Action of Nucleosides and Nucleotides at 7 Transmembrane-Spanning Receptors

Ribose ring-constrained nucleosides and nucleotides to act at cell-surface purine recesptors have been designed and synthesized. At the P2Y₁ nucleotide receptor and the A₃ adenosine receptor (AR) the North envelope conformation of ribose is highly preferred. We have applied mutagenesis and rhodopsin-based homology modeling to the study of purine receptors and used the structural insights gained to assist in the design of novel ligands. Two subgroups of P2Y receptors have been defined, containing different sets of cationic residues for coordinating the phosphate groups. Modeling/mutagenesis of adenosine receptors has focused on determinants of intrinsic efficacy in adenosine derivatives and on a conserved Trp residue (6.48) which is involved in the activation process. The clinical use of adenosine agonists as cytoprotective agents has been limited by the widespread occurrence of ARs, thus, leading to undesirable side effects of exogenously administered adenosine derivatives. In order to overcome the inherent nonselectivity of activating the native receptors, we have introduced the concept of neoceptors. By this strategy, intended for eventual use in gene therapy, the putative ligand binding site of a G protein-coupled receptor is reengineered for activation by synthetic agonists (neoligands) built to have a structural complementarity. Using a rational design process we have identified neoceptor-neoligand pairs which are pharmacologically orthogonal with respect to the native species.     

Purine receptors: GPCR structure and agonist design

An integrated approach to the study of drug-receptor interactions has been applied to adenosine receptors (ARs) and P2Y nucleotide receptors. This approach includes probing the receptor structure through site-directed mutagenesis and molecular modeling, in concert with altering the structure of the agonist ligands. Goals of this structural approach are to generate a testable hypothesis for location of the binding site and subsequently to enable the rational design of new agonists and antagonists. In this manner, receptor subtype selectivity has been increased, and agonists have been converted into partial agonists and antagonists. An approach to receptor engineering (neoceptors) has been explored, in which synthetic small molecule agonists (neoligands) are specifically tailored to activate only receptors in which the putative binding sites have been modified. This orthogonal approach to receptor activation, intended for eventual gene therapy, has been demonstrated for A3 and A2A ARs.     

Structural and functional evolution of the P2Y12-like receptor group

Metabotropic pyrimidine and purine nucleotide receptors (P2Y receptors) belong to the superfamily of G protein-coupled receptors (GPCR). They are distinguishable from adenosine receptors (P1) as they bind adenine and/or uracil nucleotide triphosphates or diphosphates depending on the subtype. Over the past decade, P2Y receptors have been cloned from a variety of tissues and species, and as many as eight functional subtypes have been characterized. Most recently, several members of the P2Y₁₂-like receptor group, which includes the clopidogrel-sensitive ADP receptor P2Y₁₂, have been deorphanized. The P2Y₁₂-like receptor group comprises several structurally related GPCR which, however, display heterogeneous agonist specificity including nucleotides, their derivatives, and lipids. Besides the established function of P2Y₁₂ in platelet activation, expression in macrophages, neuronal and glial cells as well as recent results from functional studies implicate that several members of this group may have specific functions in neurotransmission, inflammation, chemotaxis, and response to tissue injury. This review focuses specifically on the structure-function relation and shortly summarizes some aspects of the physiological relevance of P2Y₁₂-like receptor members.     

Ribose modified nucleosides and nucleotides as ligands for purine receptors

Molecular modeling of receptors for adenosine and nucleotide (P2) receptors with docked ligand, based on mutagenesis, was carried out. Adenosine 3',5'-bisphosphate derivatives act as selective P2Y1 antagonists/partial agonists. The ribose moiety was replaced with carbocyclics, smaller and larger rings, conformationally constrained rings, and acyclics, producing compounds that retained receptor affinity. Conformational constraints were built into the ribose rings of nucleoside and nucleotide ligands using the methanocarba approach, i.e. fused cyclopropane and cyclopentane rings in place of ribose, suggesting a preference for the Northern (N) conformation among ligands for P2Y1 and A1 and A3ARs.     

