R-etodolac is a more potent Wnt signaling inhibitor than enantiomer, S-etodolac

Abstract

Etodolac is an FDA-approved nonsteroidal anti-inflammatory drug (NSAID) used to treat a variety of inflammatory diseases. The drug is administered as a racemate (50/50 mixture of R- and S- enantiomers), however, studies have shown that the two enantiomers have distinct biologic and pharmacokinetic differences. Wnt signaling, which plays key roles in cell proliferation, polarity, and differentiation, has been shown to be inhibited by R-etodolac; however, comparative analyses of R- and S-etodolac in this function have not been conducted. We used in silico molecular docking and TOPflash functional biologic assays to compare R- and S-enantiomers effect on Wnt signaling inhibition. Further, we used a cultivated limbal stem epithelial cell (cLSCs) model to investigate enantiospecific changes in the colony-forming efficiency (CFE) of cLSCs. The data shows that R-etodolac is a more potent inhibitor of Wnt signaling. In addition, consistently, while both enantiomers demonstrate a dose-dependent decrease in CFE of cLSCs, R-etodolac is a more potent inhibitor.

Keywords: Limbal stem epithelial cells; R-etodolac; S-etodolac; Wnt signaling.

Fig. 1

In silico molecular docking of R- and S-etodolac with Frizzled receptors. A. Docking scores and predicted glide energy of R- and S-Etodolac with Fzd 8, 4, and 7. B. Surface modeling of R- and S-etodolac molecules in the CRD of Fzd 8, 4, and 7 (pdb codes 4F0A, 5CM4, and 5T44, respectively). The Fzd 8 surface model depicts the Wnt ligand in the CRD binding pocket as reference for the inhibitory binding site [9].

Fig. 2

Dose-dependent inhibition of Wnt signaling. TOPflash Wnt activity assays were performed after treatment (0–500 μM) with A. R-etodolac, B. S-etodolac, and C. Racemic etodolac. All figures show each of the two biological samples separately, N1 (magenta) and N2 (green), as well as the average of the two samples, Average (black). A. IC_{50(Average R-Etodolac)}: 102.5 μM, 95% CI: 86.08 to 120.7 μM; IC_{50(N1 R-Etodolac)}: 101.1 μM, 95% CI: 82.90 to 121.5 μM; and IC_{50(N2 R-Etodolac)}: 103.9 μM, 95% CI: 88.84 to 120.4 μM. B. IC_{50(Average S-Etodolac)}: 218.3 μM, 95% CI: 203.7 to 232.8 μM; IC_{50(N1 S-Etodolac)}: 226.9 μM, 95% CI: 209.4 to 243.6 μM; and IC_{50(N2 S-Etodolac)}: 209.7 μM, 95% CI: 192.2 to 227.3 μM. C. IC_{50(Average Racemic Etodolac)}: 191.7 μM, 95% CI: 157.0 to 227.1 μM; IC_{50(N1 Racemic Etodolac)}: 155.6 μM, 95% CI: 111.3 to 198.9 μM; and IC_{50(N2 Racemic Etodolac)}: 222.9 μM, 95% CI: 204.8 to 240.5 μM. Figures and IC₅₀ were determined using Graphpad Prism (version 9.3.1). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Functional assay of etodolac-induced Wnt inhibition using a cultivated human limbal epithelial stem cell model. A. Representative images of rhodamine-stained cLSC colonies: untreated, R-etodolac, and S-etodolac (25, 85, or 200 μM). B. Quantitative analysis of colony forming efficiency of cLSCs. C. Micrographs of cLSC colonies treated with R- and S-etodolac (4× magnification). Mean ± SD; N = 3 biological replicates *p < 0.05, **p < 0.01, ***p < 0.001.