Clerodane Diterpenes from Casearia corymbosa as Allosteric GABAA Receptor Modulators

Journal of Natural Products
2022.0

Abstract

An EtOAc extract of <i>Casearia corymbosa</i> 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<sub>A</sub> receptors with an α<sub>1</sub>β<sub>2</sub>γ<sub>2</sub> subunit composition. The activity was tracked by HPLC-based activity profiling, and four known (<b>2</b>, <b>3</b>, <b>4</b>, and <b>8</b>) and five new clerodane-type diterpenoids (<b>1</b>, <b>5</b>-<b>7</b>, and <b>9</b>) were isolated. Compounds <b>1</b>-<b>8</b> were obtained from the active time window. The absolute configuration of all compounds was established by ECD. Compounds <b>3</b>, <b>7</b>, and <b>8</b> exhibited EC<sub>50</sub> values of 0.5, 4.6, and 1.4 μM, respectively. To explore possible binding sites at the receptor, the most abundant diterpenoid <b>8</b> 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 <b>8</b> was not inhibited by flumazenil, a negative allosteric modulator at the benzodiazepine binding site. Finally, the activity was validated in voltage clamp studies on <i>Xenopus laevis</i> oocytes transiently expressing GABA<sub>A</sub> receptors of the α<sub>1</sub>β<sub>2</sub>γ<sub>2</sub>S and α<sub>1</sub>β<sub>2</sub> subtypes. Compound <b>8</b> potentiated GABA-induced currents with both receptor subunit compositions [EC<sub>50</sub> (α<sub>1</sub>β<sub>2</sub>γ<sub>2</sub>S) = 43.6 μM; <i>E</i><sub>max</sub> = 809% and EC<sub>50</sub> (α<sub>1</sub>β<sub>2</sub>) = 57.6 μM; <i>E</i><sub>max</sub> = 534%]. The positive modulation of GABA-induced currents was not inhibited by flumazenil, thereby confirming an allosteric modulation independent of the benzodiazepine binding site.

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