Cluster of differentiation 73 (CD73) converts adenosine 5'-monophosphate to immunosuppressive adenosine, and its inhibition was proposed as a new strategy for cancer treatment. We synthesized 5'- O-[(phosphonomethyl)phosphonic acid] derivatives of purine and pyrimidine nucleosides, which represent nucleoside diphosphate analogues, and compared their CD73 inhibitory potencies. In the adenine series, most ribose modifications and 1-deaza and 3-deaza were detrimental, but 7-deaza was tolerated. Uracil substitution with N3-methyl, but not larger groups, or 2-thio, was tolerated. 1,2-Diphosphono-ethyl modifications were not tolerated. N4-(Aryl)alkyloxy-cytosine derivatives, especially with bulky benzyloxy substituents, showed increased potency. Among the most potent inhibitors were the 5'- O-[(phosphonomethyl)phosphonic acid] derivatives of 5-fluorouridine (4l), N4-benzoyl-cytidine (7f), N4-[ O-(4-benzyloxy)]-cytidine (9h), and N4-[ O-(4-naphth-2-ylmethyloxy)]-cytidine (9e) ( Ki values 5-10 nM at human CD73). Selected compounds tested at the two uridine diphosphate-activated P2Y receptor subtypes showed high CD73 selectivity, especially those with large nucleobase substituents. These nucleotide analogues are among the most potent CD73 inhibitors reported and may be considered for development as parenteral drugs.

Both agonists and antagonists of the UDP-activated P2Y6 receptor (P2Y6R) have been proposed for therapeutic use, in conditions such as cancer, inflammation, neurodegeneration and diabetes. Uracil nucleotides containing a South-bicyclo[3.1.0]hexane ((S)-methanocarba) ring system in place of the ribose ring were synthesized and shown to be potent P2Y6R agonists in a calcium mobilization assay. The (S)-methanocarba modification was compatible with either a 5-iodo or 4-methoxyimino group on the pyrimidine, but not with a α,β-methylene 5´-diphosphate. (S)-Methanocarba dinucleotide potency was compatible with a N4-methoxy modification on the proximal nucleoside that is assumed to bind at the P2Y6R similarly to UDP; (N)-methanocarba was preferred on the distal nucleoside moiety. This suggests that the distal dinucleotide P2Y6R binding site prefers a ribose-like group that can attain a (N) conformation, rather than (S). Dinucleotide binding was modeled by homology modeling, docking and molecular dynamics simulations, which suggested the same ribose conformational preferences found empirically.

UDP and UDP-glucose activate the P2Y(14) receptor (P2Y(14)R) to modulate processes related to inflammation, diabetes, and asthma. A computational pipeline suggested alternatives to naphthalene of a previously reported P2Y14R antagonist (3, PPTN) using docking and molecular dynamics simulations on a hP2Y(14)R homology model based on P2Y(12)R structures. By reevaluating the binding of 3 to P2Y(14)R computationally, two alternatives, i.e., alkynyl and triazolyl derivatives, were identified. Improved synthesis of fluorescent antagonist 4 enabled affinity quantification (IC(50)s, nM) using flow cytometry of P2Y(14)R-expressing CHO cells. p-F3C-phenyl-triazole 65 (32) was more potent than a corresponding alkyne 11. Thus, additional triazolyl derivatives were prepared, as guided by docking simulations, with nonpolar aryl substituents favored. Although triazoles were less potent than 3 (6), simpler synthesis facilitated further structural optimization. Additionally, relative P2Y(14)R affinities agreed with predicted binding of alkynyl and triazole analogues. These triazoles, designed through a structure-based approach, can be assessed in disease models.

