A Randomized, Controlled Comparison of NCX 470 (0.021%, 0.042%, and 0.065%) and Latanoprost 0.005% in Patients With Open-angle Glaucoma or Ocular Hypertension: The Dolomites Study.

PRCIS: NCX 470 0.042% and 0.065% were statistically superior in intraocular pressure (IOP) lowering to latanoprost 0.005%, and NCX 470 0.021% was noninferior. All NCX 470 concentrations were safe and well tolerated. PURPOSE: The purpose of this study was to compare varying concentrations of NCX 470 (a nitric oxide-donating bimatoprost) to latanoprost in a dose-response safety and efficacy trial. PATIENTS AND METHODS: Adult patients with bilateral open-angle glaucoma or ocular hypertension were randomized to NCX 470 0.021% (n=111), 0.042% (n=108), 0.065% (n=107), or latanoprost 0.005% (n=107) once daily in the evening. IOP was measured at 8:00 am, 10:00 am, and 4:00 pm at weeks 1, 2, and 4. The primary efficacy endpoint was the reduction from baseline in mean diurnal IOP at week 4. Secondary efficacy endpoints included reductions from baseline in mean diurnal IOP at weeks 1 and 2, and reductions from baseline in time-matched IOP at 8:00 am, 10:00 am, and 4:00 pm at weeks 1, 2, and 4. Adverse events were evaluated. RESULTS: All concentrations of NCX 470 resulted in significant reductions of mean diurnal IOP. The 0.042% and 0.065% concentrations were statistically superior to latanoprost 0.005%, and 0.021% was noninferior to latanoprost for change from baseline in mean diurnal IOP at week 4. The 0.065% concentration was also superior to latanoprost by up to 1.4 mm Hg for reduction from baseline at 8:00 am, 10:00 am, and 4:00 pm at week 4. NCX 470 was safe and well tolerated; conjunctival hyperemia was the most frequently reported adverse event. CONCLUSIONS: NCX 470 demonstrated dose-dependent reductions in IOP. The 0.042% and 0.065% concentrations demonstrated significantly greater reductions from baseline in mean diurnal IOP than latanoprost 0.005% at week 4, suggesting that higher concentrations may show even greater efficacy.

P2Y2 receptor activation decreases blood pressure and increases renal Na⁺ excretion.

ATP and UTP are endogenous agonists of P2Y(2/4) receptors. To define the in vivo effects of P2Y(2) receptor activation on blood pressure and urinary excretion, we compared the response to INS45973, a P2Y(2/4) receptor agonist and UTP analog, in wild-type (WT) and P2Y(2) receptor knockout (P2Y(2)-/-) mice. INS45973 was administered intravenously as a bolus injection or continuous infusion to determine effects on blood pressure and renal function, respectively. Within seconds, bolus application of INS45973 (0.1 to 3 mg/kg body wt) dose-dependently decreased blood pressure in WT (maximum response -35 ± 2 mmHg) and to a similar extent in endothelial nitric oxide synthase knockout mice. By contrast, blood pressure increased in P2Y(2)-/- (maximum response +18 ± 1 mmHg) but returned to basal levels within 60 s. Continuous infusion of INS45973 (25 to 750 µg·min(-1)·kg(-1) body wt) dose-dependently increased urinary excretion of Na(+) in WT (maximum response +46 ± 15%) but reduced Na(+) excretion in P2Y(2)-/- (maximum responses of -45 ± 15%) mice. In renal clearance experiments, INS45973 did not affect glomerular filtration rate but lowered blood pressure and increased fractional excretion of fluid, Na(+), and K(+) in WT relative to P2Y(2)-/- mice. The blood pressure responses to INS45973 are consistent with P2Y(2) receptor-mediated NO-independent vasodilation and implicate responses to endothelium-derived hyperpolarizing factor, and P2Y(2) receptor-independent vasoconstriction, probably via activation of P2Y(4) receptors on smooth muscle. Systemic activation of P2Y(2) receptors thus lowers blood pressure and inhibits renal Na(+) reabsorption, effects suggesting the potential utility of P2Y(2) agonism in the treatment of hypertension.

Transformation by a nucleotide-activated P2Y receptor is mediated by activation of Galphai, Galphaq and Rho-dependent signaling pathways.

