Docking for EP4R antagonists active against inflammatory pain

Abstract

The lipid prostaglandin E₂ (PGE₂) mediates inflammatory pain by activating G protein-coupled receptors, including the prostaglandin E2 receptor 4 (EP4R). Nonsteroidal anti-inflammatory drugs (NSAIDs) reduce nociception by inhibiting prostaglandin synthesis, however, the disruption of upstream prostanoid biosynthesis can lead to pleiotropic effects including gastrointestinal bleeding and cardiac complications. In contrast, by acting downstream, EP4R antagonists may act specifically as anti-inflammatory agents and, to date, no selective EP4R antagonists have been approved for human use. In this work, seeking to diversify EP4R antagonist scaffolds, we computationally dock over 400 million compounds against an EP4R crystal structure and experimentally validate 71 highly ranked, de novo synthesized molecules. Further, we show how structure-based optimization of initial docking hits identifies a potent and selective antagonist with 16 nanomolar potency. Finally, we demonstrate favorable pharmacokinetics for the discovered compound as well as anti-allodynic and anti-inflammatory activity in several preclinical pain models in mice.

Fig. 1. Computational docking and in vitro testing for EP4R antagonists.

a The crystal structure of the EP4R-ONO-AE3-208 complex (PDB 5YWY) was targeted for docking. To reflect the low dielectric of the hydrophobic lipids surrounding the receptor, we equilibrated a POPC lipid bilayer around the protein using molecular dynamics simulations. About 440 million molecules were then docked against the EP4R structure. b Displacement of radiolabeled ³H-PGE₂ by docking hits at 10 µM. c Concentration-response curves of docking hits using a PRESTO-Tango β-arrestin2 recruitment assay. d Docking hits demonstrating antagonist activity at EP4R. Data in b) represents mean ± SEM of three (for compounds from the ZINC15 library) or four (for compounds from the additional anion library) technical repetitions (see Supplementary Data 1). Data in c) represents mean ± SEM of three independent experiments. Source data are provided as a Source Data file.

Fig. 2. Predicted binding poses of docking hits and their structure-based optimization.

a, b Docked poses of 66 and 71, respectively. c Docked pose of 74. Dotted lines indicate hydrogen bonds. Note that while the carboxylate of 71 is out of hydrogen-bond range of Arg316, the two groups maintain an ionic interaction, contributing to the overall positive electrostatic potential in this region. d Structure-based optimization of 71. e Concentration-response curves of compounds shown in d) using BRET-based EP4R-arrestin interaction reporter assay. f Calculated Ki values (using the Cheng-Prusoff approximation) of compounds shown in d). Data in e) represents mean ± SEM of three independent experiments. Source data are provided as a Source Data file.

Fig. 3. In vivo activity of the discovered EP4R antagonist.

“n” denotes number of independent animals. a Carrageenan-mediated mechanical allodynia (von Frey test) is reduced by systemic administration of EP4R antagonists. 77 exhibits significant anti-allodynic properties through i.p. (n = 5) or oral (p.o., n = 10) administration of 30 mg/kg of the molecule. b CJ-42794 and 77 equally reduce carrageenan-mediated mechanical allodynia (von Frey test) upon i.p. injections of 10 mg/kg (n = 10 per group). c Pre-treatment with 10 or 30 mg/kg 77 by i.p. administration (n = 5), 30 minutes prior to intraplantar PGE₂ injection, dose-dependently reduces mechanical allodynia (von Frey test). d CFA-induced mechanical allodynia (von Frey test) is reduced by i.p. administration of 10 (n = 5) and 30 mg/kg (n = 5) of 74 after three and four days. e Carrageenan-induced paw edema was reduced upon i.p. administration of 30 mg/kg 77 (n = 10 per cohort). f Injections of 77 on four consecutive days significantly reduced the progression of CFA-induced inflammation (n = 5 per cohort). p-values between indicated groups in a)-d) were calculated with two-tailed unpaired t-tests. p-values in e) and f) were calculated with multiple unpaired two-tailed t-tests at each timepoint. In a)-f) data is shown as mean ± SEM. Source data are provided as a Source Data file.