3 activity, and cycloheximide (CHX)-induced morphological changes typical of apoptosis in PC-3 cells, which were blocked by a selective A2B AR antagonist PSB603. NECA-induced proliferation of PC-3 cells was diminished by siRNA specific for the A2B AR. The selective A2B AR antagonist PSB603 was shown to inhibit cell growth in all three cell lines. Thus, A2B AR blockade inhibits growth of prostate cancer cells, suggesting selective A2B AR antagonists as potential novel therapeutics.
Prostate cancer; Cancer; Adenosine receptor; A2B; G protein-coupled receptor (GPCR); Cell proliferation
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.
G protein-coupled receptor; purines; molecular modeling; structure activity relationship; radioligand binding; adenosine receptor
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.
purines; fluorescence; G protein-coupled receptor; A3 adenosine receptor; flow cytometry
(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
Gold nanoparticles (AuNPs) allow the tuning of pharmacokinetic and pharmacodynamic properties by active or passive targeting of drugs for cancer and other diseases. We have functionalized gold nanoparticles by tethering specific ligands, agonists and antagonists, of adenosine receptors (ARs) to the gold surface as models for cell surface interactions with G protein-coupled receptors (GPCRs). The AuNP conjugates with chain-extended AR ligands alone (PEGylated nucleosides and nonnucleosides, anchored to the Au via thioctic acid) were found to be insoluble in water due to hydrophobic entities in the ligand. Therefore, we added a second, biologically inactive pendant moiety to increase the water solubility, consisting of a PEGylated chain terminating in a carboxylic or phosphate group. The purity and stability of the immobilized biologically active ligand were examined by ultrafiltration and HPLC. Pharmacological receptor binding studies on these GPCR ligand-derivatized AuNPs (2–5 nm in diameter), performed using membranes of mammalian cells stably expressing human A1, A2A, and A3ARs, showed that the desired selectivity was retained with Ki values (nanomolar) of A3AR agonist 21b and A2AAR antagonists 24 and 26a of 14 (A3), 34 (A2A), and 69 (A2A), respectively. The corresponding monomers displayed Ki values of 37, 61, and 1,420 nM, respectively. In conclusion, we have synthesized stable, water-soluble AuNP derivatives of tethered A3 and A2AAR ligands that retain the biological properties of their monomeric ligands and are intended for therapeutic and imaging applications. This is the first prototypical application to gold carriers of small molecule (nonpeptide) GPCR ligands, which are under investigation for treatment of cancer and inflammatory diseases.
Electronic supplementary material
The online version of this article (doi:10.1007/s11302-012-9338-z) contains supplementary material, which is available to authorized users.
G protein-coupled receptor; Nanoparticle; Nucleoside; Adenosine; Radioligand binding
The objective of this study was to create constitutively active mutant human A3 adenosine receptors (ARs) using single amino acid replacements, based on findings from other G protein-coupled receptors. A3 ARs mutated in transmembrane helical domains (TMs) 1, 3, 6, and 7 were expressed in COS-7 cells and subjected to agonist radioligand binding and phospholipase C (PLC) and adenylyl cyclase (AC) assays. Three mutant receptors, A229E in TM6 and R108A and R108K in the DRY motif of TM3, were found to be constitutively active in both functional assays. The potency of the A3 agonist Cl-IB-MECA (2–chloro-N6-(3–iodobenzyl)adenosine-5′-N-methyluronamide) in PLC activation was enhanced by at least an order of magnitude over wild type (EC50 951 nM) in R108A and A229E mutant receptors. Cl-IB-MECA was much less potent (>10-fold) in C88F, Y109F and Y282F mutants or inactive following double mutation of the DRY motif. The degree of constitutive activation was more pronounced for the AC signaling pathway than for the PLC signaling pathway. The results indicated that specific locations within the TMs proximal to the cytosolic region were responsible for constraining the receptor in a G protein-uncoupled conformation.