Purinergic mechanisms in the control of gastrointestinal motility

For many years, ATP and adenosine have been implicated in movement regulation of the gastrointestinal tract. They act through three major receptor subtypes: adenosine or P1 receptors, P2X receptors and P2Y receptors. Each of these major receptor types can be subdivided into several different classes and is widely distributed amongst various neurons, muscle types, glia and interstitial cells that regulate intestinal functions. Several key roles for the different receptors and their endogenous ligands have been identified in physiological and pharmacological studies. For example, adenosine acting at A(1) receptors appears to inhibit intestinal motility in various pathological conditions. Similarly, ATP acting at P2Y receptors is an important component of inhibitory neuromuscular transmission, acting as a cotransmitter with nitric oxide. ATP acting at P2X and P2Y(1) receptors is important for synaptic transmission in simple descending excitatory and inhibitory reflex pathways. Some P2Y receptor subtypes prefer uridine nucleotides over purine nucleotides. Thus, roles for UTP and UDP as enteric transmitters in place of ATP cannot be excluded. ATP also appears to be important for sensory transduction, especially in chemosensitive pathways that initiate local inhibitory reflexes. Despite this evidence, data are lacking about the roles of either adenosine or ATP in more complex motility patterns such as segmentation or the interdigestive migrating motor complex. Clarification of roles for purinergic transmission in these common, but understudied, motility patterns will depend on the use of subtype-specific antagonists that in some cases have not yet been developed.     

Regulation of pharmacology by hetero-oligomerization between A1 adenosine receptor and P2Y2 receptor

Adenosine and ATP/UTP are main components of the purinergic system that modulate cellular and tissue functions via specific adenosine and P2 receptors, respectively. Here, we explored the possibility that A(1) adenosine receptor (A(1)R) and P2Y(2) receptor (P2Y(2)R) form heterodimers with novel pharmacological properties. Coimmunoprecipitation showed these receptors directly associate in A(1)R/P2Y(2)R-cotransfected HEK293T cells. Agonist binding by the A(1)R was significantly inhibited by P2Y(2)R agonists only in membranes from cotransfected cells. The functional activity of A(1)R, as indicated by the G(i/o)-mediated inhibition of adenylyl cyclase, in the cotransfected cells was attenuated by the simultaneous addition of A(1)R and P2Y(2)R agonists. The increase in intracellular Ca(2+) levels induced by P2Y(2)R activation of G(q/11) was synergistically enhanced by the simultaneous addition of an A(1)R agonist in the coexpressing cells. These results suggest that oligomerization of A(1)R and P2Y(2)R generates a unique complex in which the simultaneous activation of the two receptors induces a structural alteration that interferes signaling via G(i/o) but enhances signaling via G(q/11).     

Characterization and channel coupling of the P2Y(12) nucleotide receptor of brain capillary endothelial cells

Rat brain capillary endothelial (B10) cells express an unidentified nucleotide receptor linked to adenylyl cyclase inhibition. We show that this receptor in B10 cells is identical in sequence to the P2Y(12) ADP receptor ("P2Y(T)") of platelets. When expressed heterologously, 2-methylthio-ADP (2-MeSADP; EC(50), 2 nm), ADP, and adenosine 5'-O-(2-thio)diphosphate were agonists of cAMP decrease, and 2-propylthio-D-beta,gamma-difluoromethylene-ATP was a competitive antagonist (K(B), 28 nm), as in platelets. However, 2-methylthio-ATP (2-MeSATP) (EC(50), 0.4 nm), ATP (1.9 microm), and 2-chloro-ATP (190 nm), antagonists in the platelet, were also agonists. 2-MeSADP activated (EC(50), 0.1 nm) GIRK1/GIRK2 inward rectifier K(+) channels when co-expressed with P2Y(12) receptors in sympathetic neurons. Surprisingly, P2Y(1) receptors expressed likewise gave that response; however, a full inactivation followed, absent with P2Y(12) receptors. A new P2Y(12)-mediated transduction was found, the closing of native N-type Ca(2+) channels; again both 2-MeSATP and 2-MeSADP are agonists (EC(50), 0.04 and 0.1 nm, respectively). That action, like their cAMP response, was pertussis toxin-sensitive. The Ca(2+) channel inhibition and K(+) channel activation are mediated by beta gamma subunit release from a heterotrimeric G-protein. G alpha subunit types in B10 cells were also identified. The presence in the brain capillary endothelial cell of the P2Y(12) receptor is a significant extension of its functional range.     

