Identification of γ-aminobutyric acid type A (GABAA) receptor modulators: HPLC-based activity profiling of Asarum heterotropoides

Abstract

γ-Aminobutyric acid type A (GABAA) receptors are the major mediators of fast synaptic inhibition in the central nervous system and thus play a crucial role in controlling the excitability of the brain. Diverse types of these ligand-gated chloride ion channels are a target for several therapeutically relevant drugs, such as benzodiazepines, barbiturates, neurosteroids and various general anaesthetics. However, these drugs interact in a non-selective way with several GABAA receptor subtypes and hence cause side effects such as reduced coordination, cognitive impairment, increased accident proneness, and development of dependence and abuse liability [1, 2]. In a search for new natural product derived scaffolds for GABAA receptor modulators we screened a plant extract library for GABAA receptor activity by means of a two-microelectrode voltage clamp assay using Xenopus laevis oocytes, which transiently express the target receptors of desired subunit composition. With the aid of an HPLC-based activity profiling approach [3], an active dichloromethane extract of Asarum heterotropoides herb (Aristolochiaceae) was fractionated in a time-based manner and submitted to the assay. In a next step, some of the compounds of the active time-based fractions were isolated and identified by on-line high-resolution mass spectrometry and off-line microprobe 1D and 2D NMR spectroscopy, using only milligram amounts. Dose-response experiments were carried out with these compounds in order to determine EC50 values and maximum potentiation of GABA-induced chloride influx, with the aid of the oocyte functional assay.