The P2Y14R is a G(i/o)-coupled receptor of the P2Y family of purinergic receptors that is activated by extracellular UDP and UDP-glucose (UDPG). In an earlier report we described a P2Y14R fluorescent probe, MRS4174, based on the potent and selective antagonist PPTN, a naphthoic acid derivative. Here, we report the design, preparation, and activity of an agonist-based fluorescent probe MRS4183 (11) and a shorter P2Y14R agonist congener, which contain a UDP-glucuronic acid pharmacophore and BODIPY fluorophores conjugated through diaminoalkyl linkers. The design relied on both docking in a P2Y14R homology model and established structure activity relationship (SAR) of nucleotide analogs. 11 retained P2Y14R potency with EC50 value of 0.96 nM (inhibition of adenylyl cyclase), compared to parent UDPG (EC50 47 nM) and served as a tracer for microscopy and flow cytometry, displaying minimal nonspecific binding. Binding saturation analysis gave an apparent binding constant for 11 in whole cells of 21.4±1.1 nM, with a t1/2 of association at 50 nM 11 of 23.9 min. Known P2Y14R agonists and PPTN inhibited cell binding of 11 with the expected rank order of potency. The success in the identification of a new P2Y14R fluorescent agonist with low nonspecific binding illustrates the advantages of rational design based on recently determined GPCR X-ray structures. Such conjugates will be useful tools in expanding the SAR of this receptor, which still lacks chemical diversity in its collective ligands.

The A 2B adenosine receptor (AR) is involved in many important functions and couples to several G proteins, but the endogenous effects have not been well documented partly due to the lack of selective agonists at this subtype until recently. In the present study, we probed the potential partial and/or biased agonism at the A 2B AR in several types of cells, including HEK293 cells and T24 bladder cancer cells, using a series of A 2B agonists of adenosine analogs substituted at 5’, N 6 and C2 positions, including NECA, MRS5911 and MRS3997, and the non‐nucleoside A 2B agonist, BAY60‐6583. All A 2B agonists tested induced accumulation of cyclic AMP fully or partially in all cells tested, with NECA being the most efficacious. However, only 5’‐substituted analogs are full agonists in calcium mobilization and ERK1/2 phosphorylation. Over‐expression of the A 2B AR in HEK293 cells increased the agonist potency and efficacy in inducing the Gs‐mediated accumulation of cyclic AMP, but not the ability to induce calcium mobilization by the C2‐substituted adenosine derivatives or non‐adenosine A 2B agonists. Results from an arrestin translocation assay showed that the 5’‐substituted NECA is more efficacious than adenosine derivatives substituted at C2 and N6 positions and non‐adenosine agonists. BAY actually did not induce arrestin translocation in this assay. The data suggest that 5’‐substituted adenosine analogs are balanced agonists of different pathways, whereas the C2‐ and N 6 ‐substituted adenosine analogs and the class of non‐nucleoside A 2B agonists are Gs‐biased agonists. The data also suggest that BAY and some N 6 ‐ and C2‐substituted adenosine analogs may be full agonist, partial agonist or antagonist under various conditions. Indeed, BAY behaves as an A 2B antagonist to induce insulin release in MIN6 pancreatic β cells. Thus, differences in efficacy and effector pathways might explain some of the contradictory results reported for A 2B AR activation. Grant Funding Source : Supported by NIDDK Intramural Research, NIH

G protein-coupled A2B adenosine receptor (AR) regulates numerous important physiological functions, but its activation by diverse A2BAR agonists is poorly profiled. We probed potential partial and/or biased agonism in cell lines expressing variable levels of endogenous or recombinant A2BAR. In cAMP accumulation assays, both 5′-substituted NECA and C2-substituted MRS3997 are full agonists. However, only 5′-substituted adenosine analogs are full agonists in calcium mobilization, ERK1/2 phosphorylation and β-arrestin translocation. A2BAR overexpression in HEK293 cells markedly increased the agonist potency and maximum effect in cAMP accumulation, but less in calcium and ERK1/2. A2BAR siRNA silencing was more effective in reducing the maximum cAMP effect of non-nucleoside agonist BAY60-6583 than NECA's. A quantitative 'operational model' characterized C2-substituted MRS3997 as either balanced (cAMP accumulation, ERK1/2) or strongly biased agonist (against calcium, β-arrestin). N6-substitution biased against ERK1/2 (weakly) and calcium and β-arrestin (strongly) pathways. BAY60-6583 is ERK1/2-biased, suggesting a mechanism distinct from adenosine derivatives. BAY60-6583, as A2BAR antagonist in MIN-6 mouse pancreatic β cells expressing low A2BAR levels, induced insulin release. This is the first relatively systematic study of structure–efficacy relationships of this emerging drug target.

