Discovery of new scaffolds for GABA(A) receptor modulators from natural origin

Gamma-aminobutyric acid type A (GABAA) receptors are the major inhibitory neurotransmitter receptors in the central nervous system (CNS). These heteropentameric transmembrane proteins act as chloride ion channel upon activation by the endogenous ligand γ-amino butyric acid (GABA). Until now, 11 distinct GABAA receptor subtypes have been identified in the human brain. They differ in their subunit stoichiometry, tissue localization, functional characteristics, and pharmacological properties. Many CNS depressant drugs, such as the benzodiazepines exert their action via enhancement of the GABAergic neuronal inhibition. However, therapy may be accompanied by unwanted side-effects and specific clinical action is precluded due to the lack of GABAA receptor subtype selectivity.
In a preliminary screen the lipophilic extracts of Piper nigrum fruits, Angelica pubescens roots, Acorus calamus roots, Biota orientalis leaves and twigs, and Kadsura longipedunculata fruits had shown positive GABAA receptor modulating activity in an in vitro functional, automated two-microelectrode voltage clamp assay with Xenopus laevis oocytes, which transiently expressed α1β2γ2S GABAA receptors.
Aiming at the discovery of new scaffolds which act at the GABAA receptor, the active constituents of these five plant extracts were identified by means of an HPLC-based activity profiling approach. In total, we discovered 28 secondary metabolites with positive GABAA receptor modulating properties belonging to the structural classes of coumarins, monoterpenes, sesquiterpenes, diterpenes, phenylpropanes, piperamides, and lignans. Their structures were elucidated by a combination of powerful analytical methods such as HPLC-PDA-TOF-MS, highly sensitive microprobe NMR, and for chiral compounds, polarimetry and ECD. Determination of relative and absolute configuration was supported by conformational analysis and quantum chemical calculations. Furthermore, three yet unknown natural products could be identified.
HPLC-based activity profiling with P. nigrum enabled the identification of 13 structurally related piperamides with minimum amount of extract. This allowed us to draw preliminary structure activity considerations for the scaffold of piperine, which was the main α1β2γ2S GABAA receptor modulator in this plant (EC50: 52.4 ± 9.4 μM, maximal stimulation of GABA induced chloride currents (IGABA) by 302% ± 27%).
Sandaracopimaric acid and isopimaric acid from B. orientalis were tested for subtype selectivity at α1�3,5β1-3γ2S subtypes which revealed a comparatively high efficiency of both compounds at α2/3-subunit containing receptors. Additionally, sandaracopimaric acid exerted superior efficiency at receptors comprising β2-subunits. It showed EC50 values from 24.9 ± 6.3 μM to 82.2 ± 46.6 μM, and efficiencies ranging between 502% ± 56% to 1101% ± 98% potentiation of IGABA at the subtypes of investigation. A decrease of locomotor activity in the Open Field behavioral model was observed after intraperitoneal injection of 3 to 30 mg sandaracopimaric acid per kg bodyweight in mice. A trend towards anxiolytic-like activity could be observed with 1 and 3 mg/kg.
Further “drug-like” GABAA receptor modulating scaffolds were discovered among the lignans from K. longipedunculata (potencies down to 12.8 ± 3.1 μM and efficiencies up to 886 ± 291% stimulation of IGABA) and among the sesquiterpenes from A. calamus (potencies down to 34.0 ± 6.7 μM and efficiencies up to 886 ± 105% stimulation of IGABA). These substances have potential for the further development as therapeutics acting at the GABAA receptor.