BACKGROUND: Nucleotide-actived P2Y receptors play critical roles in the growth of tumor cells by regulating cellular proliferation, differentiation and survival. RESULTS: Here we demonstrate that an avian P2Y purinoceptor (tP2YR) with unique pharmacological and signal transduction properties induces morphologic and growth transformation of rodent fibroblasts. tP2YR induced a transformed phenotype similar to the mas oncogene, a G protein-coupled receptor which causes transformation by activation of Rac-dependent pathways. tP2YR-transformed cells exhibited increased steady-state activation of Rac1 and RhoA. Like activated Rho GTPases, tP2YR cooperated with activated Raf and caused synergistic transformation of NIH3T3 cells. Our data indicate that the ability of tP2YR to cause transformation is due to its unique ability among purinergic receptors to simultaneously activate Galphaq and Galphai. Co-expression of constitutively activated mutants of these two Galpha subunits caused the same transformed phenotype as tP2YR and Mas. Furthermore, transformation by both tP2YR and Mas was blocked by pharmacological inhibition of GalphaI by pertussis toxin (PTX) indicating an essential role for Galphai in transformation by these G-protein coupled receptors. CONCLUSIONS: Our data suggest that coordinated activation of Galphaq and Galphai may link the tP2YR and possibility the Mas oncogene with signaling pathways resulting in activation of Rho family proteins to promote cellular transformation.

Ocular surface distribution and pharmacokinetics of a novel ophthalmic 1% azithromycin formulation.

PURPOSE: To investigate the ocular distribution of 1% azithromycin ophthalmic solution and the effect of polycarbophil-based mucoadhesive formulation on ocular tissue levels of azithromycin after single and multiple topical administrations in the rabbit eye. METHODS: Rabbits were treated with either a single administration of 1% azithromycin solution with or without polycarbophil, or with multiple administrations of 1% azithromycin solution in polycarbophil. Drug concentrations were measured using LC/MS/MS. Conjunctiva, cornea, aqueous humor, and tear samples were analyzed over a period of 144 h after a single administration of azithromycin with or without polycarbophil. Eyelid, conjunctiva, cornea, aqueous humor, and tear samples were collected over a period of 288 h during and after multiple administrations of azithromycin. RESULTS: Azithromycin was rapidly absorbed and distributed in the ocular tissues, reaching within 5 min, concentrations of 10,539 microg/mL in tear film, 108 microg/g in conjunctiva, and 40 microg/g in the cornea. The drug demonstrated tissue-specific half-lives of 15, 63, and 67 h, respectively. Following multiple administrations, the drug gradually accumulated. The polycarbophil formulation increased the bioavailability of the drug, producing peak concentrations that were between 5- and 12-fold higher than those without polycarbophil. Azithromycin also distributed rapidly in the eyelids, reaching peak concentrations of 180 mug/g at the end of the 7-day treatment, and was eliminated with a half-life of 125 h. Six days after treatment was discontinued, eyelid levels of azithromycin were above 40 microg/g. CONCLUSIONS: Sustained and high concentrations were encountered with 7-day approved administration of 1% azithromycin formulation (AzaSite, Inspire Pharmaceuticals, Inc., Durham, NC) within all ocular surface tissues, particularly the lids. Many ocular surface disorders involving the tear film, eyelids, and adnexal structures are associated with chronic, low-grade bacterial infection and may potentially lead to decreased vision secondary to corneal scarring. Various topical antibiotic and steroid combinations with or without oral tetracyclines are commonly used with variable clinical response and known potential side effects. The clinical relevance of this study is unknown; however, the long-lasting antibacterial and additional anti-inflammatory properties of topical azithromycin might offer an effective alternative treatment option and should be explored further in clinical studies.

Lipophilic modifications to dinucleoside polyphosphates and nucleotides that confer antagonist properties at the platelet P2Y12 receptor.

Platelet P2Y12 receptors play a central role in the regulation of platelet function and inhibition of this receptor by treatment with drugs such as clopidogrel results in a reduction of atherothrombotic events. We discovered that modification of natural and synthetic dinucleoside polyphosphates and nucleotides with lipophilic substituents on the ribose and base conferred P2Y12 receptor antagonist properties to these molecules producing potent inhibitors of ADP-mediated platelet aggregation. We describe methods for the preparation of these functionalized dinucleoside polyphosphates and nucleotides and report their associated activities. By analysis of these results and by deconstruction of the necessary structural elements through selected syntheses, we prepared a series of highly functionalized nucleotides, resulting in the selection of an adenosine monophosphate derivative (62) for further clinical development.

