5’-AMP-activated protein kinase (AMPK) and its pharmacological modulators have been targeted for treating type 2 diabetes. Extracellular uridine 5’-diphosphate (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 in MIN6 mouse pancreatic β-cells and insulin secretion. Treatment with a potent P2Y6R dinucleotide agonist MRS2957 (500 nM) activated AMPK, which was blocked by P2Y6R-selective antagonist MRS2578. 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 Ca2+ pathways. P2Y6R agonist induced insulin secretion at high glucose, which was reduced by AMPK siRNA. Thus, P2Y6R has a crucial role in β-cell function, suggesting its potential as a therapeutic target in diabetes.
nucleotides; G protein-coupled receptor; insulin; AMPK; diabetes; P2Y6 receptor
Allosteric modulators of A1 and A2A adenosine receptors have been described; however, for the A3 adenosine receptor, neither an allosteric site nor a compound with allosteric effects has been described. In this study, the allosteric modulation of human A3 adenosine receptors by a series of 3-(2-pyridinyl)isoquinoline derivatives was investigated by examining their effects on the dissociation of the agonist radioligand, [125I]N6-(4-amino-3-iodobenzyl)-5′ -N-methylcarboxamidoadenosine (I-AB-MECA), from the receptor. Several 3-(2-pyridinyl)isoquinoline derivatives, including VUF5455, VUF8502, VUF8504, and VUF8507, slowed the dissociation of the agonist radioligand [125I]I-AB-MECA in a concentration-dependent manner, suggesting an allosteric interaction. These compounds had no effect on the dissociation of the radiolabeled antagonist [3H]PSB-11 from the A3 adenosine receptor, suggesting a selective enhancement of agonist binding. By comparison, compounds of similar structure (VUF8501, VUF8503, VUF8505), the classical adenosine receptor antagonist CGS15943 and the A1 receptor allosteric enhancer PD81723 did not significantly influence the dissociation rate of [125I]I-AB-MECA. The effect of agonist on forskolin-induced cAMP production was significantly enhanced by VUF5455. When the subtype-selectivity of the allosteric enhancement was tested the compounds had no effect on the dissociation of either [3H]N6-[(R)-phenylisopropyl]adenosine from the A1 adenosine receptor or [3H]CGS21680 from the A2A adenosine receptor. Probing of structure-activity relationships suggested that a carbonyl group is essential for allosterism but preferred only for competitive antagonism. The presence of a 7-methyl group decreased the competitive binding affinity without a major loss of the allosteric enhancing activity, suggesting that the structural requirements for allosteric enhancement might be distinct from those for competitive antagonism.
We have identified a series of 1H-imidazo-[4,5-c]quinolines as selective allosteric enhancers of human A3 adenosine receptors. Several of these compounds potentiated both the potency and maximal efficacy of agonist-induced responses and selectively decreased the dissociation of the agonist N6-(4-amino-3-[125I]iodobenzyl)-5′-N-methylcarboxamidoadenosine from human A3 adenosine receptors. There was no effect on the dissociation of the antagonist [3H]8-ethyl-4-methyl-2-phenyl-(8R)-4,5,7,8-tetrahydro-1H-imidazo[2.1-i]purin-5-one (PSB-11) from the A3 receptors, as well as [3H]N6-[(R)-phenylisopropy-l]adenosine from rat brain A1 receptors and [3H]2-[p-(2-carboxyethyl)phenyl-ethylamino]-5′-N-ethylcarboxamidoad-enosine from rat striatal A2A receptors, suggesting the selective enhancement of agonist binding at A3 receptors. The analogs were tested as antagonists of competitive binding at human A3 receptors, and Ki values ranging from 120 nM to 101 μM were observed; as for many allosteric modulators of G protein-coupled receptors, an orthosteric effect was also present. The most promising leads from the present set of analogs seem to be the 2-cyclopentyl-1H-imidazo[4,5-c]quinoline derivatives, of which the 4-phenylamino analog DU124183 had the most favorable degree of allosteric modulation versus receptor antagonism. The inhibition of forskolin-stimulated cyclic AMP accumulation in intact cells that express human A3 receptors was employed as a functional index of A3 receptor activation. The enhancer DU124183 caused a marked leftward shift of the concentration-response curve of the A3 receptor agonists in the presence of antagonist and, surprisingly, a potentiation of the maximum agonist efficacy by approximately 30%. Thus, we have identified a novel structural lead for developing allosteric enhancers of A3 adenosine receptors; such enhancers may be useful for treating brain ischemia and other hypoxic conditions.
