Elsevier

Biochemical Pharmacology

Volume 71, Issue 4, 14 February 2006, Pages 540-549
Biochemical Pharmacology

Structure activity and molecular modeling analyses of ribose- and base-modified uridine 5′-triphosphate analogues at the human P2Y2 and P2Y4 receptors

https://doi.org/10.1016/j.bcp.2005.11.010Get rights and content

Abstract

With the long-term goal of developing receptor subtype-selective high affinity agonists for the uracil nucleotide-activated P2Y receptors we have carried out a series of structure activity and molecular modeling studies of the human P2Y2 and P2Y4 receptors. UTP analogues with substitutions in the 2′-position of the ribose moiety retained capacity to activate both P2Y2 and P2Y4 receptors. Certain of these analogues were equieffective for activation of both receptors whereas 2′-amino-2′-deoxy-UTP exhibited higher potency for the P2Y2 receptor and 2′-azido-UTP exhibited higher potency for the P2Y4 receptor. 4-Thio substitution of the uracil base resulted in a UTP analogue with increased potency relative to UTP for activation of both the P2Y2 and P2Y4 receptors. In contrast, 2-thio substitution and halo- or alkyl substitution in the 5-position of the uracil base resulted in molecules that were 3–30-fold more potent at the P2Y2 receptor than P2Y4 receptor. 6-Aza-UTP was a P2Y2 receptor agonist that exhibited no activity at the P2Y4 receptor. Stereoisomers of UTPαS and 2′-deoxy-UTPαS were more potent at the P2Y2 than P2Y4 receptor, and the R-configuration was favored at both receptors. Molecular docking studies revealed that the binding mode of UTP is similar for both the P2Y2 and P2Y4 receptor binding pockets with the most prominent dissimilarities of the two receptors located in the second transmembrane domain (V90 in the P2Y2 receptor and I92 in the P2Y4 receptor) and the second extracellular loop (T182 in the P2Y2 receptor and L184 in the P2Y4 receptor). In summary, this work reveals substitutions in UTP that differentially affect agonist activity at P2Y2 versus P2Y4 receptors and in combination with molecular modeling studies should lead to chemical synthesis of new receptor subtype-selective drugs.

Introduction

Pharmacological effects of UTP (uridine 5′-triphosphate) and other uracil nucleotides on second messenger signaling pathways and on various tissue responses provided the initial indications of the existence of cell surface receptors that specifically recognize extracellular pyrimidines [1], [2]. This concept was confirmed and extended over the past decade with the cloning of three different G protein-coupled receptors, the P2Y2, P2Y4 and P2Y6 receptors [3], [4], [5], [6] that are activated by uracil nucleotides, and by the direct demonstration of regulated release of UTP from a variety of cell types [7], [8].

The P2Y2 receptor is activated equipotently by both UTP and ATP (adenosine 5′-triphosphate) and is distributed in a broad range of tissues. For example, this receptor plays important physiological roles in epithelial cells of the lung, gastrointestinal tract, eye, and other tissues [9], [10]. The human P2Y4 receptor is selectively activated by UTP, and ATP is a potent competitive antagonist at this receptor [11]. However, the P2Y4 receptor of several other species is activated by both UTP and ATP [11], [12], [13], and therefore, it has proven difficult to differentiate the P2Y4 receptor from the P2Y2 receptor on the basis of its cognate agonists in, for example, rat and mouse tissues. The P2Y6 receptor is selectively activated by UDP (uridine 5′-diphosphate), and UTP is a weak agonist or inactive at this receptor [6], [14].

The existence of three different G protein-coupled receptors that recognize uracil nucleotides has made difficult the pharmacological characterization or selective activation of these receptors in native tissues. As has proved to be the case with the P2Y receptors, i.e. P2Y1, P2Y11, P2Y12, and P2Y13 receptors, that are activated by adenine nucleotides, the metabolism and interconversion of extracellular nucleotides add complexities to the study of uracil nucleotide-activated receptors in native tissues [15]. For example, the ectonucleoside triphosphate diphosphohydrolase, NTPDase2, converts extracellular UTP to UDP [16], whereas ectonucleoside diphosphokinase forms UTP from UDP with the transfer of the γ-phosphate from ATP [17].