Adenosine analogues as inhibitors of P2Y12 receptor mediated platelet aggregation.

Modified adenosine derivatives may lead to the development of P2Y(12) antagonists that are potent, selective, and bind reversibly to the receptor. Analogues of 2',3'-trans-styryl acetal-N6-ureido-adenosine monophosphate were prepared by modification of the 5'-position. The resulting analogues were tested for P2Y(12) antagonism in a platelet aggregation assay.

Rapid and reversible modulation of platelet function in man by a novel P2Y(12) ADP-receptor antagonist, INS50589.

P2Y(12) receptors participate in ADP-induced activation and aggregation of human platelets. INS50589, a selective P2Y(12) receptor antagonist, is being developed for use where controlled, reversible modulation of the platelet hemostatic function is needed. The tolerability, pharmacokinetics, and pharmacodynamics of INS50589 were tested in healthy human volunteers. Thirty-six subjects received intravenous infusions of placebo or INS50589 at 0.1-3 mg/kg/h for four hours. Platelet function, clotting parameters, bleeding time, safety assessments, and plasma concentrations of INS50589 and its major metabolite were monitored for 24 hours. Near-steady state plasma concentrations of INS50589 and effects on platelet function were achieved rapidly. The average maximal plasma concentration of INS50589 was linearly related to the dose administered. Intravenous INS50589 produced dose-dependent inhibition of platelet activation and aggregation in response to ADP in vitro until nearly full inhibition was achieved at the higher doses. Bleeding time was correspondingly increased, without any effect on activated clotting time, prothrombin time, or activated partial thromboplastin time. Platelet response to ADP had returned to at least 75% of the baseline value within 0.25-4 h after cessation of the intravenous infusion of INS50589, depending upon the dose and ADP challenge concentration. Infusions were well tolerated up to the highest dose tested. There was no evidence that the principal metabolite (INS51088) contributed to these effects. INS50589 is a well-tolerated, reversible, competitive antagonist of ADP at the P2Y(12) human platelet receptor, and its potential therapeutic utility in various cardiovascular settings is discussed.

International Union of Pharmacology LVIII: update on the P2Y G protein-coupled nucleotide receptors: from molecular mechanisms and pathophysiology to therapy.

There have been many advances in our knowledge about different aspects of P2Y receptor signaling since the last review published by our International Union of Pharmacology subcommittee. More receptor subtypes have been cloned and characterized and most orphan receptors de-orphanized, so that it is now possible to provide a basis for a future subdivision of P2Y receptor subtypes. More is known about the functional elements of the P2Y receptor molecules and the signaling pathways involved, including interactions with ion channels. There have been substantial developments in the design of selective agonists and antagonists to some of the P2Y receptor subtypes. There are new findings about the mechanisms underlying nucleotide release and ectoenzymatic nucleotide breakdown. Interactions between P2Y receptors and receptors to other signaling molecules have been explored as well as P2Y-mediated control of gene transcription. The distribution and roles of P2Y receptor subtypes in many different cell types are better understood and P2Y receptor-related compounds are being explored for therapeutic purposes. These and other advances are discussed in the present review.

Synthesis of potential purinoceptor antagonists: application of P1-tBU phosphazene base for alkylation of adenine in solution and on solid phase.

Alkylation of adenine in solution and on solid phase was accelerated by phosphazene base P1-tBu compared to mineral bases. The reactions in solution afforded regioselectively the appropriate N9-alkylated adenines with high preparative yields while the reaction with polystyrene resin-bound N-bromoacetylated peptides gave three regioisomers (alkylated at the N9, N7, and N3 position of adenine) in a 4:2:1 molar ratio. Ten novel nonphosphate nucleotide analogues were tested in an ADP-induced platelet aggregation assay.

Structure-activity relationships of dinucleotides: Potent and selective agonists of P2Y receptors.

