HPLC-based activity profiling approach for the discovery of GABAA receptor ligands using a functional assay with Xenopus oocytes

An approach for rapid HPLC-based profiling for new GABAA ligands of natural origin has been developed. Active extracts are separated by a single injection of 3 - 10 mg of extract onto a semi-preparative (150 × 10 mm i. d.) HPLC column with gradient elution and time-based fractionation. The microfractions are tested in an automated two-microelectrode voltage-clamp assay on Xenopus oocytes expressing recombinant GABAA channels composed of α1, β2 and γ2S subunits. The protocol has been validated by spiking experiments with inactive extract and the GABAA receptor ligand magnolol, and by profiling of active extracts such as valerian extract containing the known GABAA receptor ligand valerenic acid. For dereplication of GABA containing extracts, we established a rapid and simple procedure by which GABA is analyzed as OPA derivative by reversed-phase HPLC. This dereplication protocol was validated with plant and fungal extracts which had been previously tested active or inactive in the oocyte assay and with spiking experiments.