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Discovery of new muscarinic acetylcholine receptor antagonists from Scopolia tangutica

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.


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Label-free cell phenotypic profiling guided compound preparation and identification.(a) Chromatography of the first dimensional preparation and fraction collection. (b) Representative dynamic mass redistribution (DMR) traces of fraction 8 (F8) and buffer (control) in HT-29 cells (pm represented picometer, shift in resonant wavelength of the biosensor after poststimulation by fraction) (c) The DMR traces of 16 μM acetylcholine after the pretreatment with F8 or buffer for 1 hr. DMR traces in (b,c) represent the mean ± s.d. (n = 4). (d) DMR heat map of 23 fractions and probes in HT-29 and A549 cell lines. The heat map was obtained by cluster analysis of the DMR profiles of the 23 fractions in both cell lines. For each fraction, real responses of both the fraction and the probe after the fraction pretreatment, each at six discrete time points post-stimulation (3, 6, 9, 15, 30, 45 min), were used for the cluster analysis. All fractions were assayed at 1.25 mg/L. The probe was acetylcholine (Ach) for M3 receptor in HT-29, and histamine (His) for histamine receptors in A549. The control was buffer. Color code is green, negative; red, positive; and black, zero response.
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f1: Label-free cell phenotypic profiling guided compound preparation and identification.(a) Chromatography of the first dimensional preparation and fraction collection. (b) Representative dynamic mass redistribution (DMR) traces of fraction 8 (F8) and buffer (control) in HT-29 cells (pm represented picometer, shift in resonant wavelength of the biosensor after poststimulation by fraction) (c) The DMR traces of 16 μM acetylcholine after the pretreatment with F8 or buffer for 1 hr. DMR traces in (b,c) represent the mean ± s.d. (n = 4). (d) DMR heat map of 23 fractions and probes in HT-29 and A549 cell lines. The heat map was obtained by cluster analysis of the DMR profiles of the 23 fractions in both cell lines. For each fraction, real responses of both the fraction and the probe after the fraction pretreatment, each at six discrete time points post-stimulation (3, 6, 9, 15, 30, 45 min), were used for the cluster analysis. All fractions were assayed at 1.25 mg/L. The probe was acetylcholine (Ach) for M3 receptor in HT-29, and histamine (His) for histamine receptors in A549. The control was buffer. Color code is green, negative; red, positive; and black, zero response.

Mentions: To perform the activity-guided purification, the alkaloids enriched from S. tangutica using the SPE method19 were the first subject to separation on an XCharge C18 column. Results showed that the enriched alkaloids gave rises to a series of well separated and symmetric peaks even at an overloading amount on the column (Fig. 1a). Twenty-three fractions (F1 to F23) were collected sequentially according to visible peaks and these fractions have little peak overlapping (Fig. S1).


Discovery of new muscarinic acetylcholine receptor antagonists from Scopolia tangutica
Label-free cell phenotypic profiling guided compound preparation and identification.(a) Chromatography of the first dimensional preparation and fraction collection. (b) Representative dynamic mass redistribution (DMR) traces of fraction 8 (F8) and buffer (control) in HT-29 cells (pm represented picometer, shift in resonant wavelength of the biosensor after poststimulation by fraction) (c) The DMR traces of 16 μM acetylcholine after the pretreatment with F8 or buffer for 1 hr. DMR traces in (b,c) represent the mean ± s.d. (n = 4). (d) DMR heat map of 23 fractions and probes in HT-29 and A549 cell lines. The heat map was obtained by cluster analysis of the DMR profiles of the 23 fractions in both cell lines. For each fraction, real responses of both the fraction and the probe after the fraction pretreatment, each at six discrete time points post-stimulation (3, 6, 9, 15, 30, 45 min), were used for the cluster analysis. All fractions were assayed at 1.25 mg/L. The probe was acetylcholine (Ach) for M3 receptor in HT-29, and histamine (His) for histamine receptors in A549. The control was buffer. Color code is green, negative; red, positive; and black, zero response.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Label-free cell phenotypic profiling guided compound preparation and identification.(a) Chromatography of the first dimensional preparation and fraction collection. (b) Representative dynamic mass redistribution (DMR) traces of fraction 8 (F8) and buffer (control) in HT-29 cells (pm represented picometer, shift in resonant wavelength of the biosensor after poststimulation by fraction) (c) The DMR traces of 16 μM acetylcholine after the pretreatment with F8 or buffer for 1 hr. DMR traces in (b,c) represent the mean ± s.d. (n = 4). (d) DMR heat map of 23 fractions and probes in HT-29 and A549 cell lines. The heat map was obtained by cluster analysis of the DMR profiles of the 23 fractions in both cell lines. For each fraction, real responses of both the fraction and the probe after the fraction pretreatment, each at six discrete time points post-stimulation (3, 6, 9, 15, 30, 45 min), were used for the cluster analysis. All fractions were assayed at 1.25 mg/L. The probe was acetylcholine (Ach) for M3 receptor in HT-29, and histamine (His) for histamine receptors in A549. The control was buffer. Color code is green, negative; red, positive; and black, zero response.
Mentions: To perform the activity-guided purification, the alkaloids enriched from S. tangutica using the SPE method19 were the first subject to separation on an XCharge C18 column. Results showed that the enriched alkaloids gave rises to a series of well separated and symmetric peaks even at an overloading amount on the column (Fig. 1a). Twenty-three fractions (F1 to F23) were collected sequentially according to visible peaks and these fractions have little peak overlapping (Fig. S1).

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.


Related in: MedlinePlus