Activation of a cardiac myocyte P2X4 receptor protects in heart failure. 5’-Phosphonate and 5’-phosphate analogues of AMP containing a (N)-methanocarba (bicyclo[3.1.0]hexane) system could protect from heart failure by potentially activating this cardioprotective channel. Phosphoesters and phosphonodiesters were synthesized and administered in vivo via a mini-osmotic pump in a mouse ischemic heart failure model; most significantly increased intact heart contractile function (echocardiography) compared to vehicle-infusion. Several new thio and deuterated phosphate derivatives were protective in a calsequestrin (CSQ)-overexpressing heart failure model. Diethyl (7, MRS4084) and diisopropyl (8, MRS4074) phosphotriesters were highly protective in the ischemic model. Substitution of 2-Cl with iodo reduced protection in the CSQ model. Diisopropyl ester 16 (MRS2978) of (1’S,2’R,3’S,4’R,5’S)-4’-(6-amino-2-chloropurin-9-yl)-2’,3’-(dihydroxy)-1’-(phosphonoethylene)-bicyclo[3.1.0]hexane was highly efficacious (CSQ), while lower homologue 1’-phosphonomethylene derivative 14 was inactive. Thus, we identified uncharged carbocyclic nucleotide analogues that represent potential candidates for the treatment of heart failure, suggesting this as a viable and structurally broad approach.
The natural plant alkaloids caffeine and theophylline were the first adenosine receptor (AR) antagonists described in the literature. They exhibit micromolar affinities and are non-selective. A large number of derivatives and analogs have subsequently been synthesized and evaluated as AR antagonists. Very potent antagonists have thus been developed with selectivity for each of the four AR subtypes.
adenosine receptors; A1 receptor antagonists; A2A receptor antagonists; A2B receptor antagonists; A3 receptor antagonists; caffeine; deazaxanthines; molecular probes; paraxanthine; theobromine; theophylline; tricyclic xanthine derivatives; xanthines
Extracellular nucleotides acting via P2 receptors play important roles in cardiovascular physiology/pathophysiology. Pyrimidine nucleotides activate four G protein-coupled P2Y receptors (P2YRs): P2Y2 and P2Y4 (UTP-activated), P2Y6, and P2Y14. Previously, we showed that uridine 5′-triphosphate (UTP) activating P2Y2R reduced infarct size and improved mouse heart function after myocardial infarct (MI). Here, we examined the cardioprotective role of P2Y2R in vitro and in vivo following MI using uridine-5′-tetraphosphate δ-phenyl ester tetrasodium salt (MRS2768), a selective and more stable P2Y2R agonist. Cultured rat cardiomyocytes pretreated with MRS2768 displayed protection from hypoxia [as revealed by lactate dehydrogenase (LDH) release and propidium iodide (PI) binding], which was reduced by P2Y2R antagonist, AR-C118925 (5-((5-(2,8-dimethyl-5H-dibenzo[a,d]annulen-5-yl)-2-oxo-4-thioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)-N-(1H-tetrazol-5-yl)furan-2-carboxamide). In vivo, echocardiography and infarct size staining of triphenyltetrazolium chloride (TTC) in 3 groups of mice 24 h post-MI: sham, MI, and MI+MRS2768 indicated protection. Fractional shortening (FS) was higher in MRS2768-treated mice than in MI alone (40.0 ± 3.1 % vs. 33.4 ± 2.7 %, p < 0.001). Troponin T and tumor necrosis factor-α (TNF-α) measurements demonstrated that MRS2768 pretreatment reduced myocardial damage (p < 0.05) and c-Jun phosphorylation increased. Thus, P2Y2R activation protects cardiomyocytes from hypoxia in vitro and reduces post-ischemic myocardial damage in vivo.