We establish structure activity relationships of extracellular nucleosides and nucleotides at G protein-coupled receptors (GPCRs), e.g. adenosine receptors (ARs) and P2Y receptors (P2YRs), respectively. We synthesize selective agents for use as pharmacological probes and potential therapeutic agents (e.g. A3AR agonists for neuropathic pain). Detailed structural information derived from the X-ray crystallographic structures within these families enables the design of novel ligands, guides modification of known agonists and antagonists, and helps predict polypharmacology. Structures were recently reported for the P2Y12 receptor (P2Y12R), an anti-thrombotic target. Comparison of agonist-bound and antagonist-bound P2Y12R indicates unprecedented structural plasticity in the outer portions of the transmembrane (TM) domains and the extracellular loops. Nonphosphate-containing ligands of the P2YRs, such as the selective P2Y14R antagonist PPTN, are desired for bioavailability and increased stability. Also, A2AAR structures are effectively applied to homology modeling of closely related A1AR and A3AR, which are not yet crystallized. Conformational constraint of normally flexible ribose with bicyclic analogues increased the ligand selectivity. Comparison of rigid A3AR agonist congeners allows the exploration of interaction of specific regions of the nucleoside analogues with the target and off-target GPCRs, such as biogenic amine receptors. Molecular modeling predicts plasticity of the A3AR at TM2 to accommodate highly rigidified ligands. Novel fluorescent derivatives of high affinity GPCR ligands are useful tool compounds for characterization of receptors and their oligomeric assemblies. Fluorescent probes are useful for characterization of GPCRs in living cells by flow cytometry and other methods. Thus, 3D knowledge of receptor binding and activation facilitates drug discovery.

Extended N4-(3-arylpropyl)oxy derivatives of uridine-5′-triphosphate were synthesized and potently stimulated phospholipase C stimulation in astrocytoma cells expressing G protein-coupled human (h) P2Y receptors (P2YRs) activated by UTP (P2Y2/4R) or UDP (P2Y6R). The potent P2Y4R-selective N4-(3-phenylpropyl)oxy agonist was phenyl ring-substituted or replaced with terminal heterocyclic or naphthyl rings with retention of P2YR potency. This broad tolerance for steric bulk in a distal region was not observed for dinucleoside tetraphosphate agonists with both nucleobases substituted. The potent N4-(3-(4-methoxyphenyl)-propyl)oxy analogue 19 (EC50: P2Y2R, 47 nM; P2Y4R, 23 nM) was functionalized for chain extension using click tethering of fluorophores as prosthetic groups. The BODIPY 630/650 conjugate 28 (MRS4162) exhibited EC50 values of 70, 66, and 23 nM at the hP2Y2/4/6Rs, respectively, and specifically labeled cells expressing the P2Y6R. Thus, an extended N4-(3-arylpropyl)oxy group accessed a structurally permissive region on three Gq-coupled P2YRs, and potency and selectivity were modulated by distal structural changes. This freedom of substitution was utilized to design of a pan-agonist fluorescent probe of a subset of uracil nucleotide-activated hP2YRs.

