Figure 1.
Chemical structures of hTAS2R39 agonist epicatechin gallate (ECG) (A), investigated flavanones (residues specified in Table 1) (B) and hTAS2R39 agonist denatonium benzoate (C).
The commonly applied ring-nomenclature for flavonoids (A-, B-, and C-ring) is shown in the general structure formula for flavanones.
Figure 2.
Screening of flavanones for reduction of ECG response on hTAS2R39.
Screening was performed with simultaneous application of agonist (200 μM ECG) and putative antagonist (250 μM flavanone). Inhibition was indicated when . Data are presented as mean ±SD of n separate experiments conducted in duplicate. Compounds 1, 2, 4, 5, 7, 10: n = 2, compounds 8, 9, 12, 13, 14: n = 3, compound 3: n = 4, compound 6, 11: n = 5. Significance of signal reduction is indicated by ** (p≤0.01) and * (p≤0.05).
Table 1.
Flavanones tested for reduction of activation of hTAS2R39 by ECG.
Figure 3.
Fluorescent counts of ECG-induced calcium responses in cells expressing hTAS2R39 (induced) and non-expressing hTAS2R39 (non-induced).
A: Simultaneous addition of agonist (ECG 200 μM) and antagonist (4′-fluoro-6-methoxyflavanone (6) 16 μM) (□) versus the signal elicited by ECG (antagonist replaced by buffer, final concentration ECG 200 μM) (◊). Non-induced cells: ECG 200 μM and 4′-fluoro-6-methoxyflavanone (6) 16 μM (○) and ECG 200 μM (Δ). B: Stepwise addition of first antagonist (arrow “1st addition”), and then agonist (arrow “2nd addition”) (1st 6-methoxyflavanone (11) 500 μM, 2nd ECG 200 μM) (□)) versus agonist (1st buffer, 2nd ECG 200 μM) (◊). Non-induced cells: 1st 6-methoxyflavanone (11) 500 μM, 2nd ECG 200 μM (○) versus 1st buffer, 2nd ECG 200 μM (Δ).
Figure 4.
Inhibition of response of 200 μM ECG (---) on hTAS2R39 (induced (•), non-induced (○)) by 4′-fluoro-6-methoxyflavanone (6) after simultaneous addition (n = 5) (A) and stepwise addition (n = 4) (B), by 6,3′-dimethoxyflavanone (3) after simultaneous addition (n = 4) (C) and stepwise addition (n = 3) (D), and by 6-methoxyflavanone (11) after simultaneous addition (n = 5) (E) and stepwise addition (n = 3) (F).
Data are presented as mean ±SEM of n separate experiments conducted in duplicate.
Table 2.
Thresholds and IC50 values of 6-methoxyflavanones for inhibition of hTAS2R39 responses towards 200 μM ECG and 1.7 mM denatonium benzoate.
Figure 5.
Inhibition of response of 1.7(---) on hTAS2R39 (induced (•), non-induced (○)) by 4′-fluoro-6-methoxyflavanone (6) after simultaneous addition (n = 4) (A) and stepwise addition (n = 4) (B).
Data are presented as mean ±SEM of n separate experiments conducted in duplicate.
Figure 6.
Inhibition of response of 640 μM ECG (---) on hTAS2R14 (induced (•), non-induced (○)) by 4′-fluoro-6-methoxyflavanone (6) after simultaneous addition (n = 2) (A) and stepwise addition (n = 3) (B). Data are presented as mean ±SEM of n separate experiments conducted in duplicate.
Table 3.
Thresholds and IC50 values of 6-methoxyflavanones for inhibition of hTAS2R14 responses towards 640 μM ECG and 70 μM genistein.
Figure 7.
Isoproterenol responses upon application with buffer, 4′-fluoro-6-methoxyflavanone (6), 6,3′-dimethoxyflavanone (3), and 6-methoxyflavanone (11).
Data are presented as mean ±SEM of a representative experiment conducted in quadruplicate. n.s., not significant.
Figure 8.
Dose-response curves for epicatechin gallate (ECG) (•) (n = 2) (A) and denatonium benzoate (•) (n = 2) (B) on hTAS2R39, and their modification by increasing 4′-fluoro-6-methoxyflavanone (6) concentrations (○ 50 μM, Δ 100 μM, □ 200 μM).
Antagonist and agonist were added in the stepwise way. Data are presented as mean ±SEM of n separate experiments conducted in duplicate. Wash-out experiments (n = 2) (C). Cells were stimulated with 200 μM ECG in the absence (open bars) and in the presence (filled bars) of 100 μM 4′-fluoro-6-methoxyflavanone (6), or 500 μM 6,3′-dimethoxyflavanone (3), or 500 μM 6-methoxyflavanone (11), washed with Tyrode's buffer, and again stimulated with 200 μM ECG (hatched bars; control: grey bar). Antagonist and agonist were added in the stepwise way. Data are presented as mean ±SEM of n separate experiments conducted in quadruplicate. Significance of signal reduction is indicated by *** (p≤0.001), ** (p≤0.01), and n.s. (not significant, p>0.05).
Table 4.
EC50 values of ECG and denatonium benzoate in the presence of various concentrations of 4′-fluoro-6-methoxyflavanone (6) on hTAS2R39.