Dinucleoside polyphosphates act as agonists on purinergic P2Y receptors to mediate a variety of cellular processes. Symmetrical, naturally occurring purine dinucleotides are found in most living cells and their actions are generally known. Unsymmetrical purine dinucleotides and all pyrimidine containing dinucleotides, however, are not as common and therefore their actions are not well understood. To carry out a thorough examination of the activities and specificities of these dinucleotides, a robust method of synthesis was developed to allow manipulation of either nucleoside of the dinucleotide as well as the phosphate chain lengths. Adenosine containing dinucleotides exhibit some level of activity on P2Y(1) while uridine containing dinucleotides have some level of agonist response on P2Y(2) and P2Y(6). The length of the linking phosphate chain determines a different specificity; diphosphates are most accurately mimicked by dinucleoside triphosphates and triphosphates most resemble dinucleoside tetraphosphates. The pharmacological activities and relative metabolic stabilities of these dinucleotides are reported with their potential therapeutic applications being discussed.

Regulation of P2Y1 receptor-mediated signaling by the ectonucleoside triphosphate diphosphohydrolase isozymes NTPDase1 and NTPDase2.

Ectonucleoside triphosphate diphosphohydrolases (NTPDases) control the concentration of released extracellular nucleotides, but the precise physiological roles played by these isozymes in modulation of P2 receptor signaling remain unclear. Activation of the human P2Y(1) receptor was studied in the presence of NTPDase1 or NTPDase2 expressed either in the same cell as the receptor or in P2Y(1) receptor-expressing cells cocultured with NTPDaseexpressing cells. Coexpression of NTPDase1 with the P2Y(1) receptor resulted in increases in the EC(50) for 2'-methylthioadenosine 5'-diphosphate (2MeSADP; 12-fold), ADP (50-fold), and ATP (10-fold) for activation of phospholipase C. Similar effects were observed when the P2Y(1) receptor and NTPDase1 were expressed on different cells. These results are explained by the capacity of NTPDase1 to hydrolyze both nucleoside triphosphates and diphosphates. NTPDase2 preferentially hydrolyzes nucleoside triphosphates, and the presence of NTPDase2 under either coexpression or coculture conditions did not change the EC(50) of 2MeSADP, ADP, or adenosine 5'-O-(2-thiodiphosphate) for activation of the P2Y(1) receptor. However, the EC(50) for ATP was 15-fold lower in the presence of NTPDase2 than in cells expressing the P2Y(1) receptor alone. Whereas expression of NTPDase1 decreased basal activity of the P2Y(1) receptor, the presence of the NTPDase2 resulted in P2Y(1) receptor-dependent increases in basal activity. These results suggest that basal activity of the P2Y(1) receptor is maintained by paracrine or autocrine release of receptor agonists and that the biological and/or pharmacological response mediated by P2Y receptors in target tissues is highly dependent on the types of ectonucleotidases expressed in the vicinity of the receptor.

Induction of novel agonist selectivity for the ADP-activated P2Y1 receptor versus the ADP-activated P2Y12 and P2Y13 receptors by conformational constraint of an ADP analog.

ADP is the cognate agonist of the P2Y1, P2Y12, and P2Y13 receptors. With the goal of identifying a high potency agonist that selectively activates the P2Y1 receptor, we examined the pharmacological selectivity of the conformationally constrained non-nucleotide analog (N)-methanocarba-2MeSADP [(1'S,2'R, 3'S,4'R,5'S)-4-[(6-amino-2-methylthio-9H-purin-9-yl)-1-diphosphoryloxymethyl]bicyclo[3.1.0]hexane-2,3-diol] among the three ADP-activated receptors. Each P2Y receptor was expressed transiently in COS-7 cells, and inositol lipid hydrolysis was quantified as a measure of receptor activity. In the case of the Gi-linked P2Y12 and P2Y13 receptors, a chimeric G protein, Galphaq/i, was coexpressed to confer a capacity of these Gi-linked receptors to activate phospholipase C. 2MeSADP (2-methylthio-ADP) was a potent agonist at all three receptors exhibiting EC50 values in the sub to low nanomolar range. In contrast, whereas (N)-methanocarba-2MeSADP was an extremely potent (EC50=1.2 +/- 0.2 nM) agonist at the P2Y1 receptor, this non-nucleotide analog exhibited no agonist activity at the P2Y12 receptor and very low activity at the P2Y13 receptor. (N)-Methanocarba-2MeSADP also failed to block the action of 2MeSADP at the P2Y12 and P2Y13 receptors, indicating that the (N)-methanocarba analog is not an antagonist at these receptors. The P2Y1 receptor selectivity of (N)-methanocarba-2MeSADP was confirmed in human platelets where it induced the shape change promoted by P2Y1 receptor activation without inducing the sustained platelet aggregation that requires simultaneous activation of the P2Y12 receptor. These results provide the first demonstration of a high-affinity agonist that discriminates among the three ADP-activated P2Y receptors, and therefore, introduce a potentially important new pharmacological tool for delineation of the relative biological action of these three signaling proteins.