Modulation of ion channel function by P2Y receptors

P2Y receptors are classified as P2 purinergic receptors that belong to the superfamily of G-protein coupled receptors. They are distinguishable from P1 (adenosine) receptors in that they bind adenine and/or uracil nucleotide triphosphates or diphosphates depending on the subtype. Over the past decade, P2Y receptors have been cloned from a variety of tissues and species. Eight functional subtypes have been characterized. Nucleotide binding produces activation of specific G-proteins that in turn regulate the function of membrane bound enzymes including phospholipase C and adenylyl cyclase. Certain P2Y receptor subtypes possess a PDZ domain located at the end of the C-terminal region of the receptor. PDZ domains have been established as sites for protein-protein interaction, thus providing a possible mechanism for receptor modulation of membrane protein function independent of G-protein activation. In this review we discuss recent findings that suggest that P2Y receptors can modulate the function of ion channels through multiple protein-protein interactions at the plasma membrane that do not directly involve G-protein activation.     

ADP is the cognate ligand for the orphan G protein-coupled receptor SP1999

P2Y receptors are a class of G protein-coupled receptors activated primarily by ATP, UTP, and UDP. Five mammalian P2Y receptors have been cloned so far including P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11. P2Y1, P2Y2, and P2Y6 couple to the activation of phospholipase C, whereas P2Y4 and P2Y11 couple to the activation of both phospholipase C and the adenylyl cyclase pathways. Additional ADP receptors linked to Galpha(i) have been described but have not yet been cloned. SP1999 is an orphan G protein-coupled receptor, which is highly expressed in brain, spinal cord, and blood platelets. In the present study, we demonstrate that SP1999 is a Galpha(i)-coupled receptor that is potently activated by ADP. In an effort to identify ligands for SP1999, fractionated rat spinal cord extracts were assayed for Ca(2+) mobilization activity against Chinese hamster ovary cells transiently transfected with SP1999 and chimeric Galpha subunits (Galpha(q/i)). A substance that selectively activated SP1999-transfected cells was identified and purified through a series of chromatographic steps. Mass spectral analysis of the purified material definitively identified it as ADP. ADP was subsequently shown to inhibit forskolin-stimulated adenylyl cyclase activity through selective activation of SP1999 with an EC(50) of 60 nM. Other nucleotides were able to activate SP1999 with a rank order of potency 2-MeS-ATP = 2-MeS-ADP > ADP = adenosine 5'-O-2-(thio)diphosphate > 2-Cl-ATP > adenosine 5'-O-(thiotriphosphate). Thus, SP1999 is a novel, Galpha(i)-linked receptor for ADP.     

Adenosine diphosphate receptors on blood platelets: potential new targets for antiplatelet therapy

Platelets play a key role not only in physiological haemostasis, but also under pathological conditions such as thrombosis. Platelet activation may be initiated by a variety of agonists including thrombin, collagen, thromboxane or adenosine diphosphate (ADP). Although ADP is regarded as a weak agonist of blood platelets, it remains an important mediator of platelet activation evoked by other agonists, which induce massive ADP release from dense granules, where it occurs in molar concentrations. Thus, ADP action underlies a positive feedback that facilitates further platelet aggregation and leads to platelet plug formation. Additionally, ADP acts synergistically to other, even weak, agonists such as serotonin, adrenaline or chemokines. Blood platelets express two types of P2Y ADP receptors: P2Y(1) and P2Y(12). ADP-dependent platelet aggregation is initiated by the P2Y1 receptor, whereas P2Y(12) receptor augments the activating signal and promotes platelet release reaction. Stimulation of P2Y(12) is also essential for ADP-mediated complete activation of GPIIb-IIIa and GPIa-IIa, and further stabilization of platelet aggregates. The crucial role in blood platelet biology makes P2(Y12) an ideal candidate for pharmacological approaches for anti-platelet therapy.     

