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Discovery of new muscarinic acetylcholine receptor antagonists from Scopolia tangutica

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ABSTRACT

81217: Scopolia tangutica (S. tangutica) is a traditional Chinese medicinal plant used for antispasmodics, anesthesia, analgesia and sedation. Its pharmacological activities are mostly associated with the antagonistic activity at muscarinic acetylcholine receptors (mAchRs) of several known alkaloids such as atropine and scopolamine. With our recent identification of four hydroxycinnamic acid amides from S. tangutica, we hypothesized that this plant may contain previously unidentified alkaloids that may also contribute to its in vivo effect. Herein, we used a bioassay-guided multi-dimension separation strategy to discover novel mAchR antagonists from S. tangutica. The core of this approach is to use label-free cell phenotypic assay to first identify active fractions, and then to guide purification of active ligands. Besides four tropanes and six cinnamic acid amides that have been previously isolated from S. tangutica, we recently identified two new tropanes, one new cinnamic acid amide, and nine other compounds. Six tropane compounds purified from S. tangutica for the first time were confirmed to be competitive antagonists of muscarinic receptor 3 (M3), including the two new ones and with IC50 values of 1.97 μM and 4.47 μM, respectively. Furthermore, the cinnamic acid amide displayed 15-fold selectivity for M1 over M3 receptors. These findings will be useful in designing lead compounds for mAchRs and elucidating mechanisms of action of S. tangutica.

No MeSH data available.


Competitive antagonism of alkaloid compounds.(a–c) The dose responses of acetylcholine at 30 min post-stimulation in the presence of 3, 11 and 2, each at different, fixed doses. Here, the compound and acetylcholine are added to co-stimulate HT-29 cells. The control is without addition of compounds. Data were represented as mean ± s.d.
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f5: Competitive antagonism of alkaloid compounds.(a–c) The dose responses of acetylcholine at 30 min post-stimulation in the presence of 3, 11 and 2, each at different, fixed doses. Here, the compound and acetylcholine are added to co-stimulate HT-29 cells. The control is without addition of compounds. Data were represented as mean ± s.d.

Mentions: We applied a co-stimulation DMR assay to determine whether active alkaloid compounds are competitive antagonists or not. Here, acetylcholine at a series of concentrations were prepared in the presence of compound 2, 3, and 11, each at a fixed dose, and then used to co-stimulate HT-29 cells. The three compounds display diverse pharmacological actions. The control without existence of compound is in the left and both 3 and 11 dose-dependently shift the acetylcholine dose curve to right (Fig. 5a and 5b), suggesting that both act as a competitive antagonist for M3 receptor. In contrast, compound 2 (anisodamine) exhibited complicated effect; at a low dose (10 nM) it increases the potency of acetylcholine, but at a high dose (1 μM) it decreases the potency of acetylcholine (Fig. 5c). Further investigation of the biochemical mechanism of these compounds will be of great interest.


Discovery of new muscarinic acetylcholine receptor antagonists from Scopolia tangutica
Competitive antagonism of alkaloid compounds.(a–c) The dose responses of acetylcholine at 30 min post-stimulation in the presence of 3, 11 and 2, each at different, fixed doses. Here, the compound and acetylcholine are added to co-stimulate HT-29 cells. The control is without addition of compounds. Data were represented as mean ± s.d.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5384254&req=5

f5: Competitive antagonism of alkaloid compounds.(a–c) The dose responses of acetylcholine at 30 min post-stimulation in the presence of 3, 11 and 2, each at different, fixed doses. Here, the compound and acetylcholine are added to co-stimulate HT-29 cells. The control is without addition of compounds. Data were represented as mean ± s.d.
Mentions: We applied a co-stimulation DMR assay to determine whether active alkaloid compounds are competitive antagonists or not. Here, acetylcholine at a series of concentrations were prepared in the presence of compound 2, 3, and 11, each at a fixed dose, and then used to co-stimulate HT-29 cells. The three compounds display diverse pharmacological actions. The control without existence of compound is in the left and both 3 and 11 dose-dependently shift the acetylcholine dose curve to right (Fig. 5a and 5b), suggesting that both act as a competitive antagonist for M3 receptor. In contrast, compound 2 (anisodamine) exhibited complicated effect; at a low dose (10 nM) it increases the potency of acetylcholine, but at a high dose (1 μM) it decreases the potency of acetylcholine (Fig. 5c). Further investigation of the biochemical mechanism of these compounds will be of great interest.

View Article: PubMed Central - PubMed

ABSTRACT

81217: Scopolia tangutica (S. tangutica) is a traditional Chinese medicinal plant used for antispasmodics, anesthesia, analgesia and sedation. Its pharmacological activities are mostly associated with the antagonistic activity at muscarinic acetylcholine receptors (mAchRs) of several known alkaloids such as atropine and scopolamine. With our recent identification of four hydroxycinnamic acid amides from S. tangutica, we hypothesized that this plant may contain previously unidentified alkaloids that may also contribute to its in vivo effect. Herein, we used a bioassay-guided multi-dimension separation strategy to discover novel mAchR antagonists from S. tangutica. The core of this approach is to use label-free cell phenotypic assay to first identify active fractions, and then to guide purification of active ligands. Besides four tropanes and six cinnamic acid amides that have been previously isolated from S. tangutica, we recently identified two new tropanes, one new cinnamic acid amide, and nine other compounds. Six tropane compounds purified from S. tangutica for the first time were confirmed to be competitive antagonists of muscarinic receptor 3 (M3), including the two new ones and with IC50 values of 1.97 μM and 4.47 μM, respectively. Furthermore, the cinnamic acid amide displayed 15-fold selectivity for M1 over M3 receptors. These findings will be useful in designing lead compounds for mAchRs and elucidating mechanisms of action of S. tangutica.

No MeSH data available.