Ethnomedicine-based discovery and characterization of plant-derived GABAΑ receptor modulators with new scaffolds

Inhibitory neurotransmission in the central nervous system (CNS) largely relies on the actions of gamma aminobutyric acid (GABA) on GABAA receptors, heteropentameric ligand-gated chloride channels assembled from 19 possible subunits (α1-6, β1-3, γ1-3, δ, ε, θ, π, ρ1-3). GABA-induced chloride influx through GABAA receptors causes neuronal hyperpolarization and inhibition of further action potentials. Therefore, impaired GABAergic function results in CNS conditions such as epilepsy, insomnia, anxiety, and mood disorders. A number of clinically important drugs like benzodiazepines, barbiturates, neuroactive steroids, anesthetics, and certain other CNS depressants bind GABAA receptors. However, these drugs lack of subunit specificity and, therefore, induce serious side effects.
In the search for GABAA receptor modulators with new scaffolds, a plant extract library was screened at Prof. Hamburger’s group by means of an automated two-microelectrode voltage clamp functional assay in Xenopus laevis oocytes. Among others, the lipophilic extracts of Bupleurum chinense roots, Pholidota chinensis stems and roots, Adenocarpus cincinnatus roots and tubers, and Boswellia thurifera resin positively modulated GABAA receptors of the subtype α1β2γ2s, the most abundant one in the human brain.
In this work, GABAergic activity in the four extracts was tracked using of an HPLC-based activity profiling approach. In total, 22 natural products, eight of them new, were isolated by diverse chromatographic methods and characterized by HR-TOF-MS and microprobe NMR. Absolute configuration of chiral compounds was determined by CD-spectroscopy and polarimetry. Fourteen of the 22 isolates showed GABAA receptor modulatory activity in the oocyte functional assay. Dihydrostilbenes, cis-pterocarpans, and abietane diterpenes were identified as new scaffolds for GABAA receptor modulators with favorable physicochemical properties for blood-brain barrier permeation.
HPLC-based activity profiling of P. chinensis enabled the identification of the dihydrostilbene batatasin III as a very efficient, non-selective GABAA receptor modulator. Two structurally related non-flexible stilbenoids, coelonin and pholidotol D, were also isolated from the extract but showed no activity in the oocyte assay, suggesting conformational flexibility to be crucial for receptor modulation. This was confirmed by a preliminary structure-activity relationship study conducted with a series of commercially available stilbenes and their dihydro derivatives.
Fifteen flavonoid and isoflavonoid derivatives, including eight new natural products, were isolated from A. cincinnatus and tested in the oocyte assay. At a concentration of 100 μM, 12 of the 15 compounds significantly enhanced the GABA-induced chloride current through GABAA receptors. Two pterocarpans and one isoflavone showed remarkably higher potency than other natural products previously isolated in this working group (EC50 below 10 μM).
B. thurifera and B. chinense yielded two more GABAA receptor modulators, dehydroabietic acid and aristolactone, respectively. However, isolation of aristolactone from a commercial sample of the traditional Chinese herbal drug Chaihu (Bupleurum chinense roots) led to detection of adulteration of the sample with roots of the nephrotoxic species Aristolochia manshuriensis. This case raised concerns about adequate quality control of TCM drugs commercialized in Europe.