Partial adenosine A1 receptor agonists for cardiovascular therapies

Adenosine, a purine nucleoside, is present in all cells in tightly regulated concentrations. It has many different physiological effects in the whole body and in the heart. Adenosine activates four G protein-coupled receptors A1, A2a, A2b, and A3. Activation of myocardial A1 receptors has been shown to inhibit a variety of myocardial pathologies associated with ischemia and reperfusion injury, including stunning, arrhythmogenesis, coronary and ventricular dysfunction, acute myocardial infarction, apoptosis, and chronic heart failure, implying several options for new cardiovascular therapies for diseases, like angina pectoris, control of cardiac rhythm, ischemic injury during an acute coronary syndrome, or heart failure. However, the main issue of using full A1 receptor agonists in such indications is the broad physiologic spectrum of cardiac and extracardiac effects. Desired A1 receptor-mediated protective and regenerative cardiovascular effects might be counter-regulated by unintended side effects when considering full A1 receptor agonists. These effects can be overcome by partial A1 agonists. Partial A1 agonists can be used to trigger only some of the physiological responses of receptor activation depending on endogenous adenosine levels and on receptor reserve in different tissues. CV-Therapeutics reported the identification of a partial A1 receptor agonist CVT-3619, and recently, another partial A1 receptor agonist VCP28 was published. Both compounds are adenosine derivatives. Adenosine-like A1 receptor agonists often have the drawback of a short half-life and low bioavailability, making them not suitable for chronic oral therapy. We identified the first non-adenosine-like partial A1 receptor agonist(s) with pharmacokinetics optimal for oral once daily treatment and characterized the qualities of the partial character of the A1 receptor agonist(s) in preclinical and clinical studies.

     

Agonist-promoted heteromeric oligomerization between adenosine A(1) and P2Y(1) receptors in living cells

We have explored the process of oligomerization of G protein-coupled purinergic receptors, adenosine A(1) receptor (A(1)R) and P2Y(1) receptor (P2Y(1)R), in intact HEK293T cells by means of modified bioluminescence resonance energy transfer technology (BRET(2)) that offers greatly improved separation of the emission spectra of the donor and acceptor moieties compared to traditional BRET. This approach identified both constitutive and agonist-promoted heteromeric oligomerization between Myc-tagged P2Y(1)R fused to a donor, Renilla luciferase (Myc-P2Y(1)R-Rluc) and HA-tagged A(1)R fused to an acceptor, a different form of green fluorescent protein (HA-A(1)R-GFP(2)). The BRET(2) signal increased in a time-dependent manner in the cells expressing HA-A(1)R-GFP(2)/Myc-P2Y(1)R-Rluc upon addition of agonists for both receptors, which could be inhibited by pretreatment with the P2Y(1)R antagonist MRS2179. A high degree of HA-A(1)R-GFP(2) and Myc-P2Y(1)R-Rluc co-localization in the co-transfected HEK293T cells was also observed by confocal laser microscopy. These results indicate that A(1)R and P2Y(1)R can form constitutive hetero-oligomers in living cells and this process is promoted by the simultaneous activation of both receptors.     

A novel pharmacological approach to treating cardiac ischemia. Binary conjugates of A1 and A3 adenosine receptor agonists

