Novel Labdane Diterpenes-Based Synthetic Derivatives: Identification of a Bifunctional Vasodilator That Inhibits CaV1.2 and Stimulates KCa1.1 Channels

Abstract

Sesquiterpenes such as leucodin and the labdane-type diterpene manool are natural compounds endowed with remarkably in vitro vasorelaxant and in vivo hypotensive activities. Given their structural similarity with the sesquiterpene lactone (+)-sclareolide, this molecule was selected as a scaffold to develop novel vasoactive agents. Functional, electrophysiology, and molecular dynamics studies were performed. The opening of the five-member lactone ring in the (+)-sclareolide provided a series of labdane-based small molecules, promoting a significant in vitro vasorelaxant effect. Electrophysiology data identified 7 as a Ca_V1.2 channel blocker and a K_Ca1.1 channel stimulator. These activities were also confirmed in the intact vascular tissue. The significant antagonism caused by the Ca_V1.2 channel agonist Bay K 8644 suggested that 7 might interact with the dihydropyridine binding site. Docking and molecular dynamic simulations provided the molecular basis of the Ca_V1.2 channel blockade and K_Ca1.1 channel stimulation produced by 7. Finally, 7 reduced coronary perfusion pressure and heart rate, while prolonging conduction and refractoriness of the atrioventricular node, likely because of its Ca²⁺ antagonism. Taken together, these data indicate that the labdane scaffold represents a valuable starting point for the development of new vasorelaxant agents endowed with negative chronotropic properties and targeting key pathways involved in the pathophysiology of hypertension and ischemic cardiomyopathy.

Keywords: CaV1.2 channels; KCa1.1 channels; Langendorff perfused heart; docking simulations; hypertension; labdane; molecular dynamics simulations; sclareolide; vasorelaxant activity.

Figure 1

Labdane diterpenes vasorelaxant agents (1–4), (+)-sclareolide (5), sclareol (6), and new semi-synthetic tools (7–10).

Figure 2

Effect of 5 and its derivatives on high KCl-induced contraction and I_Ba1.2. (A) Concentration-response curves constructed in endothelium-denuded rings pre-contracted by 60 mM KCl. In the ordinate scale, the response is reported as a percentage of the initial tension induced by KCl. Data points represent the mean ± s.e.m. (n = 3–7). (B) Traces of I_Ba1.2, recorded from a single rat tail artery myocyte, elicited by 250-ms clamp pulses to 10 mV from a V_h of −50 mV, measured in the absence (control) or presence of cumulative concentrations of 7. The block caused by 10 µM nifedipine is also shown. (C) Concentration–response curves constructed in tail artery myocytes. On the ordinate scale, the current amplitude is reported as a percentage of the value recorded just before the addition of the first concentration of each compound. Data points represent the mean ± s.e.m. (n = 5–8).

Figure 3

Current–voltage relationships of I_Ba1.2 inhibition induced by 5 and its derivatives in single rat tail artery myocytes. (A–F) Current-voltage relationships, recorded from a V_h of −50 mV, constructed prior to the addition (control) and in the presence of (A) 100 µM 5, (B) 30 µM 6, (C) 30 µM 10, (D) 30 µM 8, (E) 10 µM 7, and (A) 10 µM 9. Data points are mean ± s.e.m. (n = 5–6). * p < 0.05 vs. control, Student’s t test for paired samples.

Figure 4

Effect of 5 and its derivatives on moderate concentration of KCl- or phenylephrine-induced contractions. (A) Concentration–response curves constructed in endothelium-denuded rings pre-contracted by 25 mM KCl. In the ordinate scale, the response is reported as a percentage of the initial tension induced by KCl. Data points represent the mean ± s.e.m. (n = 3–8). (B–E) Concentration–response curves for (B) 5, (C) 8, (D) 7, and (E) 9 constructed in rings either endothelium-denuded or -intact, pre-contracted by 0.3 µM phenylephrine. In the ordinate scale, the response is reported as a percentage of the initial tension induced by phenylephrine (phe). Data points represent the mean ± s.e.m. (n = 5–7).

Figure 5

Effect of 8, 7, and 9 on the voltage dependence of Ca_v1.2 channel activation and inactivation in single rat tail artery myocytes. Steady-state inactivation curves, recorded from a V_h of −80 mV in the absence (control) or presence of various concentrations of (A) 8, (B) 7, and (C) 9, were fitted to the Boltzmann equation. Peak current values were used. The current measured during the test pulse is plotted against membrane potential and expressed as availability. Steady-state activation curves were obtained from the current–voltage relationships of Figure 3 and fitted to the Boltzmann equation. Data points are mean ± s.e.m. (n = 5–6).

