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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.


Label-free pharmacological profiling of tropane alkaloids on M3 receptor in HT-29 cells.(a) Real-time DMR response of 16 μM acetylcholine after pretreatment with compound 8 at different doses. (b–d) The dose-dependent inhibition of twelve tropane compounds on the DMR amplitudes at 30 min post-stimulation with 16 μM acetylcholine. Data were represented as mean ± s.d.
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f3: Label-free pharmacological profiling of tropane alkaloids on M3 receptor in HT-29 cells.(a) Real-time DMR response of 16 μM acetylcholine after pretreatment with compound 8 at different doses. (b–d) The dose-dependent inhibition of twelve tropane compounds on the DMR amplitudes at 30 min post-stimulation with 16 μM acetylcholine. Data were represented as mean ± s.d.

Mentions: We systematically characterized the pharmacology of twelve tropane compounds using a two-step DMR assay on M3 receptor in HT-29. Results show that all these compounds induce little DMR response in the first step (exemplified by compound 8 and compound 12 in Fig. S40), but all dose-dependently inhibit the acetylcholine-induced DMR in the second step (exemplified by compound 8 and compound 12 in Fig. 3a and Fig. S40). Among the four known tropane alkaloids, scopolamine (1), hyoscyamine (3) and anisodine (4) are potent antagonists with IC50 values of 0.04 ± 0.01 μM, 0.06 ± 0.01 μM, and 0.31 ± 0.10 μM, respectively. However, anisodamine (2) displays partial efficacy, but moderate potency, to inhibit the acetylcholine DMR, indicating that anisodamine may be an allosteric modulator of M3 receptor. The order of inhibition potency is 1 > 3 > 4 > 2, in consistent with previous report49.


Discovery of new muscarinic acetylcholine receptor antagonists from Scopolia tangutica
Label-free pharmacological profiling of tropane alkaloids on M3 receptor in HT-29 cells.(a) Real-time DMR response of 16 μM acetylcholine after pretreatment with compound 8 at different doses. (b–d) The dose-dependent inhibition of twelve tropane compounds on the DMR amplitudes at 30 min post-stimulation with 16 μM acetylcholine. 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

f3: Label-free pharmacological profiling of tropane alkaloids on M3 receptor in HT-29 cells.(a) Real-time DMR response of 16 μM acetylcholine after pretreatment with compound 8 at different doses. (b–d) The dose-dependent inhibition of twelve tropane compounds on the DMR amplitudes at 30 min post-stimulation with 16 μM acetylcholine. Data were represented as mean ± s.d.
Mentions: We systematically characterized the pharmacology of twelve tropane compounds using a two-step DMR assay on M3 receptor in HT-29. Results show that all these compounds induce little DMR response in the first step (exemplified by compound 8 and compound 12 in Fig. S40), but all dose-dependently inhibit the acetylcholine-induced DMR in the second step (exemplified by compound 8 and compound 12 in Fig. 3a and Fig. S40). Among the four known tropane alkaloids, scopolamine (1), hyoscyamine (3) and anisodine (4) are potent antagonists with IC50 values of 0.04 ± 0.01 μM, 0.06 ± 0.01 μM, and 0.31 ± 0.10 μM, respectively. However, anisodamine (2) displays partial efficacy, but moderate potency, to inhibit the acetylcholine DMR, indicating that anisodamine may be an allosteric modulator of M3 receptor. The order of inhibition potency is 1 > 3 > 4 > 2, in consistent with previous report49.

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.