Selective Activation of Nociceptor TRPV1 Channel and Reversal of Inflammatory Pain in Mice by a Novel Coumarin Derivative Muralatin L from Murraya alata

Abstract

Coumarin and its derivatives are fragrant natural compounds isolated from the genus Murraya that are flowering plants widely distributed in East Asia, Australia, and the Pacific Islands. Murraya plants have been widely used as medicinal herbs for relief of pain, such as headache, rheumatic pain, toothache, and snake bites. However, little is known about their analgesic components and the molecular mechanism underlying pain relief. Here, we report the bioassay-guided fractionation and identification of a novel coumarin derivative, named muralatin L, that can specifically activate the nociceptor transient receptor potential vanilloid 1 (TRPV1) channel and reverse the inflammatory pain in mice through channel desensitization. Muralatin L was identified from the active extract of Murraya alata against TRPV1 transiently expressed in HEK-293T cells in fluorescent calcium FlexStation assay. Activation of TRPV1 current by muralatin L and its selectivity were further confirmed by whole-cell patch clamp recordings of TRPV1-expressing HEK-293T cells and dorsal root ganglion neurons isolated from mice. Furthermore, muralatin L could reverse inflammatory pain induced by formalin and acetic acid in mice but not in TRPV1 knock-out mice. Taken together, our findings show that muralatin L specifically activates TRPV1 and reverses inflammatory pain, thus highlighting the potential of coumarin derivatives from Murraya plants for pharmaceutical and medicinal applications such as pain therapy.

Keywords: calcium; calcium imaging; drug discovery; drug screening; ion channel; neuron; pain; transient receptor potential channels (TRP channels).

FIGURE 1.

Novel coumarin derivative muralatin L identified from M. alata and its activation of TRPV1 channel in FlexStation fluorescent calcium assay. A, image of M. alata, from which muralatin L was isolated. B, chemical structure of coumarin derivative, 8-[(2S)-2-hydroxy-3-methyl-3-butenyl)]-5,6,7-trimethoxycoumarin, named muralatin L, with a molecular mass of 320 daltons. C and D, activation of TRPV1 channel expressed in HEK-293T cells in response to a mixture of fraction A (200 μg/ml), the CHCl₃ extract of M. alata, muralatin L (500 μm), and the tool compound capsaicin (5 μm) by FlexStation3 fluorescent calcium assay. The screening scheme consists of 120 or 250 s for test compounds followed by bath application of capsaicin (C) that activates TRPV1 or 10 μm ruthenium red (D) that blocks TRPV1.

FIGURE 2.

Muralatin L induces Ca²⁺ influx in TRPV1-expressing HEK-293T cells. A, left panels, live-cell fluorescent imaging of untransfected cells (top panels). Right panel, average effects of Fura-2 ratios induced by muralatin L (blue arrow) and capsaicin (red arrow) in untransfected cells (top panel, n = 50). B, left image panels, cells expressing hTRPV1 (bottom panels) in response to application of 500 μm muralatin L or 5 μm capsaicin. Right panel, average effects of Fura-2 ratios induced by muralatin L (blue arrow) and capsaicin (red arrow) in cells expressing hTRPV1 (bottom panel, n = 54). The experiment was repeated three times.

FIGURE 3.

Dose-dependent activation of TRPV1 current by muralatin L in HEK-293T cells expressing TRPV1 channel. A, left panel, whole-cell current of TRPV1 in response to 300 μm muralatin L and 1 μm capsaicin. Right panel, current-voltage curves of TRPV1 in response to voltage ramps from −100 mV to +100 mV under control condition (0) or after addition of 300 μm muralatin L (1), 1 μm capsaicin (2), and back to baseline (3). B, representative whole-cell currents of TRPV1 channels activated by increasing the concentrations of muralatin L from 0.3 μm to 3.0 mm. C, curve fitting in red represents dose-dependent activation of TRPV1 by capsaicin with an EC₅₀ of 98.1 nm (n = 6 for all data points). Curve fitting in blue represents dose-dependent activation of TRPV1 by muralatin L with EC₅₀ at 205.6 μm; each data point is expressed as mean ± S.E. of 6–9 independent tests. D, whole-cell current of TRPV1 in response to 3 mm muralatin L or a mixture of 10 μm capsaicin with 3 mm muralatin L. E, TRPV1 current evoked by 500 μm muralatin L was inhibited by each application of 0.01, 0.1, and 1 μm TRPV1 potent antagonist JNJ-17203212.

FIGURE 4.