Cardioprotection; Heart; Ischemia/hypoxia; P2Y2 receptors
Interest is increasing in developing fluorescent ligands for characterization of adenosine receptors (ARs), which hold a promise of usefulness in the drug discovery process. The size of a strategically labeled AR ligand can be greatly increased after the attachment of a fluorophore. The choice of dye moiety (e.g. Alexa Fluor 488), attachment point and linker length can alter the selectivity and potency of the parent molecule. Fluorescent derivatives of adenosine agonists and antagonists (e.g. XAC and other heterocyclic antagonist scaffolds) have been synthesized and characterized pharmacologically. Some are useful AR probes for flow cytometry, fluorescence correlation spectroscopy, fluorescence microscopy, fluorescence polarization, fluorescence resonance energy transfer, and scanning confocal microscopy. Thus, the approach of fluorescent labeled GPCR ligands, including those for ARs, is a growing dynamic research field.
purine; G protein-coupled receptor; fluorescence; membrane proteins; binding; screening
The adenosine receptors (ARs) provide an example of how to accurately predict ligand recognition, even prior to the availability of a crystallographic structure. Homology modeling has been used to gain structural insight, in conjunction with site-directed mutagenesis and structure activity relationships of small molecular ligands. Recent X-ray structures greatly improved the accuracy of knowledge of AR ligand recogntion and furthermore characterized conformational changes induced by receptor activation. Now homology modeling extends these structural insights to related GPCRs and suggests new ligand structures. This strategy is also being applied to the eight subtypes of P2Y receptors for extracellular nucleotides, which lack X-ray structures and are best modeled by homology to the CXCR4 (peptide) receptor. Neoceoptors, as studied for three of the four AR subtypes, create a molecular complementarity between a mutant receptor and a chemically tailored agonist ligand to selectively enhance affinity, implying direct physical contact and thus validating docking hypotheses.
Purines; pyrimidines; G protein-coupled receptors; mutagenesis; molecular modeling; neoceptor; G protein-coupled receptors
Agonists of a single G protein-coupled receptor (GPCR) may activate distinct signaling pathways. Functional selectivity, an emerging concept with therapeutic relevance for GPCRs, may be due to conformational selection or stabilization with respect to particular agonists, receptor dimerization, variable expression levels of GPCRs and downstream signaling molecules, and allosteric modulation. Allosteric modulators may have potential advantages over orthosteric ligands, including greater selectivity and safety. This review focuses on functional selectivity resulting from allosteric modulation.
allosteric modulation; functional selectivity; GPCR; adenosine receptor; muscarinic receptor; metabotropic glutamate receptor; calcium sensing receptor; CCR5
In the central nervous system (CNS), an antagonistic interaction has been shown between adenosine A2A and dopamine D2 receptors (A2ARs and D2Rs) that may be relevant both in normal and pathological conditions (i.e. Parkinson’s disease). Thus, the molecular determinants mediating this receptor-receptor interaction have recently been explored, since the fine tuning of this target (namely the A2AR/D2R oligomer) could possibly improve the treatment of certain CNS diseases. Here, we used a fluorescence resonance energy transfer (FRET)-based approach to examine the allosteric modulation of the D2R within the A2AR/D2R oligomer and the dependence of this receptor-receptor interaction on two regions rich in positive charges on intracellular loop 3 (IL3) of the D2R. Interestingly, we observed a negative allosteric effect of the D2R agonist quinpirole on A2AR ligand binding and activation. However, these allosteric effects were abolished upon mutation of specific arginine residues (217–222 and 267–269) on IL3 of the D2R, thus demonstrating a major role of these positively-charged residues in mediating the observed receptor-receptor interaction. Overall, these results provide structural insights to better understand the functioning of the A2AR/D2R oligomer in living cells.