purines; G protein-coupled receptor; phospholipase C; adenylyl cyclase; radioligand binding; nucleosides
Novel D- and L-4′-thioadenosine derivatives lacking the 4′-hydroxymethyl moiety were synthesized, starting from D-mannose and D-gulonic γ-lactone, respectively, as potent and selective species-independent A3 adenosine receptor (AR) antagonists. Among the novel 4′-truncated 2-H nucleosides tested, a N6-(3-chlorobenzyl) derivative 7c was the most potent at the human A3 AR (Ki = 1.5 nM), but a N6-(3-bromobenzyl) derivative 7d showed the optimal species-independent binding affinity.
Adenosine released during myocardial ischemia mediates cardioprotective preconditioning. Multivalent drugs covalently bound to nanocarriers may differ greatly in chemical and biological properties from the corresponding monomeric agents. Here, we conjugated chemically functionalized nucleosides to poly(amidoamine) (PAMAM) dendrimeric polymers and investigated their effects in rat primary cardiac cell cultures and in the isolated heart. Three conjugates of A3 adenosine receptor (AR) agonists, chain-functionalized at the C2 or N6 position, were cardioprotective, with greater potency than monomeric agonist Cl-IB-MECA. Multivalent amide-linked MRS5216 was selective for A1 and A3ARs, and triazole-linked MRS5246 and MRS5539 (optionally containing fluorescent label) were A3AR-selective. The conjugates protected ischemic rat cardiomyocytes, an effect blocked by an A3AR antagonist MRS1523, and isolated hearts with significantly improved infarct size, rate of pressure product, and rate of contraction and relaxation. Thus, strategically derivatized nucleosides tethered to biocompatible polymeric carriers display enhanced cardioprotective potency via activation of A3AR on the cardiomyocyte surface.
dendrimer; cardiomyocyte; adenosine receptor; ischemia; isolated heart; rat
The structure activity relationship (SAR) of 1,2,4-triazolo[1,5-a]-1,3,5-triazine derivatives related to ZM241385 as antagonists of the A2A adenosine receptor (AR) was explored through the synthesis of analogues substituted at the 5 position. The A2A AR X-ray structure was used to propose a structural basis for the activity and selectivity of the analogues and to direct the synthetic design strategy to provide access to solvent-exposed regions. Thus, we have identified a point of substitution for the attachment of solubilizing groups to enhance both aqueous solubility and physicochemical properties, maintaining potent interactions with the A2A AR and, in some cases, receptor subtype selectivity. Among the most potent and selective novel compounds were a long-chain ether-containing amine congener 20 (Ki 11.5 nM) and its urethane-protected derivative 14 (Ki 17.8 nM). Compounds 20 and 31 (Ki 11.5 and 16.9 nM, respectively) were readily water soluble up to 10 mM. The analogues were docked in the crystallographic structure of the hA2A AR and in a homology model of the hA3 AR, and the per residue electrostatic and hydrophobic contributions to the binding were assessed and stabilizing factors were proposed.
G protein-coupled receptor; purines; molecular modeling; structure activity relationship; radioligand binding; adenylyl cyclase
On the basis of potent and selective binding affinity of truncated 4′-thioadenosine derivatives at the human A3 adenosine receptor (AR), their bioisosteric 4′-oxo derivatives were designed and synthesized from commercially available 2,3-O-isopropylidene-d-erythrono lactone. The derivatives tested in AR binding assays were substituted at the C2 and N6 positions. All synthesized nucleosides exhibited potent and selective binding affinity at the human A3 AR. They were less potent than the corresponding 4′-thio analogues, but showed higher selectivity to other subtypes. The 2-Cl series generally were better than the 2-H series in view of binding affinity and selectivity. Among compounds tested, compound 5d (X = Cl, R = 3-bromobenzyl) showed the highest binding affinity (Ki = 13.0±6.9 nM) at the hA3 AR with high selectivity (at least 1000-fold) in comparison to other AR subtypes. Like the corresponding truncated 4′-thio series, compound 5d antagonized the action of an agonist to inhibit forskolin-stimulated adenylate cyclase in hA3 AR-expressing CHO cells. Although the 4′-oxo series were less potent than the 4′-thio series, this class of human A3 AR antagonists is also regarded as another good template for the design of A3 AR antagonists and for further drug development.
