HPLC-based activity profiling: discovery of piperine as a positive GABA(A) receptor modulator targeting a benzodiazepine-independent binding site

Abstract

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.

Figure 1

HPLC-based activity profiling of the black pepper extract for GABA_A receptor modulating properties. The HPLC chromatogram (254 nm) of a semipreparative separation of 5 mg of extract is shown in B. Peak numbering corresponds to compounds 1–13. The 22 collected time-based fractions, 90 s each, are indicated with dashed lines. The potentiation of the GABA-induced chloride current in Xenopus oocytes (I_GABA) by each fraction is shown in A. Part C shows typical traces for the modulation of GABA-induced chloride currents through GABA_A (α₁β₂γ_2S) receptors by fractions 7 and 8 of the P. nigrum EtOAc extract.

Figure 2

Part A shows the chromatogram (254 nm) of the optimized, semipreparative HPLC separation of the active P. nigrum extract (10 mg in 100 μL of DMSO). A total of 30 peaks were collected for off-line microprobe NMR. Peak labeling corresponds to compounds 1–13. Part B shows ¹H NMR spectra of selected compounds obtained by the separation mentioned above, whereas part C shows the spectra of two representative 2D NMR experiments.

Figure 3

Part A shows the concentration–response curves for compounds 4 and 5 on GABA_A receptors composed of α₁, β₂, and γ_2S subunits using a GABA EC_5–10. Part B displays typical traces for modulation of chloride currents through α₁β₂γ_2S GABA_A receptors by piperine (5). In part C representative currents illustrate the absence of direct activation of GABA_A receptors (α₁β₂γ_2S) by piperine (5), trichostachine (2), piperlonguminine (3), piperanine (4), and piperettine (7) at 100 μM in comparison to a GABA-induced current at 1 μM.

Figure 4

Effect of (5) on I_GABA in the presence of flumazenil and diazepam. (A) Stimulation of I_GABA by 5 in the presence of flumazenil (10 μM). The left bar shows the positive allosteric modulation of the GABA (EC_5–10)-induced chloride current by 300 μM piperine (5). The right bar illustrates that flumazenil does not antagonize the 5-induced enhancement of I_GABA. (B) Typical GABA-induced chloride currents in the absence and presence of the indicated concentrations of 5, or 5 and flumazenil, respectively. (C) Additive effects of 5 and diazepam on I_GABA. The left bar illustrates the enhancement of I_GABA by 100 μM (5); the bar in the middle, by 1 μM diazepam, and the right bar illustrates enhancement of I_GABA when both compounds were coapplied. (D) Representative chloride currents induced by 5 μM GABA (corresponding to EC_5–10), current enhancement by 5 (100 μM) and diazepam (1 μM), and I_GABA during coapplication of both compounds.

Chart 1

Structures of Piperamides 1–13 and Potentiation of GABA-Induced Chloride Current (I_GABA) in Xenopus Oocytes by 100 μM 2–5 and 7