Identifying GABAA receptor ligands in black pepper by activity profiling, LC-TOFMS, and offline microprobe NMR

The pharmacological treatment of insomnia, epilepsy and panic disorders today includes the use of classical benzodiazepines and nonbenzodiazepines. However, therapy is accompanied by well-known side-effects resulting from insufficient target selectivity for different GABA_A receptor subtypes. Highly selective GABA_A receptor ligands are therefore an unmet medical need [1]. Rational lead discovery approaches are not possible due to the lack of a high-resolution structure of this pentameric transmembrane receptor [2].

We combine extract library screening with HPLC based activity profiling of extracts as an effective avenue to new natural product leads [3].

In a program for discovery of new scaffolds for GABA_A receptor agonists, a library of 880 extracts was screened in a functional assay in Xenopus laevis oocytes transiently expressing recombinant GABA_A receptors of defined subunit composition (α₁β₂γ_2S). An ethyl acetate extract of black pepper (Piper nigrum L., Piperaceae) fruits significantly potentiated GABA induced chloride current. A combination of HPLC based activity profiling, LC-PDA-TOFMS and offline microprobe NMR analysis allowed rapid identification of the active compounds with mg-amounts of extract. The major active compound was identified as piperine. A set of 12 structurally related, weakly active or inactive amides was also characterized. Structural features critical for GABA_A agonistic activity of the piperine scaffold could be identified by the combination of structural and pharmacological data of this compound series.

References: [1] Whiting, P.J. (2006) Curr. Opin. Pharmacol. 6:24–29.

[2] Barrera, N.P., Edwardson, J.M. (2008) Trends Neurosci. 31:569–576.

[3] Hamburger, M., Potterat, O. (2006) Curr. Org. Chem. 10:899–920.