Adenosine released during cardiac ischemia exerts a potent, protective effect in the heart via activation of A(1) or A(3) receptors. However, the interaction between the two cardioprotective adenosine receptors and the question of which receptor is the more important anti-ischemic receptor remain largely unexplored. The objective of this study was to test the hypothesis that activation of both receptors exerted a cardioprotective effect that was significantly greater than activation of either receptor individually. This was accomplished by using a novel design in which new binary conjugates of adenosine A(1) and A(3) receptor agonists were synthesized and tested in a novel cardiac myocyte model of adenosine-elicited cardioprotection. Binary drugs having mixed selectivity for both A(1) and A(3) receptors were created through the covalent linking of functionalized congeners of adenosine agonists, each being selective for either the A(1) or A(3) receptor subtype. MRS 1740 and MRS 1741, thiourea-linked, regioisomers of a binary conjugate, were highly potent and selective in radioligand binding assays for A(1) and A(3) receptors (K(i) values of 0.7-3.5 nm) versus A(2A) receptors. The myocyte models utilized cultured chick embryo cells, either ventricular cells expressing native adenosine A(1) and A(3) receptors, or engineered atrial cells, in which either human A(3) receptors alone or both human A(1) and A(3) receptors were expressed. The binary agonist MRS 1741 coactivated A(1) and A(3) receptors simultaneously, with full cardioprotection (EC(50) approximately 0.1 nm) dependent on expression of both receptors. Thus, co-activation of both adenosine A(1) and A(3) receptors by the binary A(1)/A(3) agonists represents a novel general cardioprotective approach for the treatment of myocardial ischemia.     

Negative Regulation of CFTR Activity by Extracellular ATP Involves P2Y2 Receptors in CFTR-expressing CHO Cells

Extracellular nucleotides exert autocrine/paracrine effects on ion transport by activating P2 receptors. We studied the effects of extracellular ATP and UTP on the cystic fibrosis transmembrane conductance regulator (CFTR) channel stably expressed in Chinese Hamster Ovary cells (CHO-BQ1 cells). CFTR activity was measured using the (¹²⁵I) iodide efflux technique and whole-cell patch-clamp recording in response to either forskolin or xanthine derivatives. Using RT-PCR and intracellular calcium concentration ([Ca²⁺]_i) measurement, we showed that CHO-BQ1 cells express P2Y2 but not P2Y4 receptors. While ATP and UTP induced similar increases in [Ca²⁺]_i, pre-addition by one of these two agonists desensitized the response for the other, suggesting that ATP- and UTP-induced [Ca²⁺]_i increases were mediated by a common receptor, which was identified as the P2Y2 subtype. CFTR activity was reduced by ATP and UTP but not by ADP or adenosine applications. This inhibitory effect of ATP on CFTR activity was not due to a change in cAMP level. Furthermore, CFTR activation by forskolin or IBMX failed to promote [Ca²⁺]_i increase, suggesting that CFTR activation did not generate an ATP release large enough to stimulate P2Y2 receptors. Taken together, our results show that endogenous P2Y2 receptor activation downregulates CFTR activity in a cAMP-independent manner in CHO cells. B. Marcet and V. Chappe contributed equally to this work.     

Current knowledge on the nucleotide agonists for the P2Y2 receptor

P2Y receptors are G-protein-coupled receptors (GPCRs) for extracellular nucleotides. There are eight mammalian P2Y receptor subtypes (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14). P2Y2 receptors are widely expressed and play important roles in multiple functionalities. Diquafosol tetrasodium, known as INS365, which was the first P2Y2 receptor agonists that had been approved in April 2010 and launched in Japan by Santen Pharmaceuticals. Besides, a series of similar agonists for the P2Y2 receptor are undergoing development to cure different diseases related to the P2Y2 receptor. This article illustrated the structure and functions of the P2Y2 receptor and focused on several kinds of agonists about their molecular structures, research progress and chemical synthesis methods. Last but not the least, we summarized the structures-activity relationship (SAR) of agonists for the P2Y2 receptor and expected more efficient agonists for the P2Y2 receptor.     

