Pharmacological comparison of novel synthetic fenamate analogues with econazole and 2-APB on the inhibition of TRPM2 channels

Abstract

Background and purpose: Fenamate analogues, econazole and 2-aminoethoxydiphenyl borate (2-APB) are inhibitors of transient receptor potential melastatin 2 (TRPM2) channels and are used as research tools. However, these compounds have different chemical structures and therapeutic applications. Here we have investigated the pharmacological profile of TRPM2 channels by application of newly synthesized fenamate analogues and the existing channel blockers.

Experimental approach: Human TRPM2 channels in tetracycline-regulated pcDNA4/TO vectors were transfected into HEK293 T-REx cells and the expression was induced by tetracycline. Whole cell currents were recorded by patch-clamp techniques. Ca(2+) influx or release was monitored by fluorometry.

Key results: Flufenamic acid (FFA), mefenamic acid (MFA) and niflumic acid (NFA) concentration-dependently inhibited TRPM2 current with potency order FFA > MFA = NFA. Modification of the 2-phenylamino ring by substitution of the trifluoromethyl group in FFA with -CH(3), -F, -CF(3), -OCH(3), -OCH(2)CH(3), -COOH, and -NO(2) at various positions, reduced channel blocking potency. The conservative substitution of 3-CF(3) in FFA by -CH(3) (3-MFA), however, gave the most potent fenamate analogue with an IC(50) of 76 µM, comparable to that of FFA, but unlike FFA, had no effect on Ca(2+) release. 3-MFA and FFA inhibited the channel intracellularly. Econazole and 2-APB showed non-selectivity by altering cytosolic Ca(2+) movement. Econazole also evoked a non-selective current.

Conclusion and implications: The fenamate analogue 3-MFA was more selective than other TRPM2 channel blockers. FFA, 2-APB and econazole should be used with caution as TRPM2 channel blockers, as these compounds can interfere with intracellular Ca(2+) movement.

Figure 1

TRPM2 channels activated by ADP-ribose and H₂O₂. Whole-cell current in the HEK293 T-REx cells inducibly transfected with TRPM2 channels was recorded by patch clamp. (A) The time course for TRPM2 channel activation by 0.5 mM ADP-ribose (ADP-r) in pipette solution. The arrow shows the point of membrane breakthrough as whole-cell patch formation. 2-APB (100 µM) was used. (B) IV curve for (A). (C) Na⁺ was substituted by equimolar concentrations of NMDG⁺. The IV curves are shown in the inset. (D) Current recorded in the non-induced cells. (E) Summary data (means ± SEM) for the current at −80 mV in cells with induced TRPM2 channels (TRPM2) and non-induced cells (control) (n= 6). (F) TRPM2 channels activated by H₂O₂ (500 µM). (G) H₂O₂-evoked Ca²⁺ influx via TRPM2 channels. The cells without tetracycline induction (non-induced) were used as control.

Figure 2

Effect of fenamates and non-fenamate NSAIDs on TRPM2 current. Representative time course and IV curves of TRPM2 channels were shown in (A–F). (A) FFA (100 µM). (B) NFA (100 µM). (C) MFA (100 µM). (D) diclofenac (DFS; 100 µM). (E) aspirin (ASP; 100 µM). (F) indomethacin (IND; 100 µM). (G) Summary data (means ± SEM) showing the percentage of inhibition of TRPM2 current. The amplitude was normalized to that blocked by 2-APB (100 µM) (n= 3–8). ***P < 0.001. (H) Concentration–response curves for FFA, MFA and NFA for the inhibition of TRPM2 current (n= 5–6 for each point).

Figure 3

Synthetic fenamate analogues and the effect on TRPM2 current. (A) Time course showing the effect of fenamate analogues, compounds (1) to (10) at 100 µM. The structures are shown at the top of each panel. (B) Summary data (means ± SEM) for the effect on TRPM2 current. The current measured at ±80 mV was normalized to that blocked by 2-APB (100 µM). ***P < 0.001, significantly different from FFA group; anova. n= 3–6 for each group. (C) Comparison of the concentration–response curves for 3-MFA (1) and FFA. (D) Current–voltage relationship and the inhibition of TRPM2 current by 3-MFA. (E) Inhibition of ADP-ribose-induced TRPM2 current by 3-MFA (100 µM) was partly reversed after wash-out and abolished by 2-APB (100 µM).

Figure 4

Effect of 3-MFA or FFA applied intracellularly. (A) Whole-cell patch was recorded in the TRPM2 cells using pipette solution containing 0.5 mM ADP-ribose (ADP-r) with or without FFA (200 µM) or 3-MFA (200 µM) (n= 5 for each group). (B) Example of outside-out patches showing the effect of FFA (100 µM) and 2-APB (100 µM). (C) Example of the effect of 3-MFA (100 µM) and 2-APB (100 µM). (D) Single channel activity of TRPM2 recorded by outside-out patches (n= 4). (E) Mean unitary current sizes for ADP-ribose-induced TRPM2 single channel events plotted against voltage. Straight lines were fitted, and the mean unitary slope conductance was 65.76 ± 0.23 pS (0.5 mM ADP-ribose). No effect of FFA (100 µM, 65.81 pS) and 3-MFA (100 µM, 65.61 pS) on the single channel conductance in the outside-out patches.

Figure 5

Ca²⁺ release induced by FFA. Cytosolic Ca²⁺ concentrations were monitored in the T-REx cells perfused with Ca²⁺-free bath solution. (A) FFA (100 µM) induced Ca²⁺ release in the Ca²⁺-free bath solution. (B) The FFA (100 µM) -induced Ca²⁺ release decreased in cells treated with 1 µM thapsigargin (TG). (C) Perfusion with MFA (100 µM) followed by FFA (100 µM). (D) No effect of 3-MFA (1) (100 µM) on Ca²⁺ release, but FFA (100 µM) evoked Ca²⁺ release. (E) FFA induced Ca²⁺ release in the non-induced cells [Tet(–)]. (F) Summary data (means ± SEM) for the amplitude of Ca²⁺ release signal. FFA, 3-MFA and MFA at 100 µM and TG at 1 µM were applied (n= 20–26 cells). ***P < 0.001, significantly different from FFA Tet(+) group. ^#P < 0.05 significantly different from TG+FFA group; ANOVA.

Figure 6

Effect of econazole on TRPM2 currents. (A–C) Examples of the time course for TRPM2 current inhibited by econazole (10, 30, 100 µM) and followed by an increase of the whole cell current. The IV curves are shown in the inset and the traces labelled as a, b, c, and d are indicated in the corresponding time course. (D) Summary data (means ± SEM) for current evoked by econazole. (E) Cytosolic Ca²⁺ oscillations induced by econazole (100 µM) in HEK-293 T-REx cells. (F) Ca²⁺ oscillations inhibited by 2-APB (100 µM) in the T-REx cells. (G) Ca²⁺ levels in cells pretreated with thapsigargin (TG; 1 µM) for 30 min and then perfused with econazole (100 µM) and then with a bath solution containing 1.5 mM Ca²⁺.