TRPA1 Inhibition Effects by 3-Phenylcoumarin Derivatives

Abstract

Transient receptor potential ankyrin 1 (TRPA1) protein plays an important role in the inflammatory response, and it has been associated with different pain conditions and pain-related diseases, making TRPA1 a valid target for painkillers. In this study, we identified potential TRPA1 inhibitors and located their binding sites utilizing computer-aided drug design (CADD) techniques. The designed 3-phenylcoumarin-based TRPA1 inhibitors were successfully synthesized using a microwave assisted synthetic strategy. 3-(3-Bromophenyl)-7-acetoxycoumarin (5), 7-hydroxy-3-(3-hydroxyphenyl)coumarin (12) and 3-(3-hydroxyphenyl)coumarin (23) all showed inhibitory activity toward TRPA1 in vitro. Compound 5 also decreased the size and formation of breast cancer cells. Hence, targeting TRPA1 may represent a promising alternative for the treatment of pain and inflammation.

Scheme 1. Synthesis of Compounds 1–28

a) Et₃N, Ac₂O, MW, 110 °C, 5–20 min; b) CH₃OH, 6M HCl, reflux, 3–6 h.

Figure 1

(A) Antagonism of TRPA1 activity by the synthesized 3-phenylcoumarins measured at 10 and 100 μM. Compounds 3, 5, 10 and 12 decreased the TRPA1 activity in both doses the most. TRPA1 activity was induced by allyl isothiocyanate (AITC). Resveratrol, the reference compound, is indicated with Res. (B) Direct agonism of TRPA1 activity by compounds 5, 12 and 23 without or with preincubation with a control. A known TRPA1 antagonist, A-967079 was used as the control. Mean, n = 8, ± SEM.

Figure 2

Binding sites in cryoEM structures 6X2J and 7JUP with them original ligands (blue) and compounds 12 (red), 16 (green) and 23 (purple). A) Binding site of TRPA1 agonist GNE551 in the pore domain (PD) of 6X2J. B) Binding site of a known tetrahydrofuran-based TRPA1 antagonist “compound 21” in PD of 7JUP. Yellow interactions are H-bonds, blue π–π stackings. Orange indicates bad interactions.

Figure 3

Effects of compounds 3, 5, 12, and 23 and resveratrol (R) on TRPA1 protein in T47D cells. (A) TRPA1 protein expression after treatment with compounds. Data are presented as fold change + SEM while DMSO control set as 1 (n = 4). The cells were treated with 10 μM concentrations of all compounds. The data was analyzed with one-way ANOVA with Dunnett post hoc test by comparing treated groups to control group (*p < 0.05 vs control and ***p < 0.001 vs control). (B) Quantification of T47D spheroid growth using CellIQ real-time analysis. Representative images of T47D spheroids treated with compound 5 (1 μM and 10 μM concentrations). (C) Time courses of the spheroid area (μm²). Data was collected over 120 h at 6 h intervals. All images captured at 10× magnification. Each data point represents mean ± SD, n = 5 wells.