Drugs that selectively activate or block the uracil nucleotide-activated P2Y receptors would provide armamentaria for circumvention of some of the problems inherent in the study of these physiologically important signaling proteins. However, receptor subtype-selective agonists or antagonists are not available for the uracil nucleotide-activated P2Y receptors [18]. Therefore, we have undertaken a series of pharmacological studies designed to systematically evaluate the effects of various modifications of the UTP structure on the capacity of analogues to activate the UTP-activated P2Y2 and P2Y4 receptors. Since UTP activates the human P2Y2 and P2Y4 receptors with similar potencies, our first goal is to identify substitutions in UTP analogues that differentially affect activity at either of these receptors. Conversely, identification of partial agonists at these receptors would open a path to synthesis of selective high affinity antagonists of the UTP-activated P2Y receptors in a manner similar to the approach we have followed to identify a subnanomolar affinity antagonist for the ADP-activated P2Y1 receptor [19], [20], [21]. The results reveal encouraging progress in the first of these goals. We also conducted docking studies with rhodopsin-based homology models of the P2Y2 and P2Y4 receptors with the goal of extending insight gained from the empirical structure activity studies to predict optimized structures that will be pursued by application of new molecular syntheses. The combination of these structure activity analyses and molecular modeling studies provides resolution of key differences in the ligand binding pockets of the P2Y2 versus P2Y4 receptor that will be of heuristic importance for future ligand development.

Section snippets

Reagents

2′-Ara-fluoro-2′-deoxyuridine (25) was purchased from R.I. Chemical, Inc. (Orange, CA). All other reagents and solvents were purchased from Sigma–Aldrich (St. Louis, MO).

Uracil nucleotide analogues

Most of the nucleotide analogues studied (compounds 510, 1217, and 20) were purchased from TriLink Biotechnologies (San Diego, CA). Compound 19 was custom synthesized by TriLink Biotechnologies and was the gift of Dr. Victor Marquez, NCI, Frederick, MD. Compounds 14 and 3-methyluridine (26) were purchased from Sigma (St.

Chemical synthesis

Synthetic methods for the preparation of nucleoside 5′-triphosphate derivatives 11 and 18 from the nucleosides of 25 and 26, respectively, are shown in Fig. 1. The classical phosphorous oxychloride method was used for the synthesis of both derivatives [28], [29]. The 5′-mono (27) and diphosphate (28) derivatives of 26 were also isolated as side products.

Pharmacological assays and structure activity relationships

The human P2Y2 and P2Y4 receptors were stably expressed in 1321N1 human astrocytoma cells using retroviral vectors as previously described [14]

Discussion

Results from this study provide the first systematic structure activity analysis designed to distinguish the P2Y2 versus P2Y4 receptor selectivity of base- and ribose-modified analogues of UTP. The UTP receptor selectivity of molecules with phosphate side chain modified UTP, i.e. stereoisomers of UTPαS and 2′-deoxy-UTPαS, also was examined. Thio-substitution at the 4-position increases potency at both P2Y2 and P2Y4 receptors, whereas other base- or ribose-modifications were identified that

Acknowledgements

We thank Dr. Victor Marquez (NCI, Frederick, MD) for the gift of zebularine 5′-triphosphate and for helpful discussion. This research was supported in part by the Intramural Research Program of the NIH, National Institute of Diabetes and Digestive and Kidney Diseases and by NIH grants GM38213 and HL34322. Susanna Tchilibon and Pedro Besada thank the Cystic Fibrosis Foundation (Bethesda, MD) for financial support. Mass spectral measurements were carried out by Dr. John Lloyd and NMR by Wesley

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