Identification of endogenous surrogate ligands for human P2Y12 receptors by in silico and in vitro methods

doi:10.1016/j.bbrc.2005.09.052

Biochemical and Biophysical Research Communications

Volume 337, Issue 1, 11 November 2005, Pages 281-288

https://doi.org/10.1016/j.bbrc.2005.09.052 Get rights and content

Abstract

Endogenous ligands acting on a human P2Y₁₂ receptor, one of the G-protein coupled receptors, were searched by in silico screening against our own database, which contains more than 500 animal metabolites. The in silico screening using the docking software AutoDock resulted in selection of cysteinylleukotrienes (CysLTs) and 5-phosphoribosyl 1-pyrophosphate (PRPP), with high free energy changes, in addition to the known P2Y₁₂ ligands such as 2MeSADP and ADP. These candidates were subjected to an in vitro Ca²⁺ assay using the CHO cells stably expressing P2Y₁₂–G₁₆α fusion proteins. We found that CysLTE4 and PRPP acted on the P2Y₁₂ receptor as agonists with the EC₅₀ values of 1.3 and 7.8 nM, respectively. Furthermore, we analyzed the phylogenetic relationship of the P2Y, P2Y-like, and CysLT receptors based on sequence alignment followed by evolutionary analyses. The analyses showed that the P2Y₁₂, P2Y₁₃, P2Y₁₄, GPR87, CysLT-1, and CysLT-2 receptors formed a P2Y-related receptor subfamily with common sequence motifs in the transmembrane regions.

Section snippets

Methods

In silico screening. We used the AutoDock 3.0, a ligand flexible docking program [7], according to the manufacturer’s instructions with our in-house database. The number of grid points in the x-, y-, z-axis was 60 × 60 × 60 with grid points separated by 0.375 Å. The population size was set to 50. Each docking experiment consisted of a series of 200 simulations. The resulting initial set of 200 receptor–ligand configurations included crude configurations and required further refinement. To this end,

Results

The effects of the P2Y₁ agonists and antagonists on the expressed receptor were measured using the cells stably expressing P2Y₁ fusion receptors. First, the influences were investigated for 2-methylthio ADP (2MeSADP), which is a known P2Y₁ agonist, on the [Ca²⁺]_i of hP2Y₁–hG_qα and hP2Y₁–hG₁₆α cells (Fig. 1, upper). The results showed that 2MeSADP induced the [Ca²⁺]_i increases in both of the cells with dose dependency. The EC₅₀ values of 2MeSADP for the hP2Y₁–G_qα and hP2Y₁–hG₁₆α receptors were

Discussion

GPCRs are integral membrane proteins and interact with endogenous ligands such as neurotransmitters and hormones. Takeda et al. [21] have analyzed the human genome sequences using SOSUI and have reported that about 950 kinds of GPCRs exist on the human genome. GPCRs are known to be the action sites of more than half of all contemporary medicines. However, since the membrane protein does not readily crystallize, very few of the tertiary structures of GPCRs have been clarified. On the other hand,

Acknowledgment

We thank Dr. E. Cooper in Ritsumeikan University for help in preparing the manuscript.

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Besides the naturally occurring nucleotide ligands of P2YRs, over the years several other agonists and antagonist, both natural and synthetic, have been identified (Table 1). In addition, non-nucleotide ligands have also been proposed, which include cysteinyl leukotriene E4 (P2Y12R), prostaglandin E2 glyceryl ester (P2Y6R) and 5-phosphoribosyl 1-pyrophosphate (P2Y1R and P2Y12R) [30,31]. This variety and flexibility in activation indicates the importance of P2YRs in signal transduction contributing to several biological pathways.
P2Y receptors are G protein-coupled receptors whose physiological agonists are the nucleotides ATP, ADP, UTP, UDP and UDP-glucose. Eight P2Y receptors have been cloned in humans: P2Y₁R, P2Y₂R, P2Y₄R, P2Y₆R, P2Y₁₁R, P2Y₁₂R, P2Y₁₃R and P2Y₁₄R. P2Y receptors are expressed in lymphoid tissues such as thymus, spleen and bone marrow where they are expressed on lymphocytes, macrophages, dendritic cells, neutrophils, eosinophils, mast cells, and platelets. P2Y receptors regulate many aspects of immune cell function, including phagocytosis and killing of pathogens, antigen presentation, chemotaxis, degranulation, cytokine production, and lymphocyte activation. Consequently, P2Y receptors shape the course of a wide range of infectious, autoimmune, and inflammatory diseases. P2Y₁₂R ligands have already found their way into the therapeutic arena, and we envision additional ligands as future drugs for the treatment of diseases caused by or associated with immune dysregulation.
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Metabotropic pyrimidine and purine nucleotide receptors (P2Y receptors) are expressed in virtually all cells with implications in very diverse biological functions, including the well-established platelet aggregation (P2Y12), but also immune regulation and inflammation. The classical P2Y receptors bind nucleotides and are encoded by eight genes with limited sequence homology, while phylogenetically related receptors (e.g., P2Y12-like) recognize lipids and peptides, but also nucleotide derivatives. Growing lines of evidence suggest an important function of P2Y receptors in immune cell differentiation and maturation, migration, and cell apoptosis. Here, we give a perspective on the P2Y receptors' molecular structure and physiological importance in immune cells, as well as the related diseases and P2Y-targeting therapies. Extensive research is being undertaken to find modulators of P2Y receptors and uncover their physiological roles. We anticipate the medical applications of P2Y modulators and their immune relevance.

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^☆: This study was partially supported by the Grant-in-Aid for scientific research from Japanese Ministry of Education, Culture, Sports, Science and Technology.

¹: Present address: Department of Pharmacology, National Defense Medical College, Tokorozawa, Saitama 359-8513, Japan.

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Identification of endogenous surrogate ligands for human P2Y12 receptors by in silico and in vitro methods☆

Abstract

Section snippets

Methods

Results

Discussion

Acknowledgment

J. Pharmacol. Sci.

Trends Pharmacol. Sci.

J. Mol. Biol.

Biochim. Biophys. Acta

Thromb. Res.

Thromb. Res.

FEBS Lett.

Life Sci.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

A vision for the future of genomics research

Nature

Towards 3D structures of G protein-coupled receptors: multidisciplinary approach

Curr. Med. Chem.

ACoMFA analysis with conformational propensity: an attempt to analyze the SAR of a set of molecules with different conformational flexibility using a 3D-QSAR method

J. Comput. Aid. Mol. Des.

Construction of hypothetical three-dimensional structure of P2Y1 receptor based on Fourier transform analysis

J. Protein Chem.

Identification of endogenous surrogate ligands for human P2Y₁₂ receptors by in silico and in vitro methods☆

Construction of hypothetical three-dimensional structure of P2Y₁ receptor based on Fourier transform analysis