Adenosine receptors and cancer

Adenosine is a ubiquitous signaling molecule whose physiological functions are mediated by its interaction with four G-protein-coupled receptor subtypes, termed A(1), A(2A), A(2B) and A(3). As a result of increased metabolic rates, this nucleoside is released from a variety of cells throughout the body in concentrations that can have a profound impact on vasculature and immunoescape. However, as high concentrations of adenosine have been reported in cancer tissues, it also appears to be implicated in the growth of tumors. Thus, full characterisation of the role of adenosine in tumor development, by addressing the question of whether adenosine receptors are present in cancer tissues, and, if so, which receptor subtype mediates its effects in cancer growth, is a vital research goal. To this end, this review focuses on the most relevant aspects of adenosine receptor subtype activation in tumors reported so far. Although all adenosine receptors now have an increasing number of recognised biological roles in tumors, it seems that the A(2A) and A(3) subtypes are the most promising as regards drug development. In particular, activation of A(2A) receptors leads to immunosuppressive effects, which decreases anti-tumoral immunity and thereby encourages tumor growth. Due to this behavior, the addition of A(2A) antagonists to cancer immunotherapeutic protocols has been suggested as a way of enhancing tumor immunotherapy. Interestingly, the safety of such compounds has already been demonstrated in trials employing A(2A) antagonists in the treatment of Parkinson's disease. As for A(3) receptors, the effectiveness of their agonists in several animal tumor models has led to the introduction of these molecules into a programme of pre-clinical and clinical trials. Paradoxically, A(3) receptor antagonists also appear to be promising candidates in human cancer treatment of regimes. Clearly, research in this still field is still in its infancy, with several important and challenging issues remaining to be addressed, although purine scientists do seem to be getting closer to their goal: the incorporation of adenosine ligands into drugs with the ability to save lives and improve human health.     

Conserved helix 7 tyrosine acts as a multistate conformational switch in the 5HT2C receptor. Identification of a novel "locked-on" phenotype and double revertant mutations

Studies in many rhodopsin-like G-protein-coupled receptors are providing a general scheme of the structural processes underlying receptor activation. Microdomains in several receptors have been identified that appear to function as activation switches. However, evidence is emerging that these receptor proteins exist in multiple conformational states. To study the molecular control of this switching process, we investigated the function of a microdomain involving the conserved helix 7 tyrosine in the serotonin 5HT2C receptor. This tyrosine of the NPXXY motif was substituted for all naturally occurring amino acids. Three distinct constitutively active receptor phenotypes were found: moderate, high, and "locked-on" constitutive activity. In contrast to the activity of the other receptor mutants, the high basal signaling of the locked-on Y7.53N mutant was neither increased by agonists nor decreased by inverse agonists. The Y7.53F mutant was uncoupled. Computational modeling based on the rhodopsin crystal structure suggested that Y7.53 interacts with the conserved aromatic ring at position 7.60 in the recently identified helix 8 domain. This provided a basis for seeking revertant mutations to correct the defective function of the Y7.53F receptor. When the Y7.53F receptor was mutated at position 7.60, the wild-type phenotype was restored. These results suggest that Y7.53 and Y7.60 contribute to a common functional microdomain connecting helices 7 and 8 that influences the switching of the 5HT2C receptor among multiple active and inactive conformations.     

Adenosine in vertebrate retina: Localization,receptor characterization,and function

1. The uptake of [3H] adenosine into specific populations of cells in the inner retina has been demonstrated. In mammalian retina, the exogenous adenosine that is transported into cells is phosphorylated, thereby maintaining a gradient for transport of the purine into the cell. 2. Endogenous stores of adenosine have been demonstrated by localization of cells that are labeled for adenosine-like immunoreactivity. In the rabbit retina, certain of these cells, the displaced cholinergic, GABAergic amacrine cells, are also labeled for adenosine. 3. Purines are tonically released from dark-adapted rabbit retinas and cultured embryonic chick retinal neurons. Release is significantly increased with K+ and neurotransmitters. The evoked release consists of adenosine, ATP, and purine metabolites, and while a portion of this release is Ca2+ dependent, one other component may occur via the bidirectional purine nucleoside transporter. 4. Differential distributions of certain enzymes involved in purine metabolism have also been localized to the inner retina. 5. Heterogeneous distributions of the two subtypes of adenosine receptors, A1 and A2, have been demonstrated in the mammalian retina. Coupling of receptors to adenylate cyclase has also been demonstrated. 6. Adenosine A1 receptor agonists significantly inhibit the K(+)-stimulated release of [3H]-acetylcholine from the rabbit retina, suggesting that endogenous adenosine may modulate the light-evoked or tonic release of ACh.