Purification and functional reconstitution of the human P2Y12 receptor.

The human P2Y12 receptor (P2Y12-R) is a member of the G protein coupled P2Y receptor family, which is intimately involved in platelet physiology. We describe here the purification and functional characterization of recombinant P2Y12-R after high-level expression from a baculovirus in Sf9 insect cells. Purified P2Y12-R, Gbeta1gamma2, and various Galpha-subunits were reconstituted in lipid vesicles, and steady-state GTPase activity was quantified. GTP hydrolysis in proteoliposomes formed with purified P2Y12-R and Galphai2beta1gamma2 was stimulated by addition of either 2-methylthio-ADP (2MeSADP) or RGS4 and was markedly enhanced by their combined presence. 2MeSADP was the most potent agonist (EC50 = 80 nM) examined, whereas ADP, the cognate agonist of the P2Y12-R, was 3 orders of magnitude less potent. ATP had no effect alone but inhibited the action of 2MeSADP; therefore, ATP is a relatively low-affinity antagonist of the P2Y12-R. The G protein selectivity of the P2Y12-R was examined by reconstitution with various G protein alpha-subunits in heterotrimeric form with Gbeta1gamma2. The most robust coupling of the P2Y12-R was to Galphai2, but effective coupling also occurred to Galphai1 and Galphai3. In contrast, little or no coupling occurred to Galphao or Galphaq. These results illustrate that the signaling properties of the P2Y12-R can be studied as a purified protein under conditions that circumvent the complications that occur in vivo because of nucleotide metabolism and interconversion as well as nucleotide release.

Requirement of Cys399 for processing of the human ecto-ATPase (NTPDase2) and its implications for determination of the activities of splice variants of the enzyme.

Ecto-ATPase (CD39L1) corresponds to the type 2 enzyme of the ecto-nucleoside triphosphate diphosphohydrolase family (E-NTPDase). We have isolated from human ECV304 cells three cDNAs with high homology with members of the E-NTPDase family that encode predicted proteins of 495, 472, and 450 amino acids. Sequencing of a genomic DNA clone confirmed that these three sequences correspond to splice variants of the human ecto-ATPase (NTPDase2 alpha,-2 beta, and -2 gamma). Although all three enzyme forms were expressed heterologously to similar levels in Chinese hamster ovary cells clone K-1 (CHO-K1) cells, only the 495-amino acid protein (NTPDase2 alpha exhibited ecto-ATPase activity. Immunolocalization studies demonstrated that NTPDase2 alpha is fully processed and trafficked to the plasma membrane, whereas the NTPDase2 beta and -2 gamma splice variants were retained in not fully glycosylated forms in the endoplasmic reticulum. The potential roles of two highly conserved residues, Cys399 and Asn443, in the activity and cellular trafficking of the ecto-ATPase were examined. Mutation of Cys399, which is absent in NTPDase2 beta and -2 gamma, produced a protein completely devoid of nucleotidase activity, while mutation of Asn443 to Asp resulted in substantial loss of activity. Neither the Cys399 nor Asn443 mutants were fully glycosylated, and both were retained in the endoplasmic reticulum. These results indicate that the lack of ecto-nucleotidase activity exhibited by NTPDase2 beta and -2 gamma and the C399S mutant, as well as the large reduction of activity in the N443D mutant are due to alterations in the folding/maturation of these proteins.

Characterization of the UDP-glucose receptor (re-named here the P2Y14 receptor) adds diversity to the P2Y receptor family.

The cloning of a human G-protein-coupled receptor (GPCR) that specifically responds to UDP-glucose and related sugar-nucleotides has been reported recently. This receptor has important structural similarities to known members of the P2Y receptor family but also shows a distinctly different pharmacological response profile. Here, the IUPHAR Subcommittee for P2Y receptor nomenclature and classification review the current knowledge of this receptor and present their reasons for including this receptor in the P2Y receptor family as the P2Y(14) receptor.

Quantitation of the P2Y(1) receptor with a high affinity radiolabeled antagonist.