A3 Adenosine Receptor; Antagonists; Truncated Adenosine; Structure-Activity Relationships
Adenosine derivatives were modified with alkynyl groups on N6 substituents for linkage to carriers using Cu(I)-catalyzed click chemistry. Two parallel series, both containing a rigid North-methanocarba (bicyclo[3.1.0]hexane) ring system in place of ribose, behaved as A3 adenosine receptor (AR) agonists: (5′-methyluronamides) or partial agonists (4′-truncated). Terminal alkynyl groups on a chain at the 3 position of a N6-benzyl group or simply through a N6–propargyl group were coupled to azido derivatives, which included both small molecules and G4 (fourth-generation) multivalent poly(amidoamine) (PAMAM) dendrimers, to form 1,2,3-triazolyl linkers. The small molecular triazoles probed the tolerance in A3AR binding of distal, sterically bulky groups such as 1-adamantyl. Terminal 4-fluoro-3-nitrophenyl groups anticipated nucleophilic substitution for chain extension and 18F radiolabeling. N6-(4-Fluoro-3-nitrophenyl)-triazolylmethyl derivative 32 displayed a Ki of 9.1 nM at A3AR with ~1000-fold subtype selectivity. Multivalent conjugates additionally containing click-linked water-solubilizing polyethylene glycol groups potently activated A3AR in the 5′-methyluronamide, but not 4′ truncated series. N6-Benzyl nucleoside conjugate 43 (apparent Ki 24 nM) maintained binding affinity of the monomer better than a N6-triazolylmethyl derivative. Thus, the N6 region of 5′-methyluronamide derivatives, as modeled in receptor docking, is suitable for functionalization and tethering by click chemistry to achieve high A3AR agonist affinity and enhanced selectivity.
G protein-coupled receptor; PAMAM dendrimer; purines; structure activity relationship; molecular modeling; adenylate cyclase
Mast cell degranulation affects many conditions, e.g., asthma and urticaria. We explored the potential role of the P2Y14 receptor (P2Y14R) and other P2Y subtypes in degranulation of human LAD2 mast cells. All eight P2YRs were expressed at variable levels in LAD2 cells (quantitative real-time RT-PCR). Gene expression levels of ADP receptors, P2Y1R, P2Y12R, and P2Y13R, were similar, and P2Y11R and P2Y4R were highly expressed at 5.8- and 3.8-fold of P2Y1R, respectively. Least expressed P2Y2R was 40-fold lower than P2Y1R, and P2Y6R and P2Y14R were ≤50 % of P2Y1R. None of the native P2YR agonists alone induced β-hexosaminidase (β-Hex) release, but some nucleotides significantly enhanced β-Hex release induced by C3a or antigen, with a rank efficacy order of ATP > UDPG ≥ ADP >> UDP, UTP. Although P2Y11R and P2Y4R are highly expressed, they did not seem to play a major role in degranulation as neither P2Y4R agonist UTP nor P2Y11R agonists ATPγS and NF546 had a substantial effect. P2Y1R-selective agonist MRS2365 enhanced degranulation, but ~1,000-fold weaker compared to its P2Y1R potency, and the effect of P2Y6R agonist 3-phenacyl-UDP was negligible. The enhancement by ADP and ATP appears mediated via multiple receptors. Both UDPG and a synthetic agonist of the P2Y14R, MRS2690, enhanced C3a-induced β-Hex release, which was inhibited by a P2Y14R antagonist, specific P2Y14R siRNA and pertussis toxin, suggesting a role of P2Y14R activation in promoting human mast cell degranulation.
