Valerenic acid derivatives as novel subunit-selective GABAA receptor ligands - in vitro and in vivo characterization

Abstract

Background and purpose: Subunit-specific modulators of gamma-aminobutyric acid (GABA) type A (GABA(A)) receptors can help to assess the physiological function of receptors with different subunit composition and also provide the basis for the development of new drugs. Valerenic acid (VA) was recently identified as a beta(2/3) subunit-specific modulator of GABA(A) receptors with anxiolytic potential. The aim of the present study was to generate VA derivatives as novel GABA(A) receptor modulators and to gain insight into the structure-activity relation of this molecule.

Experimental approach: The carboxyl group of VA was substituted by an uncharged amide or amides with different chain length. Modulation of GABA(A) receptors composed of different subunit compositions by the VA derivatives was studied in Xenopus oocytes by means of the two-microelectrode voltage-clamp technique. Half-maximal stimulation of GABA-induced chloride currents (I(GABA)) through GABA(A) receptors (EC(50)) and efficacies (maximal stimulation of I(GABA)) were estimated. Anxiolytic activity of the VA derivatives was studied in mice, applying the elevated plus maze test.

Key results: Valerenic acid amide (VA-A) displayed the highest efficacy (more than twofold greater I(GABA) enhancement than VA) and highest potency (EC(50)= 13.7 +/- 2.3 microM) on alpha(1)beta(3) receptors. Higher efficacy and potency of VA-A were also observed on alpha(1)beta(2)gamma(2s) and alpha(3)beta(3)gamma(2s) receptors. Anxiolytic effects were most pronounced for VA-A.

Conclusions and implications: Valerenic acid derivatives with higher efficacy and affinity can be generated. Greater in vitro action of the amide derivative correlated with a more pronounced anxiolytic effect in vivo. The data give further confidence in targeting beta(3) subunit containing GABA(A) receptors for development of anxiolytics.

Figure 1

Chemical structures of VA derivatives. VA-A, valerenic acid amide; VA-MA, valerenic acid methlyamide; VA-DMA, valerenic acid dimethylamide; VA-EA, valerenic acid ethylamide; VA-DEA, valerenic acid diethylamide; VA-IPA, valerenic acid isopropylamide; VA-BA, valerenic acid butylamide; VA-PIP, valerenic acid piperidine amide; VA-MO, valerenic acid morpholine amide; VA-EE, valerenic acid ethylate.

Figure 2

Concentration–effect curves for the enhancement of I_GABA through GABA_A receptors composed of α₁β₃ subunits by (A) VA, VA-A, VA-MA, VA-EA and VA-DMA; (B) VA, VA-DEA, VA-BA, VA-IPA and VA-EE; (C) VA, VA-PIP and VA-MO, using a GABA EC_3–5 (EC₅₀ and n_H values are given in Table 1). I_GABA at 300 µM (VA and VA-A) (Figure B) (grey symbols) were excluded from the fit. (D) Typical I_GABA recordings illustrating concentration-dependent modulation by VA-A of GABA elicited chloride currents through α₁β₃ subunit-containing receptors. An open channel block at high VA-A concentrations was evident from the initial rapid current decay at 100 and 300 µM. GABA, γ-aminobutyric acid; I_GABA, GABA-induced chloride currents; VA, valerenic acid; VA-A, valerenic acid amide; VA-BA, valerenic acid butylamide; VA-DEA, valerenic acid diethylamide; VA-DMA, valerenic acid dimethylamide; VA-EA, valerenic acid ethylamide; VA-EE, valerenic acid ethyl ester; VA-IPA, valerenic acid isopropylamide; VA-MA, valerenic acid methylamide; VA-MO, valerenic acid morpholine amide; VA-PIP, valerenic acid piperidine amide.

Figure 3

(A) Concentration-dependent effects for VA-A on α₁β₁, α₁β₂ and α₁β₃ receptors using a GABA EC_3–5 concentration. (B) Typical traces for modulation of chloride currents through α₁β₁, α₁β₂ and α₁β₃ channels by 30 µM VA-A at GABA EC_3–5. Control currents (GABA, single bar) and corresponding currents elicited by co-application of GABA and the indicated VA-A concentration (double bar) are shown. GABA, γ-aminobutyric acid; I_GABA, GABA-induced chloride currents; VA-A, valerenic acid amide.

Figure 4

Concentration–effect curves for the enhancement of I_GABA through GABA_A receptors composed of (A) α₁β₂γ_2S subunits and (B) of α₃β₃γ_2S subunits by VA-A and VA using a GABA EC_3–5 (EC₅₀ and maximal stimulation are given in Table 2). I_GABA at 300 µM (grey symbols) were excluded from the fit. GABA, γ-aminobutyric acid; GABA_A, GABA type A; I_GABA, GABA-induced chloride currents; VA, valerenic acid; VA-A, valerenic acid amide.

Figure 5

Behaviour in the elevated plus maze test for control and drug-treated mice at a dose of 3 mg·kg⁻¹ of the indicated VA derivative. Bars indicate the time spent on the open arms (OA) in % of the total time. Bars represent means ± SEM from at least eight different mice. *P < 0.05; **P < 0.01, significantly different from control. VA, valerenic acid; VA-A, valerenic acid amide; VA-DEA, valerenic acid diethylamide; VA-EA, valerenic acid ethylamide; VA-EE, valerenic acid ethyl ester; VA-MO, valerenic acid morpholine amide.

Figure 6

Behaviour in the elevated plus maze test for control (shaded bars in A–F) and drug-treated mice at the indicated dose and compound. Bars indicate in (A) time spent in open arms (OA), in % of the total time after application of the indicated dose of VA, (B) time spent in open arms in % of the total time after application of the indicated dose of VA-A, (C) number of closed arm (CA) entries after application of the indicated dose of VA, (D) number of closed arm entries after application of the indicated dose of VA-A, (E) total distance after application of the indicated dose of VA and (F) total distance after application of the indicated dose of VA-A. Bars represent means ± SEM from at least eight different mice. *P < 0.05; **P < 0.01, significantly different from control. VA, valerenic acid; VA-A, valerenic acid amide.