Discovery of GABAA receptor modulators of natural origin – validation of a FLIPR assay for screening and HPLC-based activity profiling

MT Faleschini; A Maier; S Fankhauser; K Thasis; S Hebeisen; V Butterweck; M Hamburger

doi:10.1055/s-0039-3400027

Planta Medica, Table of Contents

Planta Med 2019; 85(18): 1526
DOI: 10.1055/s-0039-3400027

Main Congress Poster

Poster Session 2

Discovery of GABA_A receptor modulators of natural origin – validation of a FLIPR assay for screening and HPLC-based activity profiling

Authors

MT Faleschini

¹Department of Pharmaceutical Sciences, University of Basel,, Klingelbergstrasse 50, 4056 Basel, Switzerland
A Maier

²Institute for Pharma Technology, School of Life Sciences, University of Applied Sciences Northwestern Switzerland,, Hofackerstrasse 30, 4132 Muttenz, Switzerland
S Fankhauser

²Institute for Pharma Technology, School of Life Sciences, University of Applied Sciences Northwestern Switzerland,, Hofackerstrasse 30, 4132 Muttenz, Switzerland
K Thasis

²Institute for Pharma Technology, School of Life Sciences, University of Applied Sciences Northwestern Switzerland,, Hofackerstrasse 30, 4132 Muttenz, Switzerland
S Hebeisen

³B’Sys GmbH,, Benkenstrasse 254, 4108 Witterswil, Switzerland
V Butterweck

²Institute for Pharma Technology, School of Life Sciences, University of Applied Sciences Northwestern Switzerland,, Hofackerstrasse 30, 4132 Muttenz, Switzerland

⁴Zeller Medical AG,, Seeblickstrasse 4, 8590 Romanshorn, Switzerland
M Hamburger

¹Department of Pharmaceutical Sciences, University of Basel,, Klingelbergstrasse 50, 4056 Basel, Switzerland

Abstract

Full Text

Gamma-aminobutyric acid type A (GABA_A) receptor modulators are used to treat epilepsy, insomnia, anxiety, and mood disorders. However, currently used drugs lack receptor subtype selectivity and, therefore exhibit various side effects. Moreover, the scAF3400027fold diversity of synthetic drugs and experimental compounds targeting GABA_A receptors is limited. Natural products such as piperine have been reported as allosteric GABA_A receptor modulators interacting with a benzodiazepine-independent binding site. For screening of large extract libraries and efficient localization of active compounds via HPLC-based activity profiling we established a Fluorometric Imaging Plate Reader (FLIPR) membrane potential assay utilizing stably transfected Chinese Hamster Ovary (CHO) cells expressing GABA_A receptors of α₁β₂γ₂ subunit composition. Assay protocols for rapid screening of plant extract libraries and localization of active compounds in extracts were validated with known GABAergic natural products. An HPLC-based activity profiling protocol was developed. Extract separations (0.4 to 1.2 mg) on an analytical HPLC column were sufficient for the sensitivity of the bioassay. The protocol successfully localized the activity of magnolol in Magnolia officinalis, valerenic acid in Valeriana officinalis, and piperine in Piper nigrum extracts. EC₅₀ values of compounds (magnolol: 4.81 ± 1.0 μM, valerenic acid: 12.56 ± 1.2 μM and piperine: 5.76 ± 0.7 μM) were found to be comparable or lower than those reported using Xenopus oocyte assays. The FLIPR assay is now used for the screening of a large extract library and identification of new GABAergic natural products via HPLC-based activity profiling.