Figure 6

Biophysical and pharmacological features of 7-induced blockade of single rat tail artery myocyte Ca_V1.2 channels. (A) Effect of 7 on I_Ba1.2 kinetics. Time constant for activation (τ_act) and inactivation (τ_inact) measured in the absence or presence of different concentrations of 7 from a V_h of either −50 mV or −80 mV. Columns represent the mean ± s.e.m. (n = 5–7). * p < 0.05 vs. control, repeated measures ANOVA and Dunnett’s post-hoc test. (B) Effect of membrane potential and Bay K 8644 on I_Ba1.2 inhibition induced by various concentrations of 7. I_Ba1.2, elicited by 250-ms clamp pulses to 10 mV from a V_h of either −50 mV (in the absence or presence of 100 nM Bay K 8644) or −80 mV, measured after the addition of cumulative concentrations of 7. The concentration–response curve to 7 at a V_h of −50 mV is taken from Figure 2C. On the ordinate scale, the current amplitude is reported as a percentage of the value recorded just before the addition of the first concentration of the compound. Data points represent the mean ± s.e.m. (n = 5–7). * p < 0.05 vs. control, repeated measures ANOVA and Dunnett post-hoc test.

Figure 7

Effects of 7 on K_Ca1.1 channels. (A) Average traces (recorded from five cells) of I_KCa1.1 measured in tail artery myocytes, in the absence (control) or presence of various concentrations of 7. I_KCa1.1 was elicited with a clamp pulse to 70 mV from a V_h of −40 mV, delivered every 10 s. (B) Effect of 7 on the current–voltage relationship. On the ordinate scale, I_KCa1.1 amplitude is reported in pA/pF. Data points are mean ± s.e.m. (n = 5). * p < 0.05 vs. control, repeated measures ANOVA and Dunnett post-hoc test.

Figure 8

Effect of Bay K 8644 and TEA on the vasorelaxant activity of 7. (A) Control rings were stimulated by 60 mM KCl, or rings were pre-incubated with (B) 100 nM Bay K 8644 alone or (C) Bay K 8644 plus 10 mM TEA for 10 min, and were stimulated using 10–15 mM KCl. After a steady contraction was obtained, 7 was added cumulatively. (D) Concentration–response curve to 7. In the ordinate scale, the response is reported as a percentage of the initial tension. Data points represent the mean ± s.e.m. (n = 6–7). * p < 0.05 vs. control, repeated measures ANOVA and Dunnett post-hoc test vs. control.

Figure 9

Root mean square deviation (RMSD) profiles of compounds in complex with the Ca_V1.2 channel, and overview of the channel binding pocket docked with 5, 7, and 9. (A–D) The Ca_V1.2 channel 3D structure is depicted in a multicolor cartoon. The bilayer is depicted as grey lines, while some phospholipid heads are reported in purple spheres. Interaction network of (B) 5, (C) 7 and, (D) 9 in complex with the Ca_V1.2 binding residues after the docking simulation. The residues involved in π-stacking and hydrogen bonds are reported as red and blue ball/sticks, respectively. The hydrogen atoms are hidden for clarity. (E) RMSD profiles of Ca_V1.2 channel backbone in complex with 5, 7, and 9. The RMSD trends are represented as colored lines (see legend). The RMSD (nm) and time (ns) values of the MD run are reported on the Y- and X- axis, respectively.

Figure 10

Overview of the K_Ca1.1 channel binding pocket in complex with 7 and 9. (A) The K_Ca1.1 channel 3D structure is depicted in the cyan transparency surface and cartoon. Interaction network of (B) 7 and (C) 9 in complex with the K_Ca1.1 channel binding residues. The residues involved in π-stacking, hydrogen bond, and salt bridge, are reported as red, blue, and orange ball/sticks, respectively. The hydrogen atoms are hidden for clarity.

Figure 11

Effects of compound 7 on LVP and CPP in Langendorff-perfused rat hearts. Concentration–effect relationship of compound 7 on LVP and CPP. On the ordinate scale, the response is reported as mmHg. Each value represents mean ± s.e.m. (n = 5). * p < 0.05, ** p < 0.01 vs. CTRL, repeated measures ANOVA and Dunnett’s post-test.

Figure 12

Original ECG trace recorded under (A) control conditions and (B) in the presence of 30 µM 7.