HPLC-based activity profiling: discovery of piperine as a positive GABA(A) receptor modulator targeting a benzodiazepine-independent binding site.
A plant extract library was screened for GABA(A) receptor activity making use of a two-microelectrode voltage clamp assay on Xenopus laevis oocytes. An ethyl acetate extract of black pepper fruits [Piper nigrum L. (Piperaceae) 100 microg/mL] potentiated GABA-induced chloride currents through GABA(A) receptors (composed of alpha(1), beta(2), and gamma(2S) subunits) by 169.1 +/- 2.4%. With the aid of an HPLC-based activity profiling approach, piperine (5) was identified as the main active compound, together with 12 structurally related less active or inactive piperamides (1-4, 6-13). Identification was achieved by on-line high-resolution mass spectrometry and off-line microprobe 1D and 2D NMR spectroscopy, using only milligram amounts of extract. Compound 5 induced a maximum potentiation of the chloride currents by 301.9 +/- 26.5% with an EC(50) of 52.4 +/- 9.4 microM. A comparison of the modulatory activity of 5 and other naturally occurring piperamides enabled insights into structural features critical for GABA(A) receptor modulation. The stimulation of chloride currents through GABA(A) receptors by compound 5 was not antagonized by flumazenil (10 microM). These data show that piperine (5) represents a new scaffold of positive allosteric GABA(A) receptor modulators targeting a benzodiazepine-independent binding site.
Zaugg
Janine
J
Institute of Pharmaceutical Biology, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland.
Baburin
Igor
I
Strommer
Barbara
B
Kim
Hyun-Jung
HJ
Hering
Steffen
S
Hamburger
Matthias
M
D000470:Alkaloids / Q000737:chemistry / Q000494:pharmacology*; D000818:Animals; D001569:Benzodiazepines / Q000737:chemistry / Q000494:pharmacology*; D052117:Benzodioxoles / Q000737:chemistry / Q000494:pharmacology*; D001665:Binding Sites; D018118:Chloride Channels / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D005260:Female; D005442:Flumazenil / Q000494:pharmacology; D005638:Fruit / Q000737:chemistry; D018757:GABA Modulators / Q000494:pharmacology*; D015394:Molecular Structure; D019906:Nuclear Magnetic Resonance, Biomolecular; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D029222:Piper nigrum / Q000737:chemistry; D010880:Piperidines / Q000737:chemistry / Q000494:pharmacology*; D053284:Polyunsaturated Alkamides / Q000737:chemistry / Q000494:pharmacology*; D051381:Rats; D011963:Receptors, GABA-A / Q000378:metabolism*; D014982:Xenopus laevis / Q000378:metabolism
D000470:Alkaloids; D052117:Benzodioxoles; D018118:Chloride Channels; D018757:GABA Modulators; D010880:Piperidines; D053284:Polyunsaturated Alkamides; D011963:Receptors, GABA-A; D001569:Benzodiazepines; D005442:Flumazenil; C008922:piperine