P2Y; Mast cells; Uracil nucleotide; Degranulation; GPCR; G protein-coupled receptors
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.
G protein-coupled receptor; nucleosides; purines; radioligand binding; docking; X-ray crystallography
Truncated N6-substituted-4′-oxo- and 4′-thioadenosine derivatives with C2 or C8 substitution were studied as dual acting A2A and A3 adenosine receptor (AR) ligands. The lithiation-mediated stannyl transfer and palladium-catalyzed cross coupling reactions were utilized for functionalization of the C2 position of 6-chloropurine nucleosides. An unsubstituted 6-amino group and a hydrophobic C2 substituent were required for high affinity at the hA2AAR, but hydrophobic C8 substitution abolished binding at the hA2AAR. However, most of synthesized compounds displayed medium to high binding affinity at the hA3AR, regardless of C2 or C8 substitution, and low efficacy in a functional cAMP assay. Several compounds tended to be full hA2AAR agonists. C2 substitution probed geometrically through hA2AAR-docking, was important for binding in order of hexynyl > hexenyl > hexanyl. Compound 4g was the most potent ligand acting dually as hA2AAR agonist and hA3AR antagonist, which might be useful for treatment of asthma or other inflammatory diseases.
lithiation-mediated stannyl transfer; structure-activity relationship; adenosine receptors; truncated adenosine; palladium-catalyzed cross coupling; dual-acting ligands
Adenosine receptors (ARs), the major targets of caffeine and theophylline, comprise four receptor subtypes designated as A1, A2A, A2B and A3. Over a dozen AR agonists are currently in clinical trials for various conditions, including cardiac arrhythmias, neuropathic pain, myocardial perfusion imaging, cardiac ischemia, inflammatory diseases and cancer. Adenosine (non-selective), regadenoson (A2A) and dipyridamole (act indirectly via ARs) have received regulatory approval for clinical use. The present editorial will give a brief update on the current status of AR agonists in clinical trials.
adenosine receptor; agonist; cardiac arrhythmia; cardiac perfusion imaging; cancer; G protein-coupled receptor; inflammation; nucleoside; pain; rheumatoid arthritis
The dedifferentiation agent ‘reversine’ (2-(4-morpholinoanilino)-N6-cyclohexyladenine 2) was found to be a moderately potent antagonist for the human A3 adenosine receptor (AR) with a Ki value 0.66 μM. This result prompted an exploration of the structure-activity relationship of related derivatives, synthesized via sequential substitution of 6-chloro-2-fluoropurine with selected nucleophiles. Optimization of substituents at these two positions identified 2-phenylamino-N6-(cyclohexyl)adenine 12, 2-phenylamino-N6-(cycloheptyl)adenine 19, and 2-phenylamino-N6-(endo-norbornyl)adenine 21 as potent A3 AR ligands with Ki values of 51, 42 and 37 nM, respectively, with 30 – 200-fold selectivity in comparison to A1 and A2A ARs. The most selective A3 AR antagonist (>200-fold) was 2-phenyloxy-N6-(cyclohexyl)adenine 22. 9-Methylation of 12, but not 19, was well tolerated in A3 AR binding. Extension of the 2-phenylamino group to 2-benzyl- and 2-(2-phenylethylamino) reduced affinity. In the series of 2-phenylamino, 2-phenyloxy, and 2-phenylthio substitutions, the order of affinity at the A3 AR was oxy ≥ amino > thio. Selected derivatives, including reversine (KB value of 466 nM in Schild analysis), competitively antagonized the functional effects of a selective A3 AR agonist, i.e. inhibition of forskolin-stimulated cAMP production in stably transfected Chinese hamster ovary (CHO) cells. These results are in agreement with other studies suggesting the presence of a lipophilic pocket in the AR binding site that is filled by moderately sized cycloalkyl rings at the N6 position of both adenine and adenosine derivatives. Thus, the compound series reported herein comprise an important new series of selective A3 AR antagonists. We were unable to reproduce the dedifferentiation effect of reversine, previously reported, or to demonstrate any connection between A3 AR antagonist effects and dedifferentiation.
