Extracellular purines and pyrimidines bind to several families of transmembrane-spanning (7TM) receptors. These receptors are G protein-coupled receptors of the Group A rhodopsin-like family, which includes many important targets for pharmaceutical development. We have applied mutagenesis and rhodopsin-based homology modeling to the study of purine and pyrimidine receptors and used the structural insights gained to assist in the design of novel ligands. Because crystallographic structural determination of nearly all 7TM receptors is not yet feasible, we achieved the structure-function analysis of adenosine and P2Y receptors by indirect means, using mutagenesis and homology modeling, based on a template of the high-resolution structure of rhodopsin. 3D homology models of the human A2A (1UPE), A3 (1OEA), P2Y1 (1Y36), P2Y2 (1Z8E), P2Y6 (2B6R) and P2Y12 (1Y9C) receptors from our laboratory and others have been deposited in the RCSB Protein Data Bank (Rutgers, The State University of New Jersey, USA). The nucleotide receptors, denoted P2, comprise both ligand-gated ion channels (seven subtypes of P2X receptors) and 7TM receptors (eight subtypes of P2Y receptors). The distribution of P2Y receptors is broad, and the therapeutic interests include antithrombotic therapy, modulation of the immune system and cardiovascular system, and treatment of cystic fibrosis and other pulmonary diseases. There are two subgroups of P2Y receptors, containing different sets of cationic residues for coordinating the phosphate groups. The two subgroups have been defined based on clustering of sequences (in general, low similarity among subtypes), ligand preference, second messenger coupling, and receptor sequence analysis. The P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptors couple preferentially to the stimulation of phospholipase C-beta, and the P2Y12, P2Y13, and P2Y14 receptors couple preferentially to the inhibition of adenylate cyclase. P2Y1 and P2Y12 receptors occur on the surface of platelets and both act in concert to promote aggregation in response to ADP, and consequently, their antagonists are of interest as antiplatelet agents. We used molecular modeling based on a rhodopsin template in conjunction with mutagenesis to identify recognition elements important for nucleotide binding at the P2Y1 and other P2Y receptors. In order to ascertain which residues of the P2Y1 receptor were involved in ligand recognition and activation, individual residues of the TMs (3, 5, 6, and 7) and ELs 2 and 3 were mutated to Ala and various charged residues. A cluster of positively charged lysine and arginine residues near the exofacial side of TMs 3, 6 and 7 putatively coordinate the phosphate moieties of nucleotide agonists and antagonists. The two subgroups of P2Y receptors (either P2Y1-like or P2Y12-like) contain different sets of cationic residues that function in the coordination of the 5-di- or triphosphate groups. At the P2Y1 receptor, it has been possible to convert nucleotide agonists (5-diphosphate derivatives) into antagonists by separating the diphosphate moiety into bisphophates attached at the 3 and 5 positions, or alternately at the 2 and 5 positions. Based on structure activity relationship (SAR) studies, there is a small hydrophobic pocket present at the N6 binding region of the P2Y1 receptor. The native ligands for the most recently cloned member of the P2Y receptor family, the P2Y14 receptor, are UDP-sugars, such as UDP-glucose. Recently, we have explored the pharmacology of UDP and its derivatives as P2Y14 receptor ligands. This receptor is involved in immune function. We have modeled the P2Y14 receptor by homology to rhodopsin and docked various nucleotide ligands. Compared to other P2Y receptors, the P2Y14 receptor has an atypical binding mode of the nucleobase, ribose and phosphate moieties. The ligand binding modes obtained in this study demonstrated that two key cationic residues, namely Arg6.55 and Lys7.35, together with two anionic residues (Glu166 and Glu174 located in EL2), can interact with the hexose moiety. A third conserved key cationic residue, namely Lys171, interacts with the -phosphate group of the ligand. The lower activity of 4 and its receptor docking suggest limited steric tolerance at the 2 position of the hexose moiety ARs are involved in many of the bodys cytoprotective functions. Modeling and mutagenesis of adenosine receptors has focused on determinants of binding of nucleoside and nonnucleoside heterocyclic derivatives and also on determinants of the intrinsic efficacy in