Clerodane Diterpenes from Casearia corymbosa as Allosteric GABA A Receptor Modulators.

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 α 1 β 2 γ 2 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 EC 50 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 α 1 β 2 γ 2 S and α 1 β 2 subtypes. Compound 8 potentiated GABA-induced currents with both receptor subunit compositions [EC 50 (α 1 β 2 γ 2 S) = 43.6 μM; E max = 809% and EC 50 (α 1 β 2 ) = 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.