An alternative approach to overcome the inherent lack of specificity of conventional agonist therapy can be the reengineering of the GPCRs and their agonists. A reengineered receptor (neoceptor) could be selectively activated by a modified agonist, but not by the endogenous agonist. Assisted by rhodopsin-based molecular modeling, we pinpointed mutations of the A3 adenosine receptor (AR) for selective affinity enhancement following complementary modifications of adenosine. Ribose modifications examined included, at 3′: amino, aminomethyl, azido, guanidino, ureido; and at 5′: uronamido, azidodeoxy. N6-variations included: 3-iodobenzyl, 5-chloro-2-methyloxybenzyl, and methyl. An N6-3-iodobenzyl-3′-ureido adenosine derivative 10 activated phospholipase C in COS-7 cells (EC50=0.18 μM) or phospholipase D in chick primary cardiomyocytes mediated by a mutant (H272E), but not the wild-type, A3AR. The affinity enhancements for 10 and the corresponding 3′-acetamidomethyl analogue 6 were >100-fold and >20-fold, respectively. 10 concentration-dependently protected cardiomyocytes transfected with the neoceptor against hypoxia. Unlike 10, adenosine activated the wild-type A3AR (EC50 of 1.0 μM), but had no effect on the H272E mutant A3AR (100 μM). Compound 10 was inactive at human A1, A2A, and A2BARs. The orthogonal pair comprising an engineered receptor and a modified agonist should be useful for elucidating signaling pathways and could be therapeutically applied to diseases following organ-targeted delivery of the neoceptor gene.
We studied the structural determinants of binding affinity and efficacy of adenosine receptor (AR) agonists. Substituents at the 2-position of adenosine were combined with N6-substitutions known to enhance human A3AR affinity. Selectivity of binding of the analogues and their functional effects on cAMP production were studied using recombinant human A1, A2A, A2B, and A3ARs. Mainly sterically small substituents at the 2-position modulated both the affinity and intrinsic efficacy at all subtypes. The 2-cyano group decreased hA3AR affinity and efficacy in the cases of N6-(3-iodobenzyl) and N6-(trans-2-phenyl-1-cyclopropyl), for which a full A3AR agonist was converted into a selective antagonist; the 2-cyano-N6-methyl analogue was a full A3AR agonist. The combination of N6-benzyl and various 2-substitutions (chloro, trifluoromethyl, and cyano) resulted in reduced efficacy at the A1AR. The environment surrounding the 2-position within the putative A3AR binding site was explored using rhodopsin-based homology modeling and ligand docking.
Purines; Cyclic AMP; Binding; Antagonists; Agonists; GPCR; Molecular modeling
Adenosine receptors are major targets of caffeine, the most commonly consumed drug in the world. There is growing evidence that they could also be promising therapeutic targets in a wide range of conditions, including cerebral and cardiac ischaemic diseases, sleep disorders, immune and inflammatory disorders and cancer. After more than three decades of medicinal chemistry research, a considerable number of selective agonists and antagonists of adenosine receptors have been discovered, and some have been clinically evaluated, although none has yet received regulatory approval. However, recent advances in the understanding of the roles of the various adenosine receptor subtypes, and in the development of selective and potent ligands, as discussed in this review, have brought the goal of therapeutic application of adenosine receptor modulators considerably closer.