adenosine derivatives and on amino acid residues involved in the activation process. It should be noted that the only current crystallographic structures of rhodopsin to serve as a template for 7TM receptor structure are of the inactive (resting) state, which is more applicable to modeling antagonist docking. However, the model has prooven useful for studying agonist ligands as well, and has helped to elucidate which local conformational changes may be involved in activation of the receptor. For example, for docking of NECA and other agonists to the A3AR model, binding in the region of the 5-substituent induced a movement of the side-chain of W243 (6.48). This conserved aromatic residue has been described as a rotamer switch for activation of a variety of 7TM receptors, including the beta-adrenergic receptor, A3AR, and P2Y1 receptors. Its mutation to Ala in all of the latter (purinergic) subtypes has been shown to preclude activation of the receptor by agonist but not binding of the same ligands. We recently introduced the first molecular models of dimeric adenosine receptors, i.e. the A3 and A2A homodimers. A macromolecular ligand was docked to the A2A dimer, to demonstrate the feasibility of binding simultaneously to multiple sites. In silico quantitative predictions are explored to facilitate lead optimization. Molecular modeling of the P2Y6 receptor correctly predicted conformational features of docked nucleotides, which were validated experimentally. Introduction of a methanocarba ring constrained in the South conformation enhances potency of nucleotide analogues exclusively at the P2Y6 receptor. We are currently extending the approach of structure based drug discovery (demonstrated for the A2A receptor) to other subtypes of purine receptors. The P2Y12 receptor (P2Y12R) is an ADP-activated G protein-coupled receptor (GPCR) that is an important target for antithrombotic drugs. Three homology models of P2Y12R were compared, based on different GPCR structural templates: bovine rhodopsin (bRHO), human A2A adenosine receptor (A2AAR), and human C-X-C chemokine receptor type 4 (CXCR4). We concluded that ligand docking to the CXCR4-based model of the P2Y12R predicted poses of both reversibly and irreversibly-binding small molecules, consistent with observed pharmacology and mutagenesis studies.

Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Zip Code
Jacobson, Kenneth A; Tosh, Dilip K; Toti, Kiran S et al. (2017) Polypharmacology of conformationally locked methanocarba nucleosides. Drug Discov Today 22:1782-1791
Matricon, Pierre; Ranganathan, Anirudh; Warnick, Eugene et al. (2017) Fragment optimization for GPCRs by molecular dynamics free energy calculations: Probing druggable subpockets of the A 2A adenosine receptor binding site. Sci Rep 7:6398
Duroux, Romain; Ciancetta, Antonella; Mannes, Philip et al. (2017) Bitopic fluorescent antagonists of the A2A adenosine receptor based on pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine functionalized congeners. Medchemcomm 8:1659-1667
Tosh, Dilip K; Janowsky, Aaron; Eshleman, Amy J et al. (2017) Scaffold Repurposing of Nucleosides (Adenosine Receptor Agonists): Enhanced Activity at the Human Dopamine and Norepinephrine Sodium Symporters. J Med Chem 60:3109-3123
Ciancetta, Antonella; O'Connor, Robert D; Paoletta, Silvia et al. (2017) Demystifying P2Y1 Receptor Ligand Recognition through Docking and Molecular Dynamics Analyses. J Chem Inf Model 57:3104-3123
Toti, Kiran S; Osborne, Danielle; Ciancetta, Antonella et al. (2016) South (S)- and North (N)-Methanocarba-7-Deazaadenosine Analogues as Inhibitors of Human Adenosine Kinase. J Med Chem 59:6860-77
Rodríguez, David; Chakraborty, Saibal; Warnick, Eugene et al. (2016) Structure-Based Screening of Uncharted Chemical Space for Atypical Adenosine Receptor Agonists. ACS Chem Biol 11:2763-2772
Tosh, Dilip K; Ciancetta, Antonella; Warnick, Eugene et al. (2016) Structure-Based Scaffold Repurposing for G Protein-Coupled Receptors: Transformation of Adenosine Derivatives into 5HT2B/5HT2C Serotonin Receptor Antagonists. J Med Chem 59:11006-11026
Junker, Anna; Balasubramanian, Ramachandran; Ciancetta, Antonella et al. (2016) Structure-Based Design of 3-(4-Aryl-1H-1,2,3-triazol-1-yl)-Biphenyl Derivatives as P2Y14 Receptor Antagonists. J Med Chem 59:6149-68
Tosh, Dilip K; Ciancetta, Antonella; Warnick, Eugene et al. (2016) Purine (N)-Methanocarba Nucleoside Derivatives Lacking an Exocyclic Amine as Selective A3 Adenosine Receptor Agonists. J Med Chem 59:3249-63

Showing the most recent 10 out of 42 publications