2-Chloro-N(6)-methyl-(N )-methanocarba-2'-deoxyadenosine-3',5'- bisphosphate (MRS2279) was developed previously as a selective high-affinity, non-nucleotide P2Y(1) receptor (P2Y1-R) antagonist (J Med Chem 43:829-842, 2002; Br J Pharmacol 135:2004-2010, 2002). We have taken advantage of the N(6)-methyl substitution in the adenine base to incorporate [(3)H]methylamine into the synthesis of [(3)H]MRS2279 to high (89 Ci/mmol) specific radioactivity and have used this molecule as a radioligand for the P2Y1-R. [(3)H]MRS2279 bound to membranes from Sf9 insect cells expressing recombinant human P2Y1-R but not to membranes from wild-type Sf9 cells or Sf9 cells expressing high levels of recombinant P2Y(2) or P2Y(12) receptors. Equilibrium binding of [(3)H]MRS2279 to P2Y1-R expressed in Sf9 membranes was with a high affinity (K(d) = 8 nM) essentially identical to the apparent affinity of MRS2279 determined previously in studies of P2Y1-R-promoted inositol phosphate accumulation or platelet aggregation. A kinetically derived K(d) calculated from independent determinations of the rate constants of association (7.15 x 10(7) M(-1) min(-1)) and dissociation (0.72 min(-1)) of [(3)H]MRS2279 also was in good agreement with the K(d) derived from equilibrium binding studies. Competition binding assays with [(3)H]MRS2279 and P2Y1-R expressing Sf9 cell membranes revealed K(i) values for the P2Y1-R antagonists MRS2279 (K(i) = 13 nM), N(6)-methyl-2'-deoxyadenosine-3',5'-bisphosphate (MRS2179; K(i) = 84 nM), adenosine-3', 5'-bisphosphate (K(i)=900 nM), and pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (K(i) = 6 microM) that were in good agreement with antagonist activities of these molecules previously determined at the P2Y1-R in intact tissues. Moreover, [(3)H]MRS2279 also bound with high affinity (K(d) = 4-8 nM) to Chinese hamster ovary (CHO) or 1321N1 human astrocytoma cells stably expressing the human P2Y1-R, but specific binding was not observed in wild-type CHO or 1321N1 cells. [(3)H]MRS2279 bound with high affinity (K(d) = 16 nM) to a binding site on out-dated human platelets (5-35 receptors/platelet) and rat brain membranes (210 fmol/mg protein) that fit the expected drug selectivity of a P2Y1-R. Taken together, these results indicate that [(3)H]MRS2279 is the first broadly applicable antagonist radioligand for a P2Y receptor.

A fusion protein of the human P2Y(1) receptor and NTPDase1 exhibits functional activities of the native receptor and ectoenzyme and reduced signaling responses to endogenously released nucleotides.

To begin to address the functional interactions between constitutively released nucleotides, ectonucleotidase activity, and P2Y receptor-promoted signaling responses, we engineered the human P2Y(1) receptor in a fusion protein with a member of the ectonucleoside triphosphate diphosphohydrolase family, NTPDase1. Membranes prepared from Chinese hamster ovary (CHO)-K1 cells stably expressing either wild-type NTPDase1 or the P2Y(1) receptor-NTPDase1 fusion protein exhibited nucleotide-hydrolytic activities that were over 300-fold greater than activity measured in membranes from empty vector-transfected cells. The molecular ratio for nucleoside triphosphate versus diphosphate hydrolysis was approximately 1:0.4 for both the wild-type NTPDase1 and P2Y(1)-NTPDase1 fusion protein. Stable expression of the P2Y(1)-NTPDase1 fusion protein conferred an ADP and 2MeSADP-promoted Ca(2+) response to CHO-K1 cells. Moreover, the maximal capacity of the nonhydrolyzable agonist ADPbetaS to stimulate inositol phosphate accumulation was similar, and the EC(50) of ADPbetaS was lower in the fusion protein than the wild-type receptor. In contrast, the substantial nucleotide-hydrolyzing activity of the fusion protein resulted in a greater than 50-fold shift to the right of the concentration-effect curve of ADP for activation of phospholipase C compared with the wild-type receptor. Heterologous expression of the P2Y(1) and other P2Y receptors results in marked increases in basal inositol phosphate levels. Given the high nucleotidase activity and apparently normal receptor signaling activity of the P2Y(1) receptor-NTPDase1 fusion protein, we quantitated basal inositol phosphate accumulation in cells stably expressing either the wild-type P2Y(1) receptor or the fusion protein. Although marked elevation of inositol phosphate levels occurred with wild-type P2Y(1) receptor expression, levels in cells expressing the fusion protein were not different from those in wild-type CHO-K1 cells.