Comparison of TRPV1 desensitization was induced by repeated applications of either muralatin L (A) or capsaicin (B). 1 mm muralatin L or 1 μm capsaicin was applied for 10 s with intervening washout periods of 1 min. Whole-cell current responses were evoked by muralatin L or capsaicin in TRPV1-transfected HEK-293T cells with Ca²⁺-free solution. C, summary of the percentage of the initial current after seven repeated applications of capsaicin or muralatin L from A and B (n = 3). **, p < 0.01, for comparison of desensitization between capsaicin- and muralatin L-induced currents.

FIGURE 5.

Selectivity of muralatin L over other members of TRPV channel family. Whole-cell currents (left panels) were recorded in HEK-293T cells expressing different TRPV channels, mouse Trpv2 (A), human TRPV3 (B), and mouse Trpv4 (C), in response to muralatin L (300 μm) and their corresponding agonists as positive controls (left panels). Cells were held at 0 mV in left panels. Representative ramp currents are shown in right panels in response to 300 μm muralatin L (labeled as 0 in the left panels), and their agonists (labeled as 1 in the left panels) and washout (labeled as 2 in the left panels). Blue and red bars in the left panels indicate the time course of muralatin L and tool compound applications, respectively. The experiment was repeated at least three times.

FIGURE 6.

Muralatin L activates native TRPV1 current in capsaicin-sensitive DRG neurons. A, 1 μm capsaicin or 1 mm muralatin L activated currents in outside-out patches from DRG neurons. Membrane patches from DRG neurons were first exposed to 1 μm capsaicin to identify TRPV1-expressing cells. Muralatin L was applied to capsaicin-responsive membrane patches after capsaicin was washed from the bath. B, representative current traces activated by capsaicin or muralatin L from A. Traces are shown from the experiments obtained by stepping the voltage from 0 to 80 mV for 600 ms. Right panel, in response to 1 μm capsaicin (labeled as 1 in the left panels) and 1 mm muralatin L (labeled as 2, 3, and 4 in the left panels), and baseline (labeled as 0 in the left panels). The experiment was repeated at least three times.

FIGURE 7.

Inhibition of inflammatory pain by muralatin L and lack of muralatin L effect in Trpv1 knock-out mice. A, induction of pain responses in C57BL/6 WT mice or Trpv1 knock-out mice by intraplantar injection of formalin, capsaicin, muralatin L, or vehicle, respectively. B, time of paw licking was measured during phase I (0–5 min post-injection) and phase II (15–30 min post-injection) following intraplantar injection of formalin in Kunming mice. C, muralatin L (10 and 40 mg/kg, intraperitoneal) and morphine (0.2%, intraperitoneal) were effective in reducing abdominal writhing induced by intraperitoneal injection of acetic acid in C57BL/6 mice (number of writhes, WT vehicle: 63 ± 4.7). D, number of writhes was measured for Trpv1 knock-out mice in different groups (Trpv1^−/− vehicle: 26 ± 2.4). E, muralatin L had no effect on threshold of thermal pain induced by heat radiation to tail (Kunming mice). Data points are mean ± S.E. (n = 6). Significance of statistical differences between the vehicle control and treatment groups is indicated by **, p < 0.01; or ***, p < 0.001.

FIGURE 8.

Putative binding sites for muralatin L in rat TRPV1 channel. A, location of four key residues mapped to the cryo-EM structure of rat TRPV1 (Protein Data Bank code 3J5R). The molecular surface of muralatin L is shown in blue, and key residues are in purple. B, side view of molecular docking of muralatin L into rat TRPV1 using Maestro Suite software. Amino acid side chains of residues Tyr-511, Met-547, Thr-550, and Glu-570 in TRPV1 are shown in pink. The helices are in gray, and the helices of the neighboring monomer are displayed as line ribbons. The ligand is depicted as a blue tube. Hydrogen bonds are black dashed lines. C, representative ichnography of muralatin L interacting with related amino acids of TRPV1. D, docked view of interactions between muralatin L and capsaicin with rat TRPV1 subunits. The binding pocket is composed of residues located in S3, S4, and S5. The key interacting residues are marked and displayed as a thin tube presenting in purple. The chemical structures of muralatin L and capsaicin are shown in blue and red, respectively. E, two compounds are both composed of two pharmacophoric regions as follows: the aromatic A-region and the double bond-participated junction B-region; in addition, capsaicin also possesses a hydrophobic side chain (C-region). F, sequence alignment and comparison of low-gate domains in TRPV1–4. Secondary structure elements are shown above the sequence alignment. Triangles (blue) are indicated as key residues. Current traces of TRPV1 Y511A mutant (G) and TRPV1 T550A mutant (H) expressed in HEK-293T cells in responses to muralatin L or capsaicin or 2-APB.