A Noncompetitive and Selective Prostaglandin EP2 Antagonist with Anti‐inflammatory Property
Authors: Jiang Chunxiang , Amaradhi Radhika , Ganesh Thota , Dingledine Ray 
Published: April 2020

The prostaglandin E2 (PGE2) receptor subtype EP2 promotes chronic inflammation, modulates tumorigenesis, and regulates immune function. Inhibition of EP2 receptors reduces inflammation in rodent models of epilepsy, endometriosis, and arthritis, and decreases tumor cell growth. Therefore, inhibiting EP2 receptors could be a promising method for treatment of inflammatory diseases. To date all reported EP2 antagonists have a purely competitive mode of action, which can be overcome by high concentrations of agonist. Herein, we report the characterization of a novel compound belongs to substituted pyrroloquinoxaline scaffold as the first EP2 antagonist that features a noncompetitive mode of action. Cell‐based time‐resolved fluorescence resonance energy transfer (TR‐FRET) assays were developed in 384‐well format to measure the accumulation of cyclic adenosine monophosphate (cAMP) induced by PGE2, on the basis of the fact that activation of the Gas‐coupled EP2 receptor induces cAMP formation. At a low concentration (3 μM) of the pyrroloquinoxaline antagonist, a saturating concentration of PGE2 (3 μM) could reverse the inhibition of EP2 receptor activation in human EP2‐overexpressing C6 glioma (C6G‐EP2) cells. At higher antagonist concentrations (10–30 μM), the inhibition of EP2 receptors could not be fully overcome by the agonist. Meanwhile, the maximal agonist response inhibition by the antagonist increased with increasing concentration. The maximum effect (cAMP induction) by PGE2 was decreased to 81.9 ±1.44%, unlike 100% for a typical competitive antagonist. The antagonist itself had no effect on the TR‐FRET signal since there was no change in the TR‐FRET signal in the absence of C6G‐EP2 cells. The antagonist was reversible as demonstrated by washing out the antagonist three times each at 80% efficiency. After washing out the antagonist, the maximal response of C6G‐EP2 cells to PGE2 increased to 97.6 ± 0.69% from 86.4 ± 0.85% (p < 0.0001, one‐way ANOVA with Bonferroni). Furthermore, the antagonist exhibited little to no effect on the EC50 or maximal response of other Gas‐coupled prostanoid receptors (EP4, IP, and DP1). The agonist EC50s for EP2, EP4, IP and DP1 receptors were shifted 13.2, 2.2, 1.1 and 0.95‐fold to the right, respectively, in 30 μM antagonist. To determine whether the noncompetitive antagonist exerts an anti‐inflammatory action we studied the expression of IL‐6 and IL‐1β in the murine BV2 microglial cell line stably expressing human EP2 receptors (BV2‐hEP2). Treatment of these cells with a >EC90 concentration (30 nM) of the EP2 selective agonist, ONO‐AE1‐259, caused elevated mRNA levels of the two cytokines (measured by qRT‐PCR) that was suppressed by the antagonist (20 μM). Thus, the pyrroloquinoxaline is the first noncompetitive EP2 receptor antagonist. This compound may provide a basis for further development of novel noncompetitive EP2 antagonists.

Support or Funding Information

This work was supported by the National Institutes of Health [Grant Numbers 1R01NS097776 (RD), 1 U01 AG052460 (TG)]. C.J. is a member of the joint training program between Emory University School of Medicine and Xiangya School of Medicine, Central South University, and is supported by the China Scholarship Council. We gratefully acknowledge the gift of ONO‐AE1‐259 by Ono Pharmaceuticals.