Glucose uptake by peripheral tissues such as skeletal muscles and adipocytes is important in the maintenance of glucose homeostasis. We previously demonstrated that P2Y6 receptor (P2Y6R) agonists protect pancreatic islet cells from apoptosis and stimulate glucose-dependent insulin release. Here, we investigated the effects of P2Y6R activation on glucose uptake in insulin target tissues. An agonist of the P2Y6R, P1-(5'-uridine)-P3-(5'-N4-methoxycytidine)-triphosphate (MRS2957), significantly increased the uptake of [3H]2-deoxyglucose in mouse C2C12 myotubes and 3T3-L1 adipocytes, and this stimulation was significantly decreased by a selective P2Y6R antagonist N,N″-1,4-butanediyl-bis[N'-(3-isothiocyanatophenyl)thiourea] (MRS2578). Pre-incubation with Compound C (an inhibitor of 5'-AMP-activated protein kinase, AMPK), or AMPK siRNA abolished the stimulatory effect of MRS2957 on glucose uptake. Also, MRS2957 (60 min incubation) increased recruitment of the facilitated glucose transporter-4 (GLUT4) to the cell membrane, which was blocked by MRS2578. Treatment of C2C12 myotubes with MRS2957 induced significant phosphorylation of AMPK, which increase GLUT4 expression through histone deacetylase (HDAC)5 signaling. Glucose uptake in primary mouse adipocytes from wild-type mice was stimulated upon P2Y6R activation by either MRS2957 or native agonist UDP, and the P2Y6R effect was antagonized by MRS2578. However, in adipocytes from P2Y6R-knockout mice P2Y6R agonists had no effect on glucose uptake, and there was no change in the glucose uptake by insulin. Our results indicate that the P2Y6R promotes glucose metabolism in peripheral tissues, which may be mediated through AMPK signaling.

5′‐AMP‐activated protein kinase (AMPK) and its pharmacological modulators have been targeted for treating type 2 diabetes. Extracellular UDP activates P2Y6 receptors (P2Y6Rs) in pancreatic β cells to release insulin and reduce apoptosis, which would benefit diabetes. Here, we studied the role of P2Y6R in activation of AMPK and insulin secretion in MIN6 mouse pancreatic beta cells. Treatment with a potent P2Y6R dinucleotide agonist MRS2957 activated AMPK, which was blocked by P2Y6R‐selective antagonist. Also, MRS2957 induced phosphorylation of acetyl‐coenzyme A carboxylase (ACC), a marker of AMPK activity. Calcium chelator BAPTA‐AM, calmodulin‐dependent protein kinase kinase (CaMKK) inhibitor STO‐069 and IP3 receptor antagonist 2‐APB attenuated P2Y6R‐mediated AMPK phosphorylation revealing involvement of intracellular calcium signaling pathways. P2Y6R agonist induced insulin secretion only at high glucose, which was reduced by AMPK gene silencing suggesting an involvement of AMPK in P2Y6 receptor‐mediated insulin secretion. Thus, P2Y6R has a crucial role in β cell function, suggesting its potential as a therapeutic target in diabetes.

4-Alkyloxyimino derivatives of pyrimidine nucleotides display high potency as agonists of certain G protein-coupled P2Y receptors (P2YRs). In an effort to functionalize a P2Y6R agonist for fluorescent labeling, we probed two positions (N4 and γ-phosphate of cytidine derivatives) with various functional groups, including alkynes for click chemistry. Functionalization of extended imino substituents at the 4 position of the pyrimidine nucleobase of CDP preserved P2Y6R potency generally better than γ-phosphoester formation in CTP derivatives. Fluorescent Alexa Fluor 488 conjugate 16 activated the human P2Y6R expressed in 1321N1 human astrocytoma cells with an EC50 of 9 nM, and exhibited high selectivity for this receptor over other uridine nucleotide-activated P2Y receptors. Flow cytometry detected specific labeling with 16 to P2Y6R-expressing but not to wild-type 1321N1 cells. Additionally, confocal microscopy indicated both internalized 16 (t1/2 of 18 min) and surface-bound fluorescence. Known P2Y6R ligands inhibited labeling. Theoretical docking of 16 to a homology model of the P2Y6R predicted electrostatic interactions between the fluorophore and extracellular portion of TM3. Thus, we have identified the N4-benzyloxy group as a structurally permissive site for synthesis of functionalized congeners leading to high affinity molecular probes for studying the P2Y6R.

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.