A2AR; D2R; FRET; oligomerization; allosterism; fluorescent agonist
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
There are eight subtypes of P2Y receptors (P2YRs) that are activated, and in some cases inhibited, by a range of extracellular nucleotides. These nucleotides are ubiquitous, but their extracellular concentration can rise dramatically in response to hypoxia, ischemia, or mechanical stress, injury, and release through channels and from vesicles. Two subclasses of P2YRs were defined based on clustering of sequences, second messengers, and receptor sequence analysis. The numbering system for P2YR subtypes is discontinuous; i.e., P2Y1–14Rs have been defined, but six of the intermediate-numbered cloned receptor sequences (e.g., P2y3, P2y5, P2y7–10) are not functional mammalian nucleotide receptors. Of these two clusters, the P2Y12–14 subtypes couple via Gαi to inhibit adenylate cyclase, while the remaining subtypes couple through Gαq to activate phospholipase C. Collectively, the P2YRs respond to both purine and pyrimidine nucleotides, in the form of 5′-mono- and dinucleotides and nucleoside-5′-diphosphosugars. In recent years, the medicinal chemistry of P2Y receptors has advanced significantly, to provide selective agonists and antagonists for many but not all of the subtypes. Ligand design has been aided by insights from structural probing using molecular modelling and mutagenesis. Currently, the molecular modelling of the receptors is effectively based on the X-ray structure of the CXCR4 receptor, which is the closest to the P2Y receptors among all the currently crystallized receptors in terms of sequence similarity. It is now a challenge to develop novel and selective P2YR ligands for disease treatment (although antagonists of the P2Y12R are already widely used as antithrombotics).
The P2Y1 receptor (P2Y1R) is a G protein-coupled receptor naturally activated by extracellular ADP. Its stimulation is an essential requirement of ADP-induced platelet aggregation, thus making antagonists highly sought compounds for the development of antithrombotic agents. Here, through a virtual screening campaign based on a pharmacophoric representation of the common characteristics of known P2Y1R ligands and the putative shape and size of the receptor binding pocket, we have identified novel antagonist hits of µM affinity derived from a N,N’-bis-arylurea chemotype. Unlike the vast majority of known P2Y1R antagonists, these drug-like compounds do not have a nucleotidic scaffold or highly negatively charged phosphate groups. Hence, our compounds may provide a direction for the development of receptor probes with altered physicochemical properties.
P2Y1 receptor; G protein-coupled receptor; antagonist; virtual screening; molecular modeling
We synthesized homologated truncated 4′-thioadenosine analogues 3 in which a methylene (CH2) group was inserted in place of the glycosidic bond of a potent and selective A3 adenosine receptor antagonist 2. The analogues were designed to induce maximum binding interaction in the binding site of the A3 adenosine receptor. However, all homologated nucleosides were devoid of binding affinity at all subtypes of adenosine receptors, indicating that free rotation through the single bond allowed the compound to adopt an indefinite number of conformations, disrupting the favorable binding interaction essential for receptor recognition.
homologation; A3 adenosine receptor; binding affinity; truncated 4′-thioadenosine
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
Farnesyl pyrophosphate (FPP) is an intermediate in cholesterol biosynthesis, and it has also been reported to activate platelet LPA (lysophosphatidic acid) receptors. The aim of this study was to investigate the role of extracellular FPP in platelet aggregation. Human platelets were studied with light transmission aggregometry, flow cytometry and [35S]GTPγS binding assays. As shown previously, FPP could potentiate LPA-stimulated shape change. Surprisingly, FPP also acted as a selective insurmountable antagonist to ADP-induced platelet aggregation. FPP inhibited ADP-induced expression of P-selectin and the activated glycoprotein (Gp)llb/llla receptor. FPP blocked ADP-induced inhibition of cAMP accumulation and [35S]GTPγS binding in platelets. In Chinese hamster ovary cells expressing the P2Y12 receptor, FPP caused a right-ward shift of the [35S]GTPγS binding curve. In Sf9 insect cells expressing the human P2Y12 receptor, FPP showed a concentration-dependent, although incomplete inhibition of [3H]PSB-0413 binding. Docking of FPP in a P2Y12 receptor model revealed molecular similarities with ADP and a good fit into the binding pocket for ADP. In conclusion, FPP is an insurmountable antagonist of ADP-induced platelet aggregation mediated by the P2Y12 receptor. It could be an endogenous antithrombotic factor modulating the strong platelet aggregatory effects of ADP in a manner similar to the use of clopidogrel, prasugrel or ticagrelor in the treatment of ischaemic heart disease.
