The flavonoid scaffold as a template for the design of modulators of the vascular Ca(v) 1.2 channels

Abstract

Background and purpose: Previous studies have pointed to the plant flavonoids myricetin and quercetin as two structurally related stimulators of vascular Ca(v) 1.2 channel current (I(Ca1.2) ). Here we have tested the proposition that the flavonoid structure confers the ability to modulate Ca(v) 1.2 channels.

Experimental approach: Twenty-four flavonoids were analysed for their effects on I(Ca1.2) in rat tail artery myocytes, using the whole-cell patch-clamp method.

Key results: Most of the flavonoids stimulated or inhibited I(Ca1.2) in a concentration- and voltage-dependent manner with EC(50) values ranging between 4.4 µM (kaempferol) and 16.0 µM (myricetin) for the stimulators and IC(50) values between 13.4 µM (galangin) and 100 µM [(±)-naringenin] for the inhibitors. Key structural requirements for I(Ca1.2) stimulatory activity were the double bond between C2 and C3 and the hydroxylation pattern on the flavonoid scaffold, the latter also determining the molecular charge, as shown by molecular modelling techniques. Absence of OH groups in the B ring was key in I(Ca1.2) inhibition. The functional interaction between quercetin and either the stimulator myricetin or the antagonists resokaempferol, crysin, genistein, and 5,7,2'-trihydroxyflavone revealed that quercetin expressed the highest apparent affinity, in the low µM range, for Ca(v) 1.2 channels. Neither protein tyrosine kinase nor protein kinase Cα were involved in quercetin-induced stimulation of I(Ca1.2).

Conclusions and implications: Quercetin-like plant flavonoids were active on vascular Ca(v)1.2 channels. Thus, the flavonoid scaffold may be a template for the design of novel modulators of vascular smooth muscle Ca(v)1.2 channels, valuable for the treatment of hypertension and stroke.

Figure 4

Effect of quercetin and resokaempferol on I_Ca1.2-voltage relationship in rat tail artery myocytes. Current-voltage relationships, recorded from V_h of −50 mV, constructed prior to the addition of drug (control), in the presence of 10 µM quercetin and after the subsequent addition of 10 µM resokaempferol (A) or vice versa (B). Data points are mean ± SEM (n = 8–10). P-values for repeated measures anova are shown.

Figure 1

Time course of the effects of quercetin and resokaempferol on I_Ca1.2. Peak currents were recorded during a typical depolarization from −50 mV to 0 mV, applied every 30 s (0.033 Hz), and subsequently normalized against the current recorded just prior to drug application. I_Ca1.2 amplitudes are plotted as a function of time. During the periods indicated by the horizontal lines, cells were exposed to increasing concentrations of either quercetin (A) or resokaempferol (C). Filled symbols correspond to current traces shown in panels B and D. Data points are mean ± SEM (n = 5–6). (B and D) Average traces (recorded from 5–6 cells) of conventional whole-cell I_Ca1.2 elicited with 250-ms clamp pulses to 0 mV from a V_h of −50 mV, measured in the absence or presence of cumulative concentrations of either quercetin or resokaempferol, respectively.

Figure 2

Score plot and Loading plot for the two principal components (PC1 and PC2) of the PCA model on the flavonoids, using 128 molecular descriptors (variables, present in the loading plot). (A) Molecules are grouped according to their effect on I_Ca1.2 (see Table 2). 1, 3,4′-Dihydroxyflavone; 2, 3,6,4′-trihydroxyflavone; 3, 3-methyl galangin; 4, galangin; 5, isorhamnetin; 6, kaempferol; 7, morin; 8, myricetin; 9, quercetin; 10, resokaempferol; 11, rutin; 12, 5,7,2′-trihydroxyflavone; 13, 5-hydroxyflavone; 14, apigenin; 15, baicalein; 16, chrysin; 17, luteolin; 18, scutellarein; 19 (±)-naringenin; 20, naringin; 21, tamarixetin; 22 (±)-taxifolin; 23, daidzein; 24, genistein. (B) In the loading plot, the variables that univocally clustered are encircled by a dotted line. ‘Size’ stands for variables related to size and shape of the molecule (surface, volume, molecular weight, globularity, polarizability, etc.); ‘Polarity’ and ‘Dryness’ for variables related to the hydrophilic (hydrophilic volumes and hydrophilic ‘capacities’) and hydrophobic moieties (hydrophobic volumes and hydrophobic ‘capacities’), respectively; ‘Solubility’ for variables related to the aqueous solubility of the molecule calculated at different pH values; ‘Charge’ for variables related to different ionisation features along the pH scale; and ‘LogD’ for variables related to LogD calculated at different pH values. In panel A, PC1 and PC2 represent the scores on the first and second PCs, respectively. These are the projections of the objects on PC1 and PC2. In panel B, PC1 and PC2 represent the loadings on the first and second PC, respectively. These are the variable contributions to PC1 and PC2.

Figure 3

Effects of resokaempferol, chrysin, genistein and 5,7,2′-trihydrohyflavone on I_Ca1.2 amplitude recorded in presence of quercetin in rat tail artery myocytes. The amplitude of the current recorded during a typical depolarization from −50 mV to 0 mV in the presence of 10 µM quercetin was normalized to that recorded under control conditions, taken as 100%. Data points are mean ± SEM (n = 3–5).

Figure 5

Outlines of the structure-activity relationships emerged in the present study for the series of flavonoids investigated as vascular smooth muscle Ca_v1.2 channels modulators. Arrows indicate the C with the OH group ‘Important’, ‘Not relevant’, and ‘Detrimental’ for both the agonist and antagonist activity.