Adenine nucleotide analogues locked in a Northern methanocarba conformation: enhanced stability and potency as P2Y(1) receptor agonists.

Preference for the Northern (N) ring conformation of the ribose moiety of nucleotide 5'-triphosphate agonists at P2Y(1), P2Y(2), P2Y(4), and P2Y(11) receptors, but not P2Y(6) receptors, was established using a ring-constrained methanocarba (a 3.1.0-bicyclohexane) ring as a ribose substitute (Kim et al. J. Med. Chem. 2002, 45, 208-218.). We have now combined the ring-constrained (N)-methanocarba modification of adenine nucleotides with other functionalities known to enhance potency at P2 receptors. The potency of the newly synthesized analogues was determined in the stimulation of phospholipase C through activation of turkey erythrocyte P2Y(1) or human P2Y(1) and P2Y(2) receptors stably expressed in astrocytoma cells. An (N)-methanocarba-2-methylthio-ADP analogue displayed an EC(50) at the hP2Y(1) receptor of 0.40 nM and was 55-fold more potent than the corresponding triphosphate and 16-fold more potent than the riboside 5'-diphosphate. 2-Cl-(N)-methanocarba-ATP and its N(6)-Me analogue were also highly selective, full agonists at P2Y(1) receptors. The (N)-methanocarba-2-methylthio and 2-chloromonophosphate analogues were full agonists exhibiting micromolar potency at P2Y(1) receptors, while the corresponding ribosides were inactive. Although beta,gamma-methylene-ATP was inactive at P2Y receptors, beta,gamma-methylene-(N)-methanocarba-ATP was a potent hP2Y(1) receptor agonist with an EC(50) of 160 nM and was selective versus hP2Y(2) and hP2Y(4) receptors. The rates of hydrolysis of Northern (N) and Southern (S) methanocarba analogues of AMP by rat 5'-ectonucleotidase were negligible. The rates of hydrolysis of the corresponding triphosphates by recombinant rat NTPDase1 and 2 were studied. Both isomers were hydrolyzed by NTPDase 1 at about half the rate of ATP hydrolysis. The (N) isomer was hardly hydrolyzed by NTPDase 2, while the (S) isomer was hydrolyzed at one-third of the rate of ATP hydrolysis. This suggests that new, more stable and selective nucleotide agonists may be designed on the basis of the (N)-conformation, which greatly enhanced potency at P2Y(1) receptors.

2-Chloro N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate is a selective high affinity P2Y(1) receptor antagonist.

1. We reported previously that bisphosphate derivatives of adenosine are antagonists of the P2Y(1) receptor and that modification of the ribose in these analogues is tolerated in the P2Y(1) receptor binding pharmacophore. 2. Here we delineate the pharmacological activity of one such non-nucleotide molecule, 2-chloro N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate (MRS2279), in which the ribose is replaced by a cyclopentane ring constrained in the (N)-conformation by a cyclopropane moiety. 3. MRS2279 antagonized 2MeSADP-stimulated inositol phosphate formation in turkey erythrocyte membranes with competitive kinetics (pK(B)=7.75). High affinity competitive antagonism by MRS2279 was also observed at the human P2Y(1) receptor (pK(B)=8.10) stably expressed in 1321N1 human astrocytoma cells. Antagonism was specific for the P2Y(1) receptor since MRS2279 had no effect on activation of the human P2Y(2), P2Y(4), P2Y(6), or P2Y(11) receptors by their cognate agonists. 4. MRS2279 also did not block the capacity of ADP to act through the Gi/adenylyl cyclase linked P2Y receptor of platelets to inhibit cyclic AMP accumulation. 5. In contrast, the P2Y(1) receptor is known to be obligatory in the process of ADP-induced platelet aggregation, and MRS2279 competitively inhibited ADP-promoted platelet aggregation with an apparent affinity (pK(B)=8.05) similar to that observed at the human P2Y(1) receptor heterologously expressed in 1321N1 cells. 6. Taken together these results illustrate selective high affinity antagonism of the P2Y(1) receptor by a non-nucleotide molecule that should prove useful for pharmacological delineation of this receptor in various tissues.