A series of ring-constrained (N)-methanocarba-5′-uronamide 2,N6-disubstituted adenine nucleosides have been synthesized via Mitsunobu condensation of the nucleobase precursor with a pseudosugar ring containing a 5′-ester functionality. Following appropriate functionalization of the adenine ring, the ester group was converted to the 5′-N-methylamide. The compounds, mainly 2-chloro substituted derivatives, were tested in both binding and functional assays at human adenosine receptors (ARs), and many were found to be highly potent and selective A3AR agonists. Selected compounds were compared in binding to the rat A3AR to assess their viability for testing in rat disease models. The N6-(3-chlorobenzyl) and N6-(3-bromobenzyl) analogues displayed Ki values at the human A3AR of 0.29 and 0.38 nM, respectively. Other subnanomolar affinities were observed for the following N6 derivatives: 2,5-dichlorobenzyl, 5-iodo-2-methoxybenzyl, trans-2-phenyl-1-cyclopropyl, and 2,2-diphenylethyl. Selectivity for the human A3AR in comparison to the A1AR was (fold): the N6-(2,2-diphenylethyl) analogue 34 (1900), the N6-(2,5-dimethoxybenzyl) analogue 26 (1200), the N6-(2,5-dichlorobenzyl) and N6-(2-phenyl-1-cyclopropyl) analogues 20 and 33 (1000), and the N6-(3-substituted benzyl) analogues 17, 18, 28, and 29 (700–900). Typically, even greater selectivity ratios were obtained in comparison with the A2A and A2BARs. The (N)-methanocarba-5′-uronamide analogues were full agonists at the A3AR, as indicated by the inhibition of forskolin-stimluated adenylate cyclase at a concentration of 10 µM. The N6-(2,2-diphenylethyl) derivative was an A3AR agonist in the (N)-methanocarba-5′-uronamide series, although it was an antagonist in the ribose series. Thus, many of the previously known groups that enhance A3AR affinity in the 9-riboside series, including those that reducing intrinsic efficacy, may be adapted to the (N)-methanocarba nucleoside series of full agonists.
In this paper we investigated the influence on affinity, selectivity and intrinsic activity upon modification of the adenosine agonist scaffold at the 3′- and 5′-positions of the ribofuranosyl moiety and the 2- and N6-positions of the purine base. This resulted in the synthesis of various analogues, that is, 3–12 and 24–33, with good hA3AR selectivity and moderate-to-high affinities (as in 32, Ki = 27 nM). Interesting was the ability to tune the intrinsic activity depending on the substituent introduced at the 3′-position.
Adenosine receptors; Nucleoside analogues; Binding; Efficacy
Allosteric modulators for the Gi-coupled A3 adenosine receptor (AR) are of considerable interest as therapeutic agents and as pharmacological tools to probe various signaling pathways. In this study, we initially characterized the effects of several imidazoquinolinamine allosteric modulators (LUF5999, LUF6000 and LUF6001) on the human A3 AR stably expressed in CHO cells using a cyclic AMP functional assay. These modulators were found to affect efficacy and potency of the agonist Cl-IB-MECA differently. LUF5999 (2-cyclobutyl derivative) enhanced efficacy but decreased potency. LUF6000 (2-cyclohexyl derivative) enhanced efficacy without affecting potency. LUF6001 (2-H derivative) decreased both efficacy and potency. We further compared the agonist enhancing effects of LUF6000 in several other A3 AR-mediated events. It was shown that although LUF6000 behaved somewhat differently in various signaling pathways, it was more effective in enhancing the effects of low-efficacy than of high-efficacy agonists. In an assay of cyclic AMP accumulation, LUF6000 enhanced the efficacy of all agonists examined, but in the membrane hyperpolarization assay, it only enhanced the efficacy of partial agonists. In calcium mobilization, LUF6000 did not affect the efficacy of the full agonist NECA but was able to switch the nucleoside antagonist MRS542 into a partial agonist. In translocation of β-arrestin2, the agonist-enhancing effect LUF6000 was not pronounced. In an assay of ERK1/2 phosphorylation LUF6000 did not show any effect on the efficacy of Cl-IB-MECA. The differential effects of LUF6000 on the efficacy and potency of the agonist Cl-IB-MECA in various signaling pathway were interpreted quantitatively using a mathematical model.
G protein-coupled receptors; purines; positive allosteric modulator; second messenger; nucleoside