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Zebrafish bioassay-guided natural product discovery: isolation of angiogenesis inhibitors from East African medicinal plants.

Crawford AD, Liekens S, Kamuhabwa AR, Maes J, Munck S, Busson R, Rozenski J, Esguerra CV, de Witte PA - PLoS ONE (2011)

Bottom Line: Natural products represent a significant reservoir of unexplored chemical diversity for early-stage drug discovery.Zebrafish bioassay-guided fractionation identified the active components of these plants as emodin, an inhibitor of the protein kinase CK2, and coleon A lactone, a rare abietane diterpenoid with no previously described bioactivity.These results suggest that the combination of zebrafish bioassays with analytical chromatography methods is an effective strategy for the rapid identification of bioactive natural products.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, University of Leuven, Leuven, Belgium. alexander.crawford@pharm.kuleuven.be

ABSTRACT
Natural products represent a significant reservoir of unexplored chemical diversity for early-stage drug discovery. The identification of lead compounds of natural origin would benefit from therapeutically relevant bioassays capable of facilitating the isolation of bioactive molecules from multi-constituent extracts. Towards this end, we developed an in vivo bioassay-guided isolation approach for natural product discovery that combines bioactivity screening in zebrafish embryos with rapid fractionation by analytical thin-layer chromatography (TLC) and initial structural elucidation by high-resolution electrospray mass spectrometry (HRESIMS). Bioactivity screening of East African medicinal plant extracts using fli-1:EGFP transgenic zebrafish embryos identified Oxygonum sinuatum and Plectranthus barbatus as inhibiting vascular development. Zebrafish bioassay-guided fractionation identified the active components of these plants as emodin, an inhibitor of the protein kinase CK2, and coleon A lactone, a rare abietane diterpenoid with no previously described bioactivity. Both emodin and coleon A lactone inhibited mammalian endothelial cell proliferation, migration, and tube formation in vitro, as well as angiogenesis in the chick chorioallantoic membrane (CAM) assay. These results suggest that the combination of zebrafish bioassays with analytical chromatography methods is an effective strategy for the rapid identification of bioactive natural products.

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Emodin and coleon AL inhibit endothelial cell migration in vitro.a, control (immediate); b, control; c, 30 µM emodin; d, 30 µM coleon AL.
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pone-0014694-g004: Emodin and coleon AL inhibit endothelial cell migration in vitro.a, control (immediate); b, control; c, 30 µM emodin; d, 30 µM coleon AL.

Mentions: Zebrafish have only recently been utilized for the systematic identification of bioactive small molecules [12], so the predictive power of zebrafish assays for drug discovery will only become clear as molecules found using this platform are advanced into the clinic. In any case, to further evaluate the therapeutic potential of natural products identified using embryonic or larval zebrafish models, bioactive compounds should subsequently be validated using a series of additional in vitro and in vivo assays. To this end, in vitro anti-angiogenesis assays were carried out to further characterize the anti-angiogenic activity of the bioactive natural products isolated in this study, revealing both emodin and coleon AL to inhibit the proliferation, migration and tube formation of mammalian endothelial cells (Table 2, Fig. 4, 5). In addition, both compounds inhibited blood vessel formation in the chick chorioallantoic membrane (CAM) assay (Fig. 6). Emodin and coleon AL inhibited the proliferation of mouse aortic endothelial cells (MAECs) with an IC50 similar to that of the vascular endothelial growth factor (VEGF) receptor inhibitor SU5416, a synthetic indoline derivative [45], and inhibited the proliferation of bovine aortic endothelial cells (BAECs) with an IC50 similar to that of the PI3K inhibitors wortmannin, a fungal furanosteroid [46], and LY294002, a synthetic chromone derivative [47]. In the CAM assay, we determined the anti-angiogenic activity of emodin and coleon AL to be similar to that of SU5416.


Zebrafish bioassay-guided natural product discovery: isolation of angiogenesis inhibitors from East African medicinal plants.

Crawford AD, Liekens S, Kamuhabwa AR, Maes J, Munck S, Busson R, Rozenski J, Esguerra CV, de Witte PA - PLoS ONE (2011)

Emodin and coleon AL inhibit endothelial cell migration in vitro.a, control (immediate); b, control; c, 30 µM emodin; d, 30 µM coleon AL.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3040759&req=5

pone-0014694-g004: Emodin and coleon AL inhibit endothelial cell migration in vitro.a, control (immediate); b, control; c, 30 µM emodin; d, 30 µM coleon AL.
Mentions: Zebrafish have only recently been utilized for the systematic identification of bioactive small molecules [12], so the predictive power of zebrafish assays for drug discovery will only become clear as molecules found using this platform are advanced into the clinic. In any case, to further evaluate the therapeutic potential of natural products identified using embryonic or larval zebrafish models, bioactive compounds should subsequently be validated using a series of additional in vitro and in vivo assays. To this end, in vitro anti-angiogenesis assays were carried out to further characterize the anti-angiogenic activity of the bioactive natural products isolated in this study, revealing both emodin and coleon AL to inhibit the proliferation, migration and tube formation of mammalian endothelial cells (Table 2, Fig. 4, 5). In addition, both compounds inhibited blood vessel formation in the chick chorioallantoic membrane (CAM) assay (Fig. 6). Emodin and coleon AL inhibited the proliferation of mouse aortic endothelial cells (MAECs) with an IC50 similar to that of the vascular endothelial growth factor (VEGF) receptor inhibitor SU5416, a synthetic indoline derivative [45], and inhibited the proliferation of bovine aortic endothelial cells (BAECs) with an IC50 similar to that of the PI3K inhibitors wortmannin, a fungal furanosteroid [46], and LY294002, a synthetic chromone derivative [47]. In the CAM assay, we determined the anti-angiogenic activity of emodin and coleon AL to be similar to that of SU5416.

Bottom Line: Natural products represent a significant reservoir of unexplored chemical diversity for early-stage drug discovery.Zebrafish bioassay-guided fractionation identified the active components of these plants as emodin, an inhibitor of the protein kinase CK2, and coleon A lactone, a rare abietane diterpenoid with no previously described bioactivity.These results suggest that the combination of zebrafish bioassays with analytical chromatography methods is an effective strategy for the rapid identification of bioactive natural products.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, University of Leuven, Leuven, Belgium. alexander.crawford@pharm.kuleuven.be

ABSTRACT
Natural products represent a significant reservoir of unexplored chemical diversity for early-stage drug discovery. The identification of lead compounds of natural origin would benefit from therapeutically relevant bioassays capable of facilitating the isolation of bioactive molecules from multi-constituent extracts. Towards this end, we developed an in vivo bioassay-guided isolation approach for natural product discovery that combines bioactivity screening in zebrafish embryos with rapid fractionation by analytical thin-layer chromatography (TLC) and initial structural elucidation by high-resolution electrospray mass spectrometry (HRESIMS). Bioactivity screening of East African medicinal plant extracts using fli-1:EGFP transgenic zebrafish embryos identified Oxygonum sinuatum and Plectranthus barbatus as inhibiting vascular development. Zebrafish bioassay-guided fractionation identified the active components of these plants as emodin, an inhibitor of the protein kinase CK2, and coleon A lactone, a rare abietane diterpenoid with no previously described bioactivity. Both emodin and coleon A lactone inhibited mammalian endothelial cell proliferation, migration, and tube formation in vitro, as well as angiogenesis in the chick chorioallantoic membrane (CAM) assay. These results suggest that the combination of zebrafish bioassays with analytical chromatography methods is an effective strategy for the rapid identification of bioactive natural products.

Show MeSH
Related in: MedlinePlus