Clerodane Diterpenes from Casearia corymbosa as Allosteric GABAA Receptor Modulators

Abstract

An EtOAc extract of Casearia corymbosa leaves led to an allosteric potentiation of the GABA signal in a fluorometric imaging plate reader (FLIPR) assay on Chinese hamster ovary (CHO) cells stably expressing GABA_A receptors with an α₁β₂γ₂ subunit composition. The activity was tracked by HPLC-based activity profiling, and four known (2, 3, 4, and 8) and five new clerodane-type diterpenoids (1, 5-7, and 9) were isolated. Compounds 1-8 were obtained from the active time window. The absolute configuration of all compounds was established by ECD. Compounds 3, 7, and 8 exhibited EC₅₀ values of 0.5, 4.6, and 1.4 μM, respectively. To explore possible binding sites at the receptor, the most abundant diterpenoid 8 was tested in combination with diazepam, etazolate, and allopregnanolone. An additive potentiation of the GABA signal was observed with these compounds, while the effect of 8 was not inhibited by flumazenil, a negative allosteric modulator at the benzodiazepine binding site. Finally, the activity was validated in voltage clamp studies on Xenopus laevis oocytes transiently expressing GABA_A receptors of the α₁β₂γ₂S and α₁β₂ subtypes. Compound 8 potentiated GABA-induced currents with both receptor subunit compositions [EC₅₀ (α₁β₂γ₂S) = 43.6 μM; E_max = 809% and EC₅₀ (α₁β₂) = 57.6 μM; E_max = 534%]. The positive modulation of GABA-induced currents was not inhibited by flumazenil, thereby confirming an allosteric modulation independent of the benzodiazepine binding site.

Figure 1

HPLC-based activity profile of the EtOAc extract. The lower panel shows the HPLC chromatogram (UV 254 nm) and the % potentiation of the GABA signal by microfractions F1–F8 is given above (n = 8, means ± SEM). The red and blue bars represent activation by 20 μM diazepam (in the presence of 2 μM GABA) and by 2 μM GABA (control), respectively. Dashed lines indicate microfractions collected for the bioassay (3 min each), and the numbers correspond to compounds 1 to 9. *** Statistical significance p ≤ 0.001.

Figure 2

Key correlations from COSY and HMBC of compounds 1, 5–7, and 9.

Figure 3

Experimental and calculated ECD spectra of 1 and 5.

Figure 4

Percentage of activation for compounds 1–9 (in the presence of 2 μM GABA), along with 2 μM GABA (control), 200 μM GABA (100%), and 20 μM diazepam (in the presence of 2 μM GABA) (n = 4, means ± SEM). The final DMSO concentration in the assay was 0.1%. The *** above the bars indicate statistical significance with p ≤ 0.001.

Figure 5

Percentage of activation for compounds 3 (A), 7 (B), and 8 (C) (in the presence of 2 μM GABA), along with 2 μM GABA (control), 200 μM GABA (100%), and 20 μM diazepam (in the presence of 2 μM GABA) (n = 10, means ± SEM). The plots above each bar graph show the corresponding concentration–response curves with calculated EC₅₀ values. The final DMSO concentration in the assay was 0.1%. The *, **, and *** above the bars indicate statistical significance with p ≤ 0.05, p ≤ 0.01, and p ≤ 0.001, respectively.

Figure 6

Concentration–effect curves for the enhancement of I_GABA through GABA_A receptors composed of α₁β₂γ_2S (A) and α₁β₂ (B) subunit compositions in Xenopus oocytes, by increasing concentrations of 8 in the presence of GABA EC_3–7. Representative traces for the enhancement of I_GABA for both subunit compositions by 50 μM of 8 (double bar indicates coapplication of GABA and 8) are depicted in the inset. Data points represent means ± SEM recorded with at least three oocytes.