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

Yosuke Nonaka, Takeshi Hiramoto, Norihisa Fujita

Research output: Contribution to journalArticle

89 Citations (Scopus)

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 Ca 2+ assay using the CHO cells stably expressing P2Y 12-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.

Original languageEnglish (US)
Pages (from-to)281-288
Number of pages8
JournalBiochemical and Biophysical Research Communications
Volume337
Issue number1
DOIs
StatePublished - Nov 11 2005
Externally publishedYes

Fingerprint

Phosphoribosyl Pyrophosphate
Computer Simulation
Screening
Ligands
CHO Cells
Sequence Alignment
Metabolites
G-Protein-Coupled Receptors
Adenosine Diphosphate
Free energy
Assays
Animals
Fusion reactions
Software
Databases
Proteins
In Vitro Techniques
diphosphoric acid

Keywords

  • 3D model of GPCR
  • 5-phosphoribosyl 3-pyrophosphate
  • Cysteinylleukotriene E4
  • Gα-mediated Ca response
  • In silico screening
  • P2Y receptor

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Identification of endogenous surrogate ligands for human P2Y12 receptors by in silico and in vitro methods. / Nonaka, Yosuke; Hiramoto, Takeshi; Fujita, Norihisa.

In: Biochemical and Biophysical Research Communications, Vol. 337, No. 1, 11.11.2005, p. 281-288.

Research output: Contribution to journalArticle

@article{b6344538132d4d269923c34e55ad4e7f,
title = "Identification of endogenous surrogate ligands for human P2Y12 receptors by in silico and in vitro methods",
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 Ca 2+ assay using the CHO cells stably expressing P2Y 12-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.",
keywords = "3D model of GPCR, 5-phosphoribosyl 3-pyrophosphate, Cysteinylleukotriene E4, Gα-mediated Ca response, In silico screening, P2Y receptor",
author = "Yosuke Nonaka and Takeshi Hiramoto and Norihisa Fujita",
year = "2005",
month = "11",
day = "11",
doi = "10.1016/j.bbrc.2005.09.052",
language = "English (US)",
volume = "337",
pages = "281--288",
journal = "Biochemical and Biophysical Research Communications",
issn = "0006-291X",
publisher = "Academic Press Inc.",
number = "1",

}

TY - JOUR

T1 - Identification of endogenous surrogate ligands for human P2Y12 receptors by in silico and in vitro methods

AU - Nonaka, Yosuke

AU - Hiramoto, Takeshi

AU - Fujita, Norihisa

PY - 2005/11/11

Y1 - 2005/11/11

N2 - 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 Ca 2+ assay using the CHO cells stably expressing P2Y 12-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.

AB - 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 Ca 2+ assay using the CHO cells stably expressing P2Y 12-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.

KW - 3D model of GPCR

KW - 5-phosphoribosyl 3-pyrophosphate

KW - Cysteinylleukotriene E4

KW - Gα-mediated Ca response

KW - In silico screening

KW - P2Y receptor

UR - http://www.scopus.com/inward/record.url?scp=25844519676&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=25844519676&partnerID=8YFLogxK

U2 - 10.1016/j.bbrc.2005.09.052

DO - 10.1016/j.bbrc.2005.09.052

M3 - Article

VL - 337

SP - 281

EP - 288

JO - Biochemical and Biophysical Research Communications

JF - Biochemical and Biophysical Research Communications

SN - 0006-291X

IS - 1

ER -