ADP receptors; platelet pharmacology; platelet physiology
The fluorescein conjugate, FITC-APEC (2-[2-[4-[2-[2-[1,3-dihydro-l,l-bis(4-hydroxyphenyl)-3-oxo-5-isobenzofuranthioureidyl]ethylaminocarbonyl]ethyl]phenyl]ethylamino]-5′-N-ethylcarboxamidoadenosine), is a novel ligand derived from a series of functionalized congeners that act as selective A2a-adenosine receptor agonists. The binding of FITC-APEC to bovine striatal A2a,-adenosine receptors measured by fluorescence techniques was saturable and of a high affinity, with a Bmax, of 2.3 ± 0.3 pmol/mg protein and KD of 57 ± 2 nM. The KD value estimated by fluorescence was consistent with the Ki (11 ± 0.3 nM) obtained by competition studies with [3H]CGS 21680. Additionally, the Bmax, value found by FITC-APEC measurement was in agreement with Bmax, values obtained using radioligand binding. FITC-APEC exhibited rapid and reversible binding to bovine striatum. The potencies of chemically diverse A2a-adenosine receptor ligands estimated by inhibition of FITC-APEC binding were in good agreement with their potencies determined using radioligand binding techniques (r = 0.97, P = 0.0003). FITC-APEC binding was not altered by purine derivatives that do not recognize A2a-adenosine receptors. These findings demonstrate that the novel fluorescent ligand FITC-APEC can be used in the quantitative characterization of ligand binding to A2a-adenosine receptors.
Fluorescence; A2a-adenosine receptors; receptor binding; bovine striatum
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
To explore the safety and efficacy of CF101, an A3 adenosine receptor agonist, in patients with moderate-to-severe dry eye syndrome
Phase 2, multicenter, randomized, double-masked, placebo-controlled, parallel-group study.
68 patients completed the study, 35 patients in the placebo group and 33 patients in the CF101 group.
Patients were orally treated with either 1 mg CF101 pills or matching vehicle-filled placebo pills, given twice daily for 12 weeks, followed by a 2-week post-treatment observation.
Main Outcome Measures
an improvement of >25% over baseline at week 12 in one of the following parameters: (a) tear break-up time (BUT); (b) superficial punctate keratitis assessed by fluorescein staining (FS); (c) Schirmer tear test 1 (ST1).
clinical laboratory safety tests, ophthalmic examinations, intraocular pressure (IOP) measurements, electrocardiographic evaluations, vital sign measurements and monitoring of adverse events.
A statistically significant increase in the proportion of patients who achieved more than 25% improvement in the corneal staining and in the clearance of corneal staining was noted between the CF101-treated group and the placebo group. Treatment with CF101 resulted in a statistically significant improvement in the mean change from baseline at week 12 of the corneal staining, BUT, and tear meniscus (TM) height in the CF101-treated group CF101 was well tolerated and exhibited an excellent safety profile with no serious adverse events. A statistically significant decrease from baseline was observed in the IOP of the CF101-treated group in comparison with the placebo group.
CF101, given orally, induced a statistically significant improvement in the corneal staining and an improvement in the BUT and TM in patients with moderate-to-severe dry eye syndrome. The drug was very well tolerated. These data and the anti-inflammatory characteristic of CF101 support further study of the drug as a potential treatment for the signs and symptoms of dry eye syndrome.
(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
The stereoselective synthesis of truncated 3’-aminocarbanucleosides 4a–d via a stereo- and regioselective conversion of a diol 9 to bromoacetate 11a and their binding affinity towards the human A3 adenosine receptor are described.