Thresholds and IC₅₀ values of 6-methoxyflavanones for inhibition of hTAS2R39 responses towards 200 μM ECG and 1.7 mM denatonium benzoate.

<p>n.b., no blocking.</p><p>n.d., not determined.</p

Thresholds and IC₅₀ values of 6-methoxyflavanones for inhibition of hTAS2R14 responses towards 640 μM ECG and 70 μM genistein.

<p>n.b., no blocking.</p><p>n.d., not determined.</p

Chemical structures of hTAS2R39 agonist epicatechin gallate (ECG) (A), investigated flavanones (residues specified in Table 1) (B) and hTAS2R39 agonist denatonium benzoate (C).

<p>The commonly applied ring-nomenclature for flavonoids (A-, B-, and C-ring) is shown in the general structure formula for flavanones.</p

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.

<p>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.</p

Fluorescent counts of ECG-induced calcium responses in cells expressing hTAS2R39 (induced) and non-expressing hTAS2R39 (non-induced).

<p><b>A</b>: Simultaneous addition of agonist (ECG 200 μM) and antagonist (4′-fluoro-6-methoxyflavanone (<b>6</b>) 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 (<b>6</b>) 16 μM (○) and ECG 200 μM (Δ). <b>B</b>: Stepwise addition of first antagonist (arrow “1^st addition”), and then agonist (arrow “2^nd addition”) (1^st 6-methoxyflavanone (<b>11</b>) 500 μM, 2^nd ECG 200 μM) (□)) versus agonist (1^st buffer, 2^nd ECG 200 μM) (◊). Non-induced cells: 1^st 6-methoxyflavanone (<b>11</b>) 500 μM, 2^nd ECG 200 μM (○) versus 1^st buffer, 2^nd ECG 200 μM (Δ).</p

Isoproterenol responses upon application with buffer, 4′-fluoro-6-methoxyflavanone (6), 6,3′-dimethoxyflavanone (3), and 6-methoxyflavanone (11).

<p>Data are presented as mean ±SEM of a representative experiment conducted in quadruplicate. n.s., not significant.</p

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).

<p>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).</p

Bitter Taste Receptor Activation by Flavonoids and Isoflavonoids: Modeled Structural Requirements for Activation of hTAS2R14 and hTAS2R39

Many flavonoids and isoflavonoids have an undesirable bitter taste, which hampers their use as food bioactives. The aim of this study was to investigate the effect of a large set of structurally similar (iso)­flavonoids on the activation of bitter receptors hTAS2R14 and hTAS2R39 and to predict their structural requirements to activate these receptors. In total, 68 compounds activated hTAS2R14 and 70 compounds activated hTAS2R39, among which 58 ligands were overlapping. Their activation threshold values varied over a range of 3 log units between 0.12 and 500 μM. Ligand-based 2D-fingerprint and 3D-pharmacophore models were created to detect structure–activity relationships. The 2D models demonstrated excellent predictive power in identifying bitter (iso)­flavonoids and discrimination from inactive ones. The structural characteristics for an (iso)­flavonoid to activate hTAS2R14 (or hTAS2R39) were determined by 3D-pharmacophore models to be composed of two (or three) hydrogen bond donor sites, one hydrogen bond acceptor site, and two aromatic ring structures, of which one had to be hydrophobic. The additional hydrogen bond donor feature for hTAS2R39 ligands indicated the possible presence of another complementary acceptor site in the binding pocket, compared to hTAS2R14. Hydrophobic interaction of the aromatic feature with the binding site might be of higher importance in hTAS2R14 than in hTAS2R39. Together, this might explain why OH-rich compounds showed different behaviors on the two bitter receptors. The combination of in vitro data and different in silico methods created a good insight in activation of hTAS2R14 and hTAS2R39 by (iso)­flavonoids and provided a powerful tool in the prediction of their potential bitterness. By understanding the “bitter motif”, introduction of bitter taste in functional foods enriched in (iso)­flavonoid bioactives might be avoided

EC₅₀ values of ECG and denatonium benzoate in the presence of various concentrations of 4′-fluoro-6-methoxyflavanone (<b>6</b>) on hTAS2R39.

<p>EC₅₀ values of ECG and denatonium benzoate in the presence of various concentrations of 4′-fluoro-6-methoxyflavanone (<b>6</b>) on hTAS2R39.</p

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).

<p>Data are presented as mean ±SEM